Developmental Biology (9th Edition)

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DEVELOPMENTAL BIOLOGY NINTH

EDITION

Companion Website www.devbio.com

devbio.com is an extensive Web companion to the textbook that is intended to supplement and enrich courses in developmental biology. Here you will find addi­ tional information for advanced students as well as historical, philosophical, and ethical perspectives on issues in developmental biology. Included are articles, movies, interviews, opinions (labeled as such), Web links, updates, and more. (There is even a developmental biology humor page!)

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The site is comprised of Web topics relevant to all areas of the textbook, organized by textbook chapter. These topics are referenced throughout the textbook, both within the chapter, and at the end of the chapter. From the home page of the site, you can browse by chapter, search all topics, or quickly jump directly to any specif­ ic topic. The site also includes the complete Literature Cited for the textbook, most with links to the PubMed database.

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DevBio Laboratory: vade mecum3

An Interactive Guide to Developmental Biology Mary

Tyler and Ronald

Kozlowski

http://labs.devbio.com S.

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Designed to complement the textbook, this unique resource helps you understand the organisms discussed in lecture and prepares you for the laboratory. DevBio laboratory:

which allows you the flexibility to use the software from any computer with Internet access. Access to the site is included with each new textbook.

Over 140 interactive videos and 300 labeled pho­ tographs take you through the life cycles of model organisms used in developmental biology labora­ tories. The easy-to-use videos provide you with the concepts, vocabulary, and motivation to enter the laboratory fully prepared. A chapter on zebrafish addresses how to raise the organism and the effects of various teratogens on embryon­ ic development. The site also includes chapters on: the slime mold Dictyostelium discoideum; planarian; sea urchin; the fruit fly Drosophila melanogaster; chick; and amphibian.

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laboratory Manual: The Third Edition of Mary S. Tyler's laboratory manual ,

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FIGURE 5.23

B

Spiral cleavage of the mol lusc Trochus v iewed from the animal pol e (AI and from one side (BI, The cells deri ved 'i-om the A blastomere are shown in color. The mitotic spindles, 5 'e tc hed in the ear ly stages, divide the cells unequally and at an angle to the vertical and horizontal axes. Each successive quartet of rnicromeres (indicated with lowercase letters) is d i sp l aced to the iight or to the left of its sister macromere (uppercase letters), creat­ ..ng the cha racteristic sp iral pattern.

placed to the right or to the left of its sister macromere, cre­ ating the characteristic spiral pattern. Looking down on the embryo from the animal pole, the upper ends of the mitotic spindles appear to alternate clockwise and coun­ :erclockwise (Figure 5.24) . This arrangement causes alter­ nate micromeres to form obliqu�ly to the left and to the right of their macromeres. At the third cleavage, the A macromere gives rise to hvo daughter cells, macromere 1A and micromere 1a. The B, C, Mid D cells behave sirnilarly, producing the first quartet of micro meres. In most species, these micromeres are to the right of their macromeres (looking down on the animal pole). At the fourth cleavage, macromere 1A divides to :orm macromere 2A and micromere 2a, and micromere 1a divides to form tvvo more micromeres, 1a1 and 1a2 (see fig­ ure 5.23). The micromeres of this second quartet are to the left of the macromeres. Further cleavage yields blastomeres 3_-'\ and 3a from macromere 2A, and micromere 1a2 divides :0 produce cells 1a21 and 1a22. In normal development, the first-quartet micromeres form the head structures, while ilie second-quartet micromeres form the statocyst (balance organ) and shell. These fates are specified both by cyto-

plasmic localization and by induction (Cather 1967; Clement 1967; Render 1991; Sweet 1998). The orientation of the cleavage plane to the left or to the right is controlled by cytoplasmic factors ir1 the oocyte. This was discovered by analyzing mutations of snail coiling. Some snails have their coils opening to the right of their shells (dextral coiling), whereas the coils of other snails open to the left (sinistral coiling). Usually the direction of coiling is the same for all members of a given species, but occasional mutants are found (i.e., in a population of right­ coiling snails, a few individuals will be found with coils that open on the left). Crampton (1894) analyzed the embryos of such aberrant snails and found that their early cleavage differed from the norm. The orientation of the cells after the second cleavage was different in the sinis­ trally coiling snails as a result of a different orientation of the mitotic apparatus (Figure 5.25). In some species (such as the pond snail Physa, an entirely sir1istral species), the sinistrally coiling cleavage patterns are mirror images of the dextrally coiling pattern of the right-handed species. In other instances (such as Lynmaea, where about 2% of the snails are lefties), sinistrality is the result of a two-step process: at each division, the initial cleavage is radial; how­ ever, as the cleavage furrow forms, the blastomeres shift to the left-hand spiral position (Shibazaki et a1. 2004). In Figure 5.25, one can see that the position of the 4d blas­ tomere (which is extremely important, as its progeny will form the mesodermal organs) iS,different in the two types of spiraling embryos.

See WEBSITE 5.2 Alfred Sturtevant and the genetics of snail coiling

1 80

CHAPTER 5

(A) FIGURE 5.24 Spiral cleavage in molluscs. (A) The spiral nature of third cleavage can be seen i n the confocal fluorescence micrograph of the 4-cell embryo of th e clam Acila cas­ trenis. Microtubules stain red, RNA stains green, and the DNA stain s yellow. Two cells and a portion of a third cell are visible; a polar body can be seen at th e top of the micro­ graph. IB-E) Cleavage in the mud snail lIyanassa obsoleta. The 0 blastomere is larger than the others, al lowing the identification of eac h cell. Cl eavage is dextral. (B) 8-cel l stage. PB, polar body. IC) Mid-fourth cleavage 11 2-cell em bryo) . The macro meres have already divided i n to large and small spirally oriented cells; 1 a-d have not divided yet. (D) 32-cell embryo. (A cou rtesy of G. von Dassow and the Center for Cell Dynamics; B-E from Craig and Morrill 1 986, courtesy of the authors.)

(B)

(D)

(C)

In snails such as Lymnaea, the direction of snail shell coil­ ing is controlled by a single pair of genes (Sturtevant 1923; Boycott et al. 1930). In Lymnaea peregra, rare mutants exhibiting sinistral coiling were found and mated with wild-type, dextrally coiling snails. These matings showed that the right-coiling allele, D, is dominant to the left-coil­ ing allele, d. However, the direction of.deavage is deter­ mined not by the genotype of the developing snail but by the genotype of the snail's mother. A dd female snail can produce only sinistrally coiling offspring, even if the off­ spring's genotype is Dd. A Dd individuaL will coil either left or right, depencling on the genotype of its mother. Such matings produce a chart like this: G e n otyp e

DD " x dd rJ

->

Dd

DD rJ x dd "

->

Dd

Dd x Dd

....

tDD:2Dd: ldd

Ph e notype

All eft oili ng All

All right-coiling

l -c

right-coiling

The genetic factors involved in snail coiling are brought to the embryo by the oocyte cytoplasm. It is the genotype

of the ovary in which the oocyte develops that determines which orientation cleavage will take. When Freeman and Lundelius (1982) injected a small amount of cytoplasm from dextrally COiling snails into the eggs of dd mothers, the resulting embryos coiled to the right. Cytoplasm from sinistrally coiling snails did not affect right-coiling embryos. These findings confirmed that the wild-type mothers were placing a factor into their eggs that was absent or defective in the dd mothers. Just as in sea urchins (and vertebrates), the right-left axis comes to be defined by the Nodal family of para crine fac­ tors. In the case of snails, Nodal activates genes on the right side of dextrally coiling embryos and on the left side of sinistrally coiling embryos. Changing the direction of cleav­ age (using glass needles) at the 8-ce11 stage changes the location of Nodal gene expresion (Grande and Patel 2009; Kuroda et aJ. 2009). Nodal appears to be expressed in the C-quadrant micromere lineages (which give rise to the ectoderm). This signal induces the expression of the gene encoding the Pitx transcription factor (a target of Nodal protein in vertebrate axis formation) in the neighboring D­ quadrant blastomeres.

FI o

., '(

7 T

EARLY DEVELOPMENT IN SELEOED INVERTEBRATES

( A)

Left-handed coiling

(E )

Right-handed coiling

A AB Zygote

B

CD D 4d

Looki ng down on the animal pole of leh-coiling and right-coiling WI snails. The origin of sinistral and dextral coiling can be traced to the orientation of the mitotic spindle at t he second cleavage. Left- and righ t-co i l i ng snails develop as mir­ ror images of each other. (Aher Morgan 1 927.1 FIGURE 5.25 AI

The snail fate map

The fa te maps of Ilyanassa obsoleta and CrepUlula Jornicafa were constructed by injecting specifi c micromeres with large polymers conj uga ted to fluorescent dyes (Render 1997; Hej no et a!. 2007). The fluorescence is maintained

l

La Left eye; left velum; apical plate 2a Left vdnm; left stomodeum; upper half left statocyst; lA mantle edge; upper left foot 3a Left velu left esophagus 3AB Velar retractor; digestive glands Ib Velum; apical plate 2b Velum; dorsal stomodeum; I B mantle edge; foot retractor muscle 3b Right vehlm; right esophagus VeJar retractor; digestive glands Ie Right eye; right velum; right tentacle; apical plate 2e Right velum; right stomodeum; upper half right statocyst Ie dorsal mantle edge; upper right foot; heart 3c velum; right statocyst; 2C Right right half of foot; mantle edge 3C Velar retractor; digestive glands; style sac Left velum near eye; apical plate 2d Mantle edge; tip of foot Left velum; left statocyst; ID 3d heart; lef! half of foot 2D MEl Left velar retractor; 4d part of intestine (ME) ME2 Right - retractor; � c cvelar 3D heart; kidney; part of intestine 4D Lumen of digest.ive glands; yolk 01;

C

4d

181

Id

32-

Fate map of ifyana" a 0650/eta. Beads containing Lucifer Yellow were i jected into individual blastomeres at the cell stage. When the embr os developed into larvae, their descen­ dants could be identified by their fluorescence. (After Render FIGURE 5.26

1 997.)

n

y

over the period of embryogenesis and can be seen in the

larval tissue derived from the n i jec ted ceUs. The results of the Tlyanassa studies, shown n F igure 5.26, indicated that the second-quartet rnicromeres (20-d) generally contribute to the shell-forming mantle, the velum, the mouth, and the heart. The third-quartet micromeres (3a-d) generate large

i

1 82

CHAPTER 5

(C)

(A)

FIGURE 5.27 Assoc iation of decapentap/egic (dpp) mRNA with specific centrosomes of lIyanassa. (A) In situ hybridization of the mRNA for the 8MP-like paracr ine factor Opp in the 4-cell snail embryo shows no Opp accumulalion. (81 At prophase of the 4- to 8 cell stage, dpp mRNA (black) accumulales at one centrosome of the pair forming the mitotic spindle. (C) As mitosis continues, -

regions of the foot, velum, esophagus, and heart. The 4d cell-the mesentoblast-contributes to the larval kidney, heart, retractor muscles, and intestine.

dpp mRNA is seen to attend the centrosome in the macromere rather than the centrosome in the micromere of each cell. The dpp message encodes a BMP-like paracrine factor critical to mol­ luscan development. (From Lambert and Nagy 2002, courtesy of L. Nagy.) rial but extrudes it again prior to second cleavage. After this division, the polar lobe is attached only to the D blastomere, which absorbs its material. From this point on, no polar lobe is formed. Crampton

The polar lobe: Cell determination and axis formation

(1896) showed that if one removes the polar

lobe at the trefoil stage, the remaining cells divide normal­ ly. However, instead of a normal trochophore larva/ the

Molluscs provide some of the most impressive examples of both mosaic development-in which the blastomeres

(intestine) and mesodermal organs (such as the heart and

are specified autonomously-and of cytoplasmic localiza­

retractor muscles), as well as some ectodermal organs (such

tion, wherein morphogenetic determinants are placed

as eyes; Figure

in a

result is an incomplete larva, wholly lacking its endoderm

5.29). Moreover, Crampton demonstrated

specific region of the oocyte (see the Part II opener).

that the same type of abnormal larva can be produced b y

Autonomous specification of early blastomeres is especial­

removing the D blastomere from the 4-ole cytop l sm (A), then m igr ti ng _p the presumptive posterior sur· ce of egg (B) ocalize in t e 84."1 l astomere C. ( rom

yt

a

in

a the and becoming d h b F Nishida and Sawada _001 , courtesy of H. Nishida and . Satoh.)

(A)

enchyme. However, FCF Signals from the endoderm prevent these mesenchyme precursors from developing into muscle celis, as we will see later. "'Macho-l mRNA is also localized in the cells that become the mes­

(B)

(e)

1 90

CHAPTER 5

(A)

(B)

(C)

staining of - a e n protein ho nd derm FIGURE 5.38 A ntib po e ,1 Ciona em b r . formation. (A) No p ca e n is seen in the nuclei i n he endoderm pre­ (6) the " O-cell stage. (e) not c or precursor bec m e endoderm and e p e s alkaline normal endoderm; the black show notochordal e pres in g endodermal enzymes. (From Imai et al. 2000, ourtesy H .

ody p c t ni s ws its involvement with e o - t ni in the animal l nuclei of a O-cell yo In contrast, nuclear p-catenin is readily seen t vegetal cursors at When p-catenin is expressed in o h dal cells, those cells will o x r s enclodermal markers such as phosphatase. The white arrows show arrows cells that are x s c of Nishi­ da and N. Satoh.) ically normal. Moreover, B4.1 blastomeres isolated from 11U1cho-I-depleted embryos failed to produce muscle tissue. Nishida and Sawada then injected Inacho-l mRNA into cells that would not normally fonn muscle, and found that these ectoderm or endoderm precursors did generate muscle cells when given macho-I mRNA. Macho-l turns out to be a transcription factor that is required for the activation of several mesodermal genes, including muscle acHl1, myosin. tbx6, an d snail (Sawada et aJ. 2005; Yagi et al. 200Sa). Of these gene products, only the Tbx6 protein produced muscle differentiation (as Macho-l did) when expressed in cells ectopically. Macho-1 thus appears to directly activate a set of tbx6 genes, and Tbx6 proteins activate the rest of muscle development (Yagi et al. 200Sb). Thus, the macho-I message is found at the right place and at the right time, and these experiments suggest that Macho-I protein is both necessary and sufficient to promote muscle differentiation in certai� ascidian cells. The Macho-1 and Tbx6 proteins also appear to activate the muscle-specific gene snail. Snail protein is important in preventing 8rachyury (T) exp ressio n in presumptive muscle cells, and is therefore needed to prevent the mus­ cle precu rsors from becoming notochord cells. It appears, then, that the Macho-l transcription factor is a critical com­ ponent of the tunicate yellow crescent, muscle-forming cytoplasm. Macho-l activates a transcription factor cas­ cade that promotes muscle differentiation while at the same time inhibiting notochord specification. AUTONOMOUS SPECI FICATION OF T H E E N DODERM: p­ CATENIN Presumptive endoderm originates from the veg­ etal A4.1 and B4.1 blastomeres. The specificati on of these cells coincides with the scription factor

localization of p-catenin, a tran­ discussed earlier in regard to sea urchin

endoderm specification. Inhibition of p-catenm results in the loss of endoderm and its replacement by ectoderm in the ascidian embryo ( Figure 5.38; Imai et aL 2000). Con­ versely, Increasing p-catenin synthesis causes an increase in the endoderm at the expense of the ectoderm (just as in sea urchins). The p-catenin transcription factor appears to function b y activating the synthesis of the homeobox tran­ scription factor Lhx-3. Inhlbition of the lhx-3 message pro­ hibits the differentiation of endoderm (Satou et al. 2001). CONDITIONAL SPECIFICATION OF T H E .MESENCHYME AND NOTOCHORD BY THE ENDODERM While most of the mus­

cles are specified autonomously from the yellow crescent cytoplasm, the most posterior muscle cells form through conditional specification by ceB interactions with the descendants of the A4.1 and b4.2 blastomeres (Nishida 1987, 1992a,b). Moreover, the notochord, brain, heart, and mesenchyme also form through inductive Interactions. In fact, the notochord and mesenchyme appear to be induced by the fibroblast growth factor that is secreted by the endo­ derm cells (Nakatani et aL 1996; Kim et aL 2000; Imai et al. 2002). The posterior cells that will become mesenchyme respond differently to the FGF signal due to the presence of Macho-1 in the posterior vegetal cytoplasm (Figure 5.39; Kobayashi et aL 2003). Macho-1 prevents notochord induc­ tion in the mesenchymal cell precursors by ac tiva ting the snnil gene (which will in turn suppress the activation of 8rachyllry). Thus, Macho-l is not only a muscle-activating determinant, it is also a factor that distinguishes cell response to the FGF Signal. These FGF-responding cells do not become muscle, because FGF also activates cascades that block muscle formation-another role that is con­ served in vertebrates. As can be seen in Figure 5.40, the

:

EARLY DEVELOPMENT I N SELECTED I N V E RTEB RATES

FIGURE 5.39 Th e two-step process for specifying the marginal cells of the tuni­ cate embryo. The first step involves th e acquis ition (or nonacquisition) by the cells of the Macho-1 transcripti on factor. The secon d step involves the reception or non reception) of the FGF signal from :he endoderm. (After Ko bayash i et al. 1003.)

Anterior ,---,---,_-,

en �� Macho-1 RNA"-erm fla nki ng Ihe venlral midline. (C) Dorsal view of a slightly older embryo, showing pole cells and posterior endoderm sinking into the embryo. (0) Dorsolateral view an embryo at fullest germ band extension, just prior to segmentation. The cephalic _trOW separates the future head region (procephalon) from Ihe germ band, which will m the thorax and abdomen. (E) lateral view, showing fu llest extension of the germ :.and and the beginnings of segmen tation . Subtle indentations mark the incipient seg· .....nts along the germ band. Ma, Mx, and Lb correspond to Ihe mandibular, max i l l ary, :-.l labial head segments; Tl-T3 are the thor.cic segment'; and A l-A8 are the axIominal segments. (F) Germ band reversing direction. The true segments are now .s.ible, as well as the other territories of the dorsal head, such as the c1ypeolabrum, ",ephalic region, optic ridge, and dorsal ridge. (G) Newly halched firsl-instar larva. otographs courtesy of F. R. Turner. D aher Campos-Ortega and Hartenstein t 98S.)

.

(G)

208

CHAPTER 6

FIGURE 6.5 Schematic representation of gastrulation in Drosophila. Anterio r is to the left; dorsal is facing upward. (A,B) Surfac e and cutaway views showing the fates of the tissues immediately prior to gastrulation. (C) The begi n ni ng of gastrul atio n as the ven tra l mesoderm i nvaginates into the embryo. (0) This view corresponds to Figure 6.4A, while (E) correspon ds to Figure 6.4B,C. I n (E), the neuroectoderm is largely differentiated into the nervous system and the epidermis. (After Campos� Ortega and Ha rtenstein 1 985.)

the anterior and posterior ends of furrow. The p ole cells are internalized a long with the endoderm ( Figure 6.4B,C) . At this time , the embryo b ends to form the cephalic furrow. The ectodermal cells on the surface and the mesoderm tmdergo convergence and extension, migrating toward the ventral midline to form the germ band, a collection of cells along the ventral midline that includes all the cells that will form the trunk of the emb ryo. The germ band extends pos­ teriorly and, p erha p s because of the egg case, w ra ps around the top (dorsal) surface of the embryo (Figure 6.40) . Thus, at the end of germ band formation, the cells destined to form the most posterior larval structures are located immediately behind the future head region (Figure 6.4E) . At this time, the b ody segments begin t o appear, dividing the ectoderm and mesoderm. The germ band then retracts, placing the presumptive posterior segments at the poste­ rior tip of the embryo ( Fi gure 6.4F) . At the dorsal su rface, the two sides of the epidermis are brought together in a process called dorsal closure. The amnioserosa, which had

Internal ectoderm

Amnioserosal covering of

( A)

to form two p ocke ts at

the ventral

(B)

(C)

(D)

been the most dorsal structure, interacts with the epider­

I t

cells to encourage their migration (reviewed in Pan­ filio 2007; Heisenbe rg 2009). While the germ band is in its extended position, sever­ al key morphogenetic processes occur: organogenesis, seg­ mentation, and the segregation of the imaginal discs.' In addition, the nervous system forms from mo regions of mal

�� ---'�

ventral ectoderm. Neuroblasts differentiate from this neu ­

rogenic ectoderm ",ithin each segment (and also from the nonsegmented region of the head ectoderm). Therefore in insects like Drosophila, the nervous system is located ven� tra lly, rather than be ing derived from a dorsal neural tube as in verteb rates (Figure 6.S) . The general body plan of Drosophila is the same in the embryo, the larva, and the a dul t each of which has a dis­ tinct head end and a distinct tail end, between which are repeating segmental units ( Figure 6.6). Three of these seg­ ments form the thorax, while another eight segments form the abdomen. Each segment of the adult fly has its own identity. The first thoracic segment, for exa mple, has only

Epidermis

,

,

*lmaginal discs are those cells set aside to produce the adult struc­

in Chapter 1 5 . For more information on Drosophila developmental

tures. The details of imaginal disc differenti(ltion will be discussed

(lnatomy, see Bate and Martinez�Arias 1993; Tyler and Schetzer 1996: and Schwatm 1997.

Nervous system

legs; the second thoracic segment has legs and wings; and the third thoracic segment has legs and halteres (balancing organs). Thoracic and abdominal segments can also be dis­ tinguished from each other by differences i.n the cu tiele of the newly hatched first-instar larvae.

GENES THAT PATTERN THE

D ROSOPH I LA BODY PLAN

Most of

the genes involved in shaping the· larval and adult 1990s u sing a powerful "forward genetics" approach. The basic strat­ egy was to randomly mutagenize flies and then screen for mutations that disrupted the normal formation of the body forms of Drosophila were identiiied in the early

THE G E N ETICS OF AXIS SPECIFICATION IN A)

DROSOPHILA

20 9

(B) Head

Metatho rax

Abdominal gments

FIGURE 6.6 Comparison of larval l eft) and adult (right) segmen­ tation in Drosophila. In th e adult, the three thoracic segments can be distinguished by their appendages: Tl (protherax) has legs o n l y; T2 m esoth e ra x) has wings and legs; T3 (metatherax has nalteres (not visible) and legs. (BI Segmen ts i n ad u lt tra nsgen ic Drosophila in which the gen e fo r green fl uorescen t protei n has n fused to the cis-regulatory region of the engrai/ed gene. Thus, GFP is produced in the areas of engraifed transcription, ... hich is active at the border of each segment and in the posterior l e es . compartment of the wing. (B courtesy of

(A)

(

(

)

A. K b 1

Primary Axis Formation during Oogenesis The processes of embryogenesis may "offiCially" begin at fertilization, but many of the molecular events critical for Drosophila embryogenesis actually occur during oogene­ sis. Each oocyte is descended from a single female germ cell-the oogonium-which is surrounded by an epithe­ lium of follicle cells. Before oogenesis begins, the oogonium

divides four times with incomplete cytokinesis, giving rise

to

16

interconnected cells:

15

nurse cells and the single

oocyte precursor. These 16 cells constitute the egg

cham­

ber (ovary) in which the oocyte will develop, and the oocyte will be the cell at the posterior end of the egg cham­ Ian. Some of these mutations were quite fantastic, and

ber (see Figure 16.4). As the oocyte precursor develops,

included embryos and adult flies in which specific body

numerous mRNAs made in the nurse cells are transport­

structures were either missing or in the wrong place.

These

:JIutant collechons were distributed to many different lab­

ed on microtubules through the cellular interconnections into the enlarging oocyte.

oratories. The genes involved in the mutant phenotypes were cloned and then characterized with respect to their expression patterns and their functions, This combined

Anterior-posterior polarity in the oocyte

effort has led to a molecular understanding of body plan

The follicular epithelium surrounding the developing

Drosophila that is unparaUeled in aU of biol­

oocyte is initially uniform with respect to cell fate, but this

y, and in 1995 the work resulted in a Nobel Prize for

uniformi ty is broken by two signals organized by the

evelopment in

E ward Lewis, Christiane Niisslein-Volhard, and Eric .. 'jeschaus,

The rest of this chapter details the genetics of Drosophi­

the same gene, gllrken. The gllrken message appears to be oocyte nucleus. Intereshngly, both of these signals involve synthesized in the nurse cells, but it becomes transported

development as we have come to upderstand it over the

specifically to the oocyte nucleus. Here it is localized

past two decades. First we will examine how the dorsal­

between the nucleus and the cell membrane and is trans­

'entral and anterior-posterior axes of the embryo are estab­

lated into Gurken protein (Caceres and Nilson

2005).

At

. hed by interactions between the developing oocyte and

this time the oocyte nucleus is very near the posterior tip

.- surrounding follicle cells. Next we will see how dorsal­

of the egg chamber, and the Gurken signal is received by

-entral patterning gradients are formed

within the embryo,

the follicle cells at that position through a receptor protein

d how these gradients specify cli!ferent tissue types. The

encoded by the torpedo gene< (Figure 6.7A). This signal

rmed along the anterior-posterior axis, and how the dif­

results in the "posteriorization" of these follicle cells ( Fig-

=w-d part of the discussion will examine how segments are

:erent segments become specialized. Finally, we will briefly !low how the positioning of embryOniC tissues along the ..'o primary axes specifies these tissues to become partic­

�,

":ar organs.

*Molecular analysis has established that gurken encodes a homo­ logue of the vertebrate epidermal growth factor (EGF), while torpe­ do encodes a homologue of the vertebrate EGF recep tor (Price et a1. 1989; Neuman-Silberberg and Sch upbach 1993).

CHAPTER 6

210

(A)

Posterior

Anteri or Nurse cells

Nucleus

o

T rpedo (Curken receptor )

e

T r min al follicle c lls

e

Uncommitted

polar fol l icle ce lls

o

j

o

o

(8)

Anterior follicle cells

Curken protein

Microtubules

o

Posterior follicle

(D)

bicoid mRNA

Anterior border cells

oskar mRNA in

associa tion with ki nesin I Nucleus

(E) Dorsal

I)

o o

Curken

0

Oskar protein (F) �-'-

An terior

FIGURE 6.7 The anterior- posteri o r axis is specified during ooge­ nesi s. (A) The oocyte moves into the posterior region of the egg chamber, while nurse cells fill the anterior portion. The oocyte nucleus moves toward the terminal follicle cells and synthesizes Gurken protein (green). The terminal follicle cells express Torpe­ do, the receptor for Gurken. (8) When Gurken binds to Torpedo, the terminal fol licle cells differentiate into posterior follicle cells and synthesize a molecule that activates protein kinase A in the egg. Protein kinase A orients the microtubules such that the grow­ ing end is at the posterior. (C) The Par-l protein (green) localizes to the cortical cytoplasm of nurse cells and to the posterior pole of the oocyte. (The Stauffen protein marking the posterior pole is labeled rcd; the red and green signals combine to Ouoresce yel­ low.) (D) The bicoid message binds to dynein, a motor protein associated with the non -growing end of microtubules. Dynein moves the bicoid message to the anterior end of the egg. The oskar message becomes complexed to kinesin I, a motor protein that moves it toward the growing end of the microtubules at the posterior region, where Oskar can bind the nanos message. (E) The nucleus (with its G u rken protein) migrates along the micro­ tubules, inducing the adjacent follicle cells to become the dorsal follicles. (F) Photomicrograph of bicoid mRNA (s tai ned black) passing from the nurse cells and localizing to the anterior end of the oocyte during oogenesis. Ie co u rtesy of H. Docrflinger; F from 5tephanscn et 01. 1 988, courtesy of the authors.) ure 6.7B). TI,e posterior follicle cells send a signal back into O,e oocyte. The identity of this signal is not yet known, but it recruits the par-l protein to t he po s ter i or edge of the

Posterior

oocyte cytoplasm (Figure 6.7C; Doerflinger et a1. 2006). Par­ I protein org aniz es microtubules specifically with their

T H E G E N ETICS OF AXIS SPECI FICATION I N DROSOPHILA

(8)

211

(C)

D)

nllilus (cap) and plus (growing) ends at the anterior and posterior ends of the oocyte, respectively (Gonzalez-Reyes

1995; Roth et a1. 1995; Januschke et a1. 2006). The orientation of the m.icrotubules is critical, because different microtubule motor proteins will transport their

et a1.

FIGURE 6.8 Expression of the gurken message and protein between the oocyte nucleus and the dorsal anterior cell mem­ brane. (A) The gurken mRNA is l ocal i zed between the oocyte nucleus and the dorsal follicle cells of the ovary. Anterior is to the left; do rsal faces upward. (8) The Gurken protein is si m ila rly locat­ ed (sh ow n here in a younger stage oocyte than A). (C) Cross sec­ tion of the egg through the reg ion of Gurken protein expressi on. (D) A more mature oocyte, showing Gurken protein (yellow) across the dorsal region. The actin is stained red, showi ng cell boundaries. As the oocyte grows, follicle cells migrate across the top of the oocyte, becomi n g exposed to Gurken. (A from Ray and Schupbach 1 996, courtesy of T. Schupbach; B,C from Peri et al.

1 999, courtesy of S. Roth; D courtesy of C. van Bu ski rk and T. Schupbach.)

A or protein cargoes in different directions. The motor

protein kinesin, for instance, is an ATPase that will use the energy of ATP to transport material to the plus end of the motor protein that will transport its cargo the opposite way.

ing event takes place. Here the gurken message becomes localized in a crescent between the oocyte nucleu::i and the

One of the messages transported by

oocyte cell membrane, and its protein product forms an

microtubule. Dynein, however, is a "minus-directed"

kinesin along the microtubules to the posterior end o f the oocyte is oskar A (Zimyanin et a1. 2008). The oskar mRNA is not able :.

be translated until it reaches the posterior cortex, at

which time it generates the Oskar protein. Oskar protein �ruits more par-l protein, thereby stabilizing the micro­ :ubule orientation and allowing more material to b e rECruited to the posterior pole o f

the oocyte (Doerflinger 2006; Zimyanin et a1. 2007). The posterior pole will :hereby have its own distinctive cytoplasm, called pol e

et .1.

?lasm, which contains the determinants for producing the �bdomen and the germ cells. This cytoskeletal rearrangement in the oocyte is accom­ :-anied by an increase in oocyte volllIT!e, owing to transfer i cytoplasmic components from

. icoid and

romponents

the nurse cells. These

include maternal messengers such as the

Hllnos

mRNAs. These mRNAs are carried by

=totor proteins along the micro tubules to the anterior and ""sterior ends of the oocyte, respectively ( Fi gure

6.7D-F) .

.\s we shall soon see, the protein products encoded by

-oid and 1IatJos are critical for establishing the anterior­

:'OSterior polarity of the eU1bryo.

( Fi gure 6.8; Neuman-Silberberg and Schupbach 1993). Since it can diffuse only a short distance, Gurken protein reaches only those follicle cells closest to the oocyte nucleus, and it signals those cells to become the more columnar dorsal follicle cells (Montell et al. 1 991; SchUp­ bach et a1. 1991; see Figure 6.7E). This establishes the dor­ anterior-posterior gradient along the dorsal surface of the

oocyte

sal-ventral polarity in the fullicle cell layer that surrounds

the growing oocyte.

Maternal deficiencies of either the gurkell or the torpedo

gene cause ventralization of the embryo. However, gurken

is active only in the oocyte, whereas torpedo is active only in the somatic follicle ce lls.

This fact was revealed by experi­

ments with germline/somatic chimeras. Tn one such exper­

iment, SchUpbach

(1987)

transplanted genm cell precursors

from wild-type embryos into embryos whose mothers car­

ried the torpedo mutahon. Cunversely, she transplanted the germ cell precursors from

embryos (Figure

torpedo mutants into wild-type

6.9). The wild-type eggs produced mutant,

ventralized embryos when they developed in a

torpedo

mutant mother's egg chamber. The torpedo mlltant eggs were able to produce normal embryos if they developed

•

:Jorsal-ventral patterning in the oocyte oocyte volume increases, the oocyte nucleus moves to m anterior dorsal position where a second major signal-

in a wild-type ovary. Thus, unlike Gurken, the Torpedo protein is needed in the follicle celis, not in the egg itself. TI,e GlUken-Torpedo signal that specifies dorsalized fol­ licle cells initiates a cascade of gene

activities that create

212

CHAPTER 6

n /-'" ;

Embryo from wild-type ! ! mother

torpedo.deficient germ cells in a wild-type female

\ \

,

,

\

)

n·- -.' V

Embryo from / mother i deficient in :. torpedo gene

(

\.

\

-+

torpedo-deficient oocyte in wild-type follicle

pOle cells ,--(germ ceU precursors)

.... .

----1.� ,

Wild-type germ ceJls ill a torpedo­ deficient female

\

- :;;­ E'--

i

V

-' .. . �

�:;s���el"ral aXIS

No dorsal-ventral -----.... axis (entire embryo ventral)

Wild-type germ cells ill a torpedodeficient follicle

FIGURE 6.9 Germline chimeras made by inlerchanging pole cells (germ cell precu rsors) between wild·lype embryos and emb ryos from mothers homozygous for a mutation of the torpedo gene. These IransplanlS produced wi ld-Iype females whose eggs came from mutant mothers, and torpedo-deficient females that laid wild-type eggs, The torpedo·defi cient eggs produced normal embryos when they developed in the wil d-type ovary, whereas the wild-type eggs produced ventra l ized embryos when they developed in the mutant mother's ovary.

and Snake is found throughout the perivitelline space sur­ rounding the embryo. Indeed,

this protein is very similar clot­

to the mammalian protease inhibitors that limit blood

ting protease cascades to the area of injury. In this way, the proteolytic cleavage of Easter and Spatzle is strictly limit­ ed to the area around the most ventral embryonic cells. The cleaved Spii tzle protein is now able to bind to its

the dorsal-ventral axis of the embryo (Figure 6.1 0) . The acti­ the pipe gene. As a result, Pipe protein i s made only in the ventral follicle cells (Sen et a1. 1998; Amiri and Stein 2(02). In some as yet unknown way (probably involVing sulfa­ tion), Pipe activates the Nudel protein, whlch is secreted

vated Torpedo receptor protein inhibits the expression o f

to the cell membrane of the neighboring ventral embryon­ ic cells (see Zhang et a!.

2009). A few hours later, activated

Nudel initiates the activation of three serine proteases that are secreted into the perivitelline fluiq (see Figure

6. 10B; 1995). These proteases are the prod­ ucts of the gastmlatiol1 defective (gd), snake (sl1k), and easter (en) genes. Like most extracellular proteases, these mole­ euJes are secre ted in an inacti ve form and. are subsequent­ ly activated by peptide cleavage. In a complex cascade of

Hong and Hashlmoto

events, activated Nudel activates the Gastrulation-defec­

tive protease, The Cd protease cleaves the Snake protein, activating the Snake protease, which in turn cleaves the Easter protein. This cleavage activates the Easter protease, whlch then cleaves the Spatzle protein (Chasan et a!.

1992; 1995; LeMosy et a!. 2001). It is obviously important that the cleavage of these three proteases be limited to the most ventral portion of the embryo. This is accomplished by the secretion of a protease Hong and Hashlmoto

inhibitor from the follicle cells of the ovary (Hashimoto et a!.

2003;

Ligoxygakis

et a!. 2003). Thls inhibitor of Easter

receptor in the oocyte cell membrane, the product of the toll gene . Toll protein is a maternal product that is evenly FIGURE 6.1 0 Generaling dorsal-ventral polarity in Drosophila. (A) The nucleus of Ihe oocyte travels 10 whal will become Ihe dor·

mRNA that becomes localized bet .... veen the oocyte nucleus and the cel l membrane, where it is translated into Gurken protei n . The sal side of I he embryo. The gurken genes of Ihe oocyte synlhesize

Gurken signal is received by the Torpedo receptor protein made by the follicle cells (see Figure 6.7). Given the short diffusibi l i ty of the signal, only the follicle cells cl osest 10 the oocyte nucleus (Le., the dorsal fol licle cells) receive Ihe Gu rken signal, which causes the fall icle cells 10 take on a characleristic dorsal foil icle morphol­ ogy and inhibits the synthesis of Pipe prolein. Therefore, Pipe pro­ tein is made only by Ihe ventral follicle cel l s. (8) The ventral region at a slightly later stage of development. Pipe modifies an unknown protei n (x) and allows it to be secreted from the ventral follicle cells. Nudel protein interacts with this modified factor to split the product of the gastrulation defective gene, which then splits the product of the snake gene to create an active enzyme that will split the inactive Easter zymogen into an active Easter protease. The Easter protease splits the Spatzle protei n into a form Ihal can bind 10 Ihe Tol l receplor (which is found Ihroughout Ihe embryonic cell membrane). This protease activity of Easter is striclly l i m ited by the protease inhib itor found in the perivitelline space. Thus. only the ventral cells receive the Tol l signal. This sig· nal separates the Cactus protein from the Dorsal protein, al lowing Dorsal to be translocated into the nuclei and ventralize the cells. (After van Eeden and st. Johnslon 1 999.)

T H E GENETICS OF AXIS SPECIFICATION I N DROSOPHILA

distributed throughout the cell membrane o f the egg ashlmoto et a1. 1988, 1991), but it becomes activated only by binding the Spatzle protein, which is produced only on the ventral side of the egg. Therefore, the Toll receptors on the ventral side of the egg are transducing a signal into the egg, while the Toll receptors on the dorsal side of the egg are not. This localized activation establishes the dorsal-ven­ tral polarity of the oocyte.

Generating the Dorsal-Ventral Pattern in the Embryo Dorsal, the ventral morphogen The protein that distinguishes dorsum (back) from ven­ trum (belly) in the fly embryo is the product 01 the dorsal gene. The mRNA transcript of the mother's dorsal gene is

(A)

(E)

\\ (porsa!) j} � �� c:/

Nucleus

cells

Oocyte

'\ Caclus ',� � .�e sections

show that the originally symmetrical organ rudiments acquire asymmetric positions by week 1 '1 . The liver moves to the right and the spleen moves to the len. (B) N ot only does the heart move to the left side of the body, but the originally symmetrical veins of the heart regress dif­ ferentially to form the superior and inferior venae cavae, which connect only to the right side of the heart. (C) The right lu n g branches Into three lobes, while the len lung (near the heart) forms only two lobes. In human males, the scrotum also forms asymmetrically. (After Kosaki and Casey 1 998.)

not move and the situs (l ateral position) of each asymmet­ rical organ was randomized.

This finding correlated extremely well with other data. long been known that humans with a dynein deficiency had immotile cilia and a random chance of hav­ ing their hearts on the left or right side of the body (Afzelius 1976). Second , when the iv gene described above was cloned/ it was found to encode the ciliary dynein pro­ tein (Supp et al. 1997) . Third, when Nonaka and colleagues (2002) cultured early mouse embryos under an artificial

First, it had

flow of medium from left to right, they obtained a rever­ of the left - right axis. Moreover, the flow was able to direct the polarity of the left-right axis in iv-mutant mice, whose cilia are otherwise immotile. Why should fluid flow be all-impor tant to establishing left-righ t asymmetry? The reason may reside in small (around 1 flm), membrane-bound pa rticl es called nodal vesicular p a rcel s (NVPs). These "parcels," which contain Sonic hedgehog protein and retinoic acid, are secreted from the node cells under the influence of FGF signals (Fig ure sal

B I R DS AND MAMMALS

oa

N d l vesicular �� �m

�\�� (B)

(A)

FGF Ca2+ Nodalflow �\ ��

•

�

�. iJ}I ' � n \ :�L.�--" �JL -'L \ �. . eili

o

Right

•

�

0

-

0

J

'�

31 9

0

��

:

. JI ':;".:....:

'

\1

o

•

.

•

•

Left

FIGURE 8.35 La tera li ty axis formation in mammals. (A) Ciliated cells of the mam­ malian node. (B) Schematic drawi ng showing the FGF-induced secretion of nodal vesicular parcels from the cells of the node, the movement of the NVPs to th e l eh side motivated by the ciliary currents, and the rise in Ca2+ concentration on the left side of the node. (e) Calcium ions (red, green) concentrated on the lefl side of the node in mice. (A courtesy of K. Sulik and G. C. Schoenwolf; B aher Tana ka et al. 2005; C courtesy of M. B u eckn er. )

(e)

,

Right

Although fish, amphibian, avian, and mammalian embryos

Left

have different patterns of cleavage and gastrulation, they

use many of the same molecules to accomplish the same goals. Each group uses gradients of Nodal proteins to

left side of the body; if FCP signaling is inhibited, the pa rcels are not secreted and left-right asymmetry fails to become established (Tanaka et a1. 2005). Such a method of

establish polarity along the dorsal-ventral axis. In Xenopus

elivering paracrine factors from one set of ceUs to anoth­

from the posterior marginal zone" while elsewhere Nodal

8.35 6). Jt appears tha t ciliary flow carries the NVPs to the

er represents a newly discovered mode of signaling.

One of the results of the transport of the NVPs is the rise of calcium ions on the left side of the node (Figure 8.35C;

how the expression of genes such as nodal become placed

:"evin 2003; McGrath et aJ. 2003). It is yet to be discovered

and zebrafish, rna ternal factors induce Nodal proteins in the vegetal hemisphere or marginal zone. In the chick, Nodal expression is induced by Wnt and Vgl emanating activity is suppressed by the hypoblast. In the mouse, the hypoblast similarly restricts Nodal activity, although the source of its ability to do so remains uncertain. Each of these vertebrate groups uses BMP inhibitors to specify the dorsal axis; however, they use them in differ­

under the control of these ion fluxes; but we are beginning

ent ways. Simila rly, Wnt inhibition and Otx2 expression

:0 tmderstand the differences between right

are important in specifying the anterior regions of the

and left.

Coda

these proteins. In

all cases, the region of the body from the is specified by Hox genes. Finally, the

embryo, but different groups of cells may be expressing hindbrain to the tail

,"aria tions on the important themes of development have

left-right axis is established through the expression of

"" olved in the different vertebrate groups (Figure 8 .36) .

Nodal on the left-hand side of the embryo. Nodal activates

-::-he maj or themes o f vertebrate gastrulation include: �. lnternalization of the endoderm and mesoderm

: Epiboly of the ectoderm around the entire embryo , Convergence of the internal cells to the midline �

Extension of the body along the anterior-posterior axis

Pitx2, leading to the differences between the left and right left side appears to differ among the vertebrate groups. But

sides of the embryo. How Nodal becomes expressed on the overall, despite their initial differences

in cleavage and gas­

trulation, the vcrtebrate� have maintained very similar ways of establishing the three body axes.

320

CHAPTER 8

Zcbrafish

Frog

Chicken

MOllse

Dan;o rerio

Xenopus /nevis

Gallus gallus

1\11115 muscuills

Early cleavage

I

Area pellucid..

Late cleavage

D ' S � � Ep;bl,,' _

Early gastrula

Blastopore

Primary

S«ondary

hypobla.u

hypoblast

�. I

Prechordal

forebrain

Epiderm.i!; Fordmun

M;db"in brain

Pharyngula

--\-t::\

ectoderm

o..SMO

�

Hindbrain $omi..

Pre-

Extra-

embryonic

Hypoblast

SMO

Late gastrula

\

'C

Epiblast

SMO

A�a opaca

--'1

SMO

somilic mesodum

Prechordal

� lGllt '

Spinal cord� SMO

Prechordal

plate m�erm Notochord

endoderm

p�.

�omilic mtsOderrn Somite

Spinal cord

BI RDS AND MAMMALS -

-"

.. .�

3

2/3 4

5 6

� ��

Subventricular zone (white matter)

60 40 20

�---,,------' Whma iter 2/3

5

4

6

)

Cortical layer s

Homigsrtanteiounral �

tO t

Vewnntericular Cel -autonomousfate ..h.. ena e transplanted after last S ph s

thre

FIGURE 9.24 Cortical neurons are generated from types of neural precursor cells: radial glia cells, short neural precursors, and intermediate pro­ genitor cells. RGCs and SNPs divide al the apical (luminal) surface of the ven· tricular layer. SNPs are committed neu­ ral precursors. IPes divide away from the luminal surface in the ventricular ?nd subventricular zones. Most IPes undergo neurogenic divisions, with a small fraction undergoing symmetrical proliferative divisions (dotted circular arrow). Through asymmetrical divisions, RGCs give rise to IPes that migrate to the subventricular layer. The ventricular zone generates lower-layer neurons; the subventricular zone generates upper­ layer neurons. (After De ay and Kennedy 2007.)

h

353

t Radial glial cell (RGC)

�

Short ncuronal precursor

(D)

Homi��gsrtanteiounral cel n e phfatse wh n _ Glial

Host (conditional) t ra spJam d in S

e

354

CHAPTER 9

SIDEL IGH TS SPECULATIONS

Adult Neural Stem Cells

O

(B) CA) ntil recently, i t was generally believed that once the mam­ malian nelVQUS system was mature, no new neurons were "born" -in other words, the neurons formed in utero and during the first few years of life were all we could ever expect to have. The good news from recent studies, however, is that the adult brain is capable of producing new neurons, and environmental stim­ ulntion can increase the n umber of these new neurons. In these experiments, researchers injected adult mice, rats, or mar­ mosets with bromodeoxyuridine (BrdU), a n ucleoside that resemb les thymidine. SrdU is incorporated into a ce l l 's DNA only if the cell is undergo­ i n g DNA replication; therefore, any cell labeled with BrdU must have been undergoing DNA synthesis dur­ ing the time it was exposed to BrdU. Figure 9.25 Evidence of adult neural stem This l abe l ing technique revealed that cells. The green s tai n i ng, which indicates newly divi ded cells, is from a fluo rescent thousands of new neurons are being antibody agai nst BrdU (a thym idi ne ana­ made each day in adult mice. More­ l ogue that is taken up only duri ng the 5 over, these new brain cells integrated phase of the cell cycle). (A) Newly gen erated with other cells of the brain, had nor­ adult mouse neurons (green cells) have a mal neuronal morphology, and exhib­ no rma l mo rpho logy and receive synaptic ited action potentials (Fi g u re 9.25A; inputs. The red spots are synaptophysin, a van Praag et a l . 2002). pro tei n found on the dendrites at the synaps· Injecting h u mans with SrdU is usu� es of axons from other neurons. (8) Newly ally unethical, since l a rge doses of generated neuron (arrow) in the adul t BrdU are often lethal. However, i n human brain. This cell is located in the den­ certain cancer patients, the progress of tate gyrus of the hippocampus. The red fluo­ chemotherapy is monitored by trans­ rescence is from an anti body that stains only fusing the patient with a small amount neural cells. Yellow indicates the overlap of of BrdU. Gage and colleagues (see red and green. Glial cells are stained purple. Erikkson et al. 1 998) took postmortem (A from van Praag et al. 2002; B from Erikks­ samples from the brains of five such son et al. 1 998, photograph courtesy of F. H. patients who died between 1 6 and Gage.) 781 days after the BrdU infusion. tn all five subjects, they saw labeled (new) neurons i n the granular cell layer of The existence of neural stem cells the h i ppocampal dentate gyrus (a part in adults is now well established for of the brain where memories may be the olfactory epithelium and the hip­ formed). The BrdU-labeled cells also pocampus {Kempermann et al. stained for neuron-specific markers 1 997a,b; Kornack and Rakic 1 999; (Figure 9.256). Thus, although the rate van Praag et al. 1 999; Kato et a l . of new neuron formation in adulthood 2001). These cells respond to Sonic may be relatively low, the human hedgehog and can proliferate to brain is not an a natomical fait accom­ become multiple ce l l types for at least pli at birth, or even after childhood. the first year of a mouse's l ife (Ahn

and Joyner 2005). I t appears that the

stem cells prodUCing these neurons are located in the ependyma (the for­ mer ventricular zone, i n which the embryonic neural stem cells once resided) or in the subventricular zone (SVZ) adjacent to it (Doetsch et al. 1 999; Johansson et al. 1 999; Cassidy and Frisen 2001). These adult neural stem cells represent only about 0.3% of the ventricle wall cell population, but they can be distinguished from more differentiated cells by their cell surface proteins* (Rietze et a l . 2001 ). In the adult mouse, thousands of new neuroblasts are generated each day, migrating from the lateral SVZ to the olfactory bulb, where they differenti­ ate into several different types of neu­ rons. Recent evidence suggests that *These neural stem cells may have partic� u l ar physiological roles as wel l. During

pregnancy, prolactin stimulates the pro� duction of neuronal progenitor celts in the subventricular zone of the adult mouse forebrain. These progenitor cells migrate to produce olfactory neurons that may be important for maternal behavior of rearing offspring (Shingo e t a l . 2003).

T H E EMERGENCE O F T H E ECTODERM

355

SIDELIGHTS & SPECULATIONS (Conti n u ed ) ste m ce l ls are n ot mult i poten t beco ming spec i fi ed only when th ey reach the o lfacto ry bulb) but i nstead are a pop u latio n of h eterogenous neu­ robl asts that a re already committed to becoming certain neuronal types ,V\e rk l e et al. 2007). These adult neu­ ral stem cells proliferate in respo nse to exercise, learni ng, and stress (Zhang et

th ese

al. 2008).

The existence of adult neural stem cells in the cortex is more controver­ sial (see Gould 2007). Some i nvestiga-

tors (Gould et al. 1 999a,b; Magavi et al. 2000) c laim to have identified them; other scie ntists (see Rakic 2002) q uestion the existence of these corti­ cal neural stem cells. The mechanisms by which neural stem cells are kept i n a state of ready qu iescence well into adulthood are c urrently being explo red . Before they become neu­ rons, neural stem cells are character­ ized by the expression of the NRSE translational inhibi tor that prevents neuronal differentiation by binding to

a si lencer region of DNA (see Chapter 2). When n eu ra l stem cells begin to differe nt i a te, they synthesize a small, double-stranded RNA that has the same sequence as the si lencer and which might bind NRSE and thereby perm it neuronal differentiation (Kuwabara et al. 2004). The use of c u l­ tured neuronal stem cells to regener­ ate or repair parts of the brain wi ll be considered in Chapter 1 7 .

CORTICAL CELL MIGRATION The first cortical neurons to be

times. One of these switches is the gene encoding the tran­

generated migrate out of the germinal zone to form the

When the mouse neuronal progen­ to form the first layer of cortical neurons, Foxgl is not exp ressed in the progenitor cells or in the first­ formed neurons. However, later, when the progenitor cells generate those neurons destined for layers 4 and 5, they express tlus gene. If tl.e FoxgI gene is conditionally knocked out of this In.eage, the neural precursor cells con­ tinually give rise to layer 1 neurons. Therefore, it seems

:ransient prep l ate (Kawauch.i and Hoshino 2008). Subse­ quently generated neurons migrate into the preplate and "'parate it into two layers: the Cajal-Retzius layer and the 5ubplate. The Cajal-Retzius layer becomes and remains the most superficial layer of the neocortex, and its cells express

the cell surface glycoprotein Reelin. The subplate remains .he deepest layer through which the successive waves of neuroblasts travel to form the cortical plate. The Reelin­ producing cells of the Cajal-Retzius layer are critical in the separation of the preplate. In Reelin-deficient mice, the pre­ plate fails to sp lit, and the neurons produced by the ger­ minal layers pile up behind the previously generated neu­

scription factor Foxg 1 . itor cells divide

that the Foxgl transcription factor is required to suppress

the "layer

1" neural fate.

Neither the vertical nor the horizontal organization of the cerebral cortex is clonally specified-that is, none of the functional units form from the progeny of a single cell.

(instead of migrating through them). By activating the

Rather, the developing cortex forms from the mixing of

'\latch pathway, Reelin on the surface of the Cajal-Retzius

cells derived from numerous stem cells. The early region­

rons

cells allows the neuronal stem cell to produce a long fiber

alization of the neocortex is

that extends through the cortical plate (Hashimoto-Torii et

paracrine factorS secreted by the epidermiS and neural crest

2008; Nomura et al. 2008). This (and the fact that it pro-

cells a t the margins of the developing brain (Rakic et al. 2009). The paracrine factors induce the expression of tran­ scription factors in the specific brain regi ons, which then mediate the survival, differentiation, proliferation, and migration of the newly generated neurons. For instance, Fgf8 protein is secreted by the anterior neu­ ral ridge and is important for specifying the telencephalon (Figure 9.26). If Fgf8 is overexpressed n. the ridge, specifi­ cation of the telencephalon is extended caudally, whereas if Fgf8 is ectopically added to the caudal region of the cortex, part of that caudal region will become anterior (Fukuchi­ Shimogori and Grove 2001, 2005). Sonic hedgehog is secret­ ed by the medial ganglioniC eminence and helps form the ventral neurons of the cortex, including those of the sub­

al.

uced some proteins thought to be glial-specific) caused the neural stem cell to be called the radial glial cell. The

process from this cell becomes cr iti.cal for

the migra tion of

the neural cells produced by the germinal zones. We also know that there are mutations that specifically affect the microtubular cytoskeleton of the migrating neu­

geJ:>e prevent neuronal with microtubule assembly; humans with such mutations have been seen to suffer from mental dysfunctions, among them autism, bipolar enchyme (Figure 1 1 .9). Ectodermal Signals appear to cause the peripheral somitic cells to undergo mesenchymal-to-epithelial transition by lowering the Cdc42 levels in these cells. Low Cdc42 levels alter the cytoskeleton, allowing epithelial cells to form a box around the remaining mesenchymal cells, which have a higher level of Cdc42. Another small GTPase, Rac1, must be at a certain level that allows it to activate Paraxi�, another tran­ scription factor involved in epithelialization (Burgess et a1. 1995; Barnes et aJ. 1997; Nakaya et aJ. 2004). The epithelialization of each somite is stabilized by syn­ thesis of the extracellular matrix protein fibronectin and the adhesion protein N-cadherin (Lash and Yamada 1986; Hatta et aJ. 1987; Saga et al. 1997; Linask et al. 1998). N-cad­ herin links the adjoining cells into an epithelium while the fibronectin matrix acts alongside the Ephrin and Eph to promote the separation of the somites from each other (Martins et a1. 2009).

Separation of somites from the unsegmented mesoderm Two proteins whose roles appear to be critical for fissure formation and somite sep aration are the Eph tyrosin e kinases and their ligands, the ephrin proteins. We saw in Chapter 10 that the Eph tyrosine kinase receptors and their ephrin ligands are able to elicit cell-cell repulsion between the posterior somite and migrating neural crest cells. The separation of the somite from the presomitic mesoderm occurs at the ephrin B2/Eph A4 border. In the zebra fish, the boundary between the most recently separated somite and the presomitic mesoderm forms between ephrin B2 in the posterior of the somite and Eph A4 in the most anteri­ or portion of the presomitic mesoderm (Figure 1 1 .8; Durbin et a1. 1998). Eph A4 is restricted to the boundary area in chick embryos as well. Interfering with this signallng (by injecting embryos with mRNA encoding dominant nega­ tive Ephs) leads to the formation of abnormal somite bOlllldaries. In addition to the posterior-ta-anterior induction of the fissure (from Eph proteins to ephrin proteins on their neighboring cells), a second signal originates from the ven­ tral posterior cells of the somite, putting all the ceUs in reg­ ister so that the cut is clean from the ventral to the doral aspects of the somite (Sato and Takahashi 200S).

,

Somite specification along the anterior­ posterior axis Although all somites look identical, they will form differ­ ent structures. For instance, the somites that form the cer­ vical vertebrae of the neck and lumbar vertebrae of the abdomen are not capable of forming ribs; ribs are generat­ ed only by the somites forming the thoracic vertebrae. Moreover, spedfication of the thoracic vertebrae occurs

Epithelialization of the somites Several studies in the chick have shown that epithelializa­ tion occurs immediately after somitic fission occurs. As

(A)

(B) Somites: Anterior Posterior

2

3

Somite number 5 4

7

-l ephriu 82 --+--""

EphA4 --+-�

Unsegmented paraxial mesoderm

FIGURE 1 1 .8 Ephrin and its receplor constitute a poss i ble fissure site for somite formation. (A) Expression pattern of the receptor tyrosine kinase Eph A4 (blue) and its ligand, ephrin 62 (red), as somites deve lop. The somite boundary forms at the junction between the region of ephrin expression on the posterior of the last somite formed and the region of Eph A4 expression on the anterior

of the next somi te to form. In the presomitic mesoderm, the pattern is created anew as each somite buds off. The posteriormosl (egion ofthe next somite to form does not express ephrin until that somite is ready to separale. (6) In situ hybridizalion showing Eph A4 (dark blue) expression as new somites form in the chick embryo. (A after Durbin el al. 1 998; 6 courtesy of J . Kaslner.)

421

PARAXIAL AND INTERMEDIATE MESODERM

(A)

Epithelial cel s ��t>1 r����������;��,! �e /�"':'J1�������.vIkQ Formed �;:..i • -1 3 �' 3 C,-' 4 3 (� 1 C)/ .,2 r 2-->1 \ 3-->2 "C/. 3 of t i n I D 4) ? •

-

I

s

7

7 F Q-..•

b--' 4

FIGURE 13.22 Regulation digit iden ti ty by BMP concentrations in the interdigital space anterior to the digit and by Gli3. (A) Scheme for removal of interdigital (10) regions. The results are shown in (B) and (C), respec ively. (B) Removal of ID region 2 between digit primordia 2 (p2) and 3 (p3) causes digit 2 to change to the structure of digit 1 . (C) Removing ID region 3 (between digit primordia 3 and causes digit 3 to form the struc­ tures digit 2. (D) Control digits and their ID spaces. (E,FIThe same transformations as in (B) and (C) can be obtained by adding

of

" 4

/

I

La •

32-->2

iii

•

t

(E )

4

b

(F)

1Q

.....

•c

4

beads containing the BMP inhibitor Noggin to the ID regions. (E) When a Noggin-containing bead (green dot) is placed in I D region 2 , digit 2 i s transformed into a copy of digit 1. (F) When the Noggin bead is placed region .1, digit 3 is transformed into a copy of digit 2. (G)The forelimb of a mouse homozygous for deletions of both gli3 and shh is characterized by extra digits of no specific type. (After Dahn and Fallon 2000; litingtung et al. 2002; B-G, photographs courtesy of R. D. Dahn and J. F. Fallon.)

4

502

CHAPTER 1 3

were placed in the webb ing between digits 3 and 4, digit 3 was anteriorly transformed into digit 2 ( F igure 1 3.22 D-F). digit has a characteristic array of nodules that form the digit skeleton, and S uz uki and olleagues (2008) have shown that the differen t levels of BMP signaling in the interdigital webbing regulate the recruitment of progress zone mesen hyma l cells into the nodules that make the

Each

(A)

c

c

digits.

(B)

vp

Generation of the Dorsal-Ventral Axis

,

The third axis of the

limb distinguishes the dorsal half of (knuckles, nails) frolll the ventral half (pads, soles). In 1974 MacCabe and co-workers demonstrated that the dorsal-ventral polarity of the limb bud is determined by the ectoderm encasing it. If the ectoderm is rotated 180 degrees with respect to the limb bud mesenchyme, the dor­ sal-ventral axis is partially reversed; the d i stal elements (digits) are "upside- � o v �

" '0 � oo 2! c � � v '"

s ;:c �

90 80 70

• o

• •

Carbamoyl phosphatetransferase synthase Orni t hi n e carbamoyl Argininosuccinate synthetase Argininosuccinate lyase

60 50

Urea excretion

40 30

20 f-

.1

t o f0

/

545

I�I

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--.J

_ _ _ _

FIGURE 15.3 me orpho

Develop e t

. (A)

the

cycle

ra

exc ete (8) The of enzyme activities cor­ with metamorphic changes in the frog Rana catesbeiana. (Aher Cohen 1 970.1

ammorna. Like most terrestrial vertebrates, many adult frogs (such as the genus Rann, although not the more aquatic Xenopus) are ureotelic: they excrete urea, wruch requires less water than arrunonia excretion. During meta­ morphosis, the liver begins to synthesize the enzymes nec­ essary to create urea from carbon dioxide and alrunonia (Figure 15.3). TJ may regulate this change by inducing a set of transcription factors that specifically activates expres­ sion of the urea-cycle genes while suppressing the genes responsible for arrunonia synthesis (Cohen 1970; Atkinson et al. 1996, 1998).

Hormonal control of amphibian metamorphosis

i

The control of metamorphosis by thyroid hormones was f rst demonstrated in 1912 by Gudernatsch, who discov­ ered that tadpoles metamorphosed prematurely when fed powdered horse thyroid glands. In a complementary study, Allen (1916) found that when he removed or destroyed the thyroid rudinlent of early tadpoles (thyroidectomy), the larvae never metamorphosed but instead grew into giant tadpoles. Subsequent studies showed that the sequential steps of anuran metamorphosis are regulated by increas­ ing amounts of thyroid hormone (see Saxen et a!. 1957; Kollros 1961; Hanken and Ha ll 1988). Some events (such as the development of limbs) occur early, when the con­ centration of thyroid hormones is low; other events (such as the resorption of the tail and remodeling of the intes-

tine) occur later, after the hormones have reached higher concentrations. These observations gave rise to a thresh· old model, wherein the different events of metamorpho­ sis are triggered by different concentrations of thyroid hor­ mones. Although the threshold model remains useful, molecular studies have shown that the timing of the events of amphibian metamorphosis is more complex than just increasing hormone concentrations. The metamorphic changes of frog development are brought about by (1) the secretion of the hormone thyrox­ ine (TJ into the blood by the thyroid gland; (2) the conver­ sion of T4 intu the more active hormone, tri-iodothyronine (TJ) by the target tissues; and (3) the degradation of TJ in the target tissues (F gure 15.4). TJ binds to the nuclear thy­ roid hormone receptors (TRs) with much higher affinity than does T¥ and causes these receptors to become tran­ scrip t onal activators of gene expression. Thus, the levels of both T, and TRs in the target tissues are essential for pro­ ducing the metamorphic response in each tissue (Kistler et a!. 1977; Robinson et aJ. 1977; Becker et a ! . 1997) .

i

i

The concentration ofT, in each tissue is regulated by the concentration of T, in the blood and by two critical intra­ cellular enzymes that remove iodine atoms from T4 and T3. Type n deiodinase removes an iodine atom from the outer ring of the precursor hormone (T4) to convert it into the more active hormone T3. Type 111 deiodinase removes an iodine atom from the inner ring of T3 to convert it into an inactive compound that will eventually be metabolized to tyrosine (Becker et aJ. 1997). Tadpoles that are genetically modified to overexpress type III deiodinase in their target tissues never complete metamorphosis (Huang et al. 1999). There are two types of thyroid hormone receptors. In Xenopus, thyroid hormone receptor a (TRa) is widely dis­ tributed throughout all tissues and is present even before the organism has thyroid gland. Thyroid honnone recep-

a

546

H0

CHAPTER 1 5

9- 9-

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0

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CH,-CH- COOH I NH,

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Thyroxine (T,) ( rela ively i nactive)

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09-'

ype II deiodinase

HO

h

---

"8 •

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:;:

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Gradient shown in �

Compartmentalization and anlerior- posterior pat­ terning i n the wing imaginal disc. (A) In the first instar l arva, the a nteri or-posterior axis has been formed and can be recognized by the expression of th e engrailed gen e in the posterior compart· ment. Engrailed, a t ranscripti on factor, activates the hedgehog gene. Hedgehog acts as a short-range paracrine factor to activate decapentaplegic (dpp) in th e anterior cells adjacent to the posteri­ or compartment, where Opp and a related protein, Glass-bottom boat (ebb), act over a l onger range. (B) Dpp and ebb proteins FIGURE 15.14

l

1990).

become the most distal structures of the leg-the claw and

(A)

i

disc epithelium (Condie et al. Using fluorescently labeled phalloidin to sta n the peripheral microfilaments of leg disc cells, they showed tha, the cells of early third instar discs are tight y arranged along the proximal-distal axis. When the hormonal Signal to dif­ ferentiate is given, the cells change their shape and Ihe leg is everted, the central cells of the disc becoming the rna distal (claw) cells of the limb. The leg structures will differ­ entiate within the pupa, so that by the time the adult fly ecloses, they are fully formed and functional. cell shape changes within the

paracrine facto r) . High concentrations of these paracrine factors cause the expressi on of the Distal-less gene. Mod­ erate concentrations cause the expression of the dachshund gene, and lower concentrations cause the expression of the

�

...... . . . . - -. . . . . .�

prOJecte gradient : ofGbb : : : + Dpp "". /'1:-- : ::

L2 L3 14

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omb

create a concentration gradi ent of BMP-like signaling, measured by the p hosphoryl ati on of Mad (pMad). High concentrations of Dpp plus Gbb near the source activate both the spail (san and oplOmolOr blind (omb) genes. Lower concentrations (near the periphery) activate omb but ot sal. When Dpp plus ebb levels

n

drop below a certain threshold, brinker (brk) is no longer repressed. L2-L5 mark the longitudinal wing veins, with L2 being the most anterior. (After Bangi and Wharton 2006.)

POSTEMBRYONIC DEVELOPMENT (A)

(B)

t

FIGURE 15.15 surface of

(e)

555

Determining the dorsol-ventral axis. (A) The p rospective ventral the wing is stained by antibodies to Ves igial protein (green), while the prospective dorsal surface is stai ned by antibodies to Apterous protein (red). The region of yellow illustrates where the two proteins overlap in the margi n . (B) Wingless protein (purple) synthesized at the marginal juncture organizes the wing disc along the dorsal-ventral axis. The expression of Vestigial (green) is seen in cells dose to those exp ressi n g Wi ngless. (C) The dorsal and ventral portions of the wing disc te lescope out to form the tvvo-Iayered wing. Gene expression patterns are indicated on the double-layered wing. (A,B courtesy of s. Carroll a ndS. Paddock.)

Dorsal (Apt o s expression) Ventral (Vestigial expression) er u

. Margm (Wingless expression)

factor Hedgehog. Hedgehog functions only when cells have the receptor (Patched) to receive it. In a complex man­ ner, the diffusion of Hedgehog activates the gene encod­ ing Oecapentaplegic (Dpp) in a na rrow stripe of cells in the anterior reg ion of the wing disc (Ho et al. 2005). Opp and a co-expressed BMP called Glass-bottom boat (Gbb) act to establish a gradient of BMP signaling activity. BMPs activate the Mad transcription factor (a Smad protein) by phosphorylating it, so this gradient can be measured by the phosphorylation of Mad. Dpp is a short-range paracrine factor, while Gbb exhibits a much longer range of diffusion to create a gradient (Figure 15.14B; Bangi and Wharton 2006). nus signaling gradient regulates the amount of cell prolif­ eration in the wing regions and also specifies cell fates (Rogulja and Irvine 2005). Several transcription factor genes respond differently to activated Mad. At high levels, the spalt (sal) and optomotor blind (omb) genes are activated, whereas at low levels (where Gbb prOVides the primary signal), only omb is activated. Below a particular level of phosphorylat­ ed Mad activity, the brinker (brk) gene is no longer inhibit­ ed; thu s brk is expressed outside the signaling domain. Spe­ cific cell fates of the wing are specified in response to the action of these transcription factors. (For example, the fifth longitudinal vein of the wing is formed at tl1e border of opto­ motor blind and brinker; see Figure 15.14B). DORSAL-VENTRAL AND PROXIMAL-DISTAL AXES The dorsal­ ventral axis of the wing is fomled at the second instar stage by the expression of the apterolls gene in the prospective

dorsal cells of the wing disc (Blair 1993; Oiaz-Benjumea

and Cohen 1993). Here, the upper l aye r of the wing is dis­ tinguished from the lower layer of the wing blade (Bryant 1970; Garcia-Bellido et aL 1973). The vestigial gene remains "on" in the ventral portion of the wing disc (Figure 15.15A). The dorsal portion of the wing synthesizes transmembrane proteins that prevent the intermixing of the dorsal and ven­ tral cells (Milan et aL 2005). At the boundary between the dorsal and ventral comparlments, the Apterous and Ves­ tigial transcrip tion factors interact to activate the gene encoding the Wn t pa racr ine factor Wingless (Figure 1 5.15B). Neumann and Cohen (1996) showed that Wing­ less protein acts as a growth factor to promote the cell pro­ liferation that extends the wing.' Wingless also helps estab­ lish the proximal-distal axis of the wing: high levels of Wingless activate the Distal-less gene, which speCifies the most distal regions of the wing (Neumann and Cohen 1996, 1997; Zecca et aL 1996):This is in the central region of the disc and "telescopes" outward as the distal margin of the wing blade (Figure 15.15C). See WEBSITE 1 5.1 The molecular biology of wing formation See WEBSITE 1 5.2 Homologous specification *The diffusion of paracrine factors such oS Wingless and Hedgehog is facilitated when these factors cluster on lipid spheres that can travel betvoJeen cells without getting caught in the extracellular matrix (Glise et a!. 2005; Gorfinkiel et al. 2005; Panakova et al. 200S).

556

CHAPTER 1 5

Hormonal control of insect metamorphosis Although the details of insect metamorphosis differ among species, the general pattern of honnonal action is very sim­ ilar. Like amphibian metamorphosis, the me tamorphosi s of insects is regulated by systemic hormonal signals, which are controlled by neurohormones from the brain (for reviews, see Gilbert and Goodman 1981; Riddiford 1996). Insect molting and metamorphosis are controlled by two effector hormones: the steroid 20-hydroxyecdysone (20E) and the lipid juvenile hormone UH) (Figure 1 5. 16A) . 20Hydroxyecdysonet initiates and coordinates each molt and regulates the changes in gene expression that occur dur­ ing metamorphosis . Juvenile hormone prevents the ecdysone-induced changes in gene expression that are nec­ essary for metamorphosis . Thus, its presence during a molt ensures that the result of that molt is another larval instar, not a pupa or an adult.

The molting process (Figure

1 5. 1 6 B) is initiated

in the

brain, where neurosecretory cells release prothoracicotrop­

ie hormon e (PTTH)

in response to neural, hormonal, or

environmental signals. PTTH is

a peptide hormone with a molecular weight of a pp roximately 40,000, and it stimu­ lates the production of ecdysone by the prothoracic gland by activating the RTK pathway in those cells (Rewitz et a1. 2009). Ecdysone is modilied in peripheral tissues to become

cHic mRNAs

are not replaced,

and new

.rn RNA

s are syn­

thesized whose protein products inhibit the transcription 0:

the larval messages.

There are two maj or p u lses of 20E d uring Drosophi:.l first pulse occurs in the third instar larva and triggers the initiation of ("prepupal") morpho­ genesis of the leg and wing imaginal discs (as well as the death of the larval hindg ut) . The larva stops eating and migra tes to find a site to begin pupation. The second 20E pulse occurs 10-12 hours later and tells the "prepup a" te become a pupa. The head inverts and the salivary gland> degenerate (Riddiford 1982; Nijhout 1994). It appears, then that the first ecdysone pulse during the last larval ins tar triggers the processes that inactivate the l arva-specific metamorphosis. The

genes and initiates the morphogenesis of imaginal disc

structures. The second pulse transcribes pupa-specific genes and initiates the molt (Nijhou t 1994). At the imagi­ nal molt, when 20E acts in the absence of juvenile hormone, the imaginal discs fully differentiate and the molt gives rise to an

adult.

See WEBSITE 1 5.3 Insect metamorphosis The molecular biology of 20-hydroxyecdysone activity

the active molting hormone 20E. Each molt is initiated by

ECDYSON E RECEPTORS 20-Hydroxyecdysone cannot bind

one or more pulses

to DNA by itself. Like amphibian thyroid hormones, 20E first binds to nuclear receptors. These proteins, called ecdysone receptors (EcRs), are almost identical n i structure to the thyroid hormone receptors of amphibians. An EcR protein forms an. active molecule by pairing with an Ultra­ spiracle (Usp) protein, the homologu e of the amphibian RXR that helps form the active thyroid hormone receptor

of 20E. For a larval molt, the first pulse produces a small rise in the 20E concentration in the larval hemolymph (blood) and elicits a change in cellular com­

mitment in the epidermis. A second, larger pulse of 20E

associated with molting. the epidermal cells to synthesize enzymes that digest the old cuticle and s ynthesize a new one. Juvenile hormone is secreted by the c orpora allata. The secretory cells of the corp ora allata are active during lar­ initiates the differentiation events

These pulses of 20E commit and stimulate

val molts but inactive during the metamorphic molt and the imaginal molt. As long as

JH is present, the 20E-stim­

ulated molts result in a new larval instar. In the last larval instar, however, the medial nerve from the bra.in to the co[­ pora allata inhibits these glands from producing JH, and there is

a simultaneous increase in the body's ability to degrade existing JH (Sa franek and Williams 1989). Both these mechanisms cause JH levels to drop below a critical threshold value, triggering the release of PTTH from the brain (Nijhout and Williams 1974; Rountree and Bollen­ bacher 1986). PTTH, in turn, stimulates the prothoracic gland to secrete a small amount of ecdysone. The result­ ing pulse of 20E, in the absence of high levels ofjH, com­ mits the epidermal cells to pupal development. Larva-spe-

tSince its discovery in ] 954, when Butenandt and Karlson isolated 25 mg of ecdysone from 500 kg of silkworm moth pupae, 20hydroxyecdysone has gone under several names, i.ncluding ccdys­ terone, p-ecdysone, and crustecdysone.

(Koelle et al. 1991; Yao et al. 1992; Thomas et al. 1993). In

the absence of the hormone-bound EcR, the Usp protein binds to the ecdysone-responsive genes and inhibits their transcription.* This inhibition is converted into activation when the ecdysone receptor binds to the Usp (Schubiger and Truman 2000).

Although there is only one gene lor EcR, the EcR mRNA be spliced in at least three different ways to form three distinct proteins. AU three EcR proteins have the same domains for 20E and DNA binding, but they differ in their amino-terminal domains. The type of EcR present in a cell may inform that cell how to act when it receives a hor­ monal signal (Talbot et aJ. 1993; 'Ihunan et al. 1994). All cells appear to have some EcRs of each type, but the strictly lar­ val tissues and neurons that die when exposed to 20E are characterized by their abundance of the EcR-BI isoforru 01 the ecdysone receptor. Imaginal discs and differentiating transcript can

neurons, by contrast, show a preponderance of the EcR-A

*The Ultraspirade protein may be a juvenile hormone receptor, or J1iR (see Figure 15.16), suggesting mechanisms whereby JH can block 20E at the level of transcription (Jones et at 2001; Jones ct al. 2001, 2007; Sa,orith et al. 2002).

557

POSTEMBRYONIC DEVELOPMENT

�I I , "" "" �

Brain

(8 )

(A) luvenHe hormone (jH)

o

'H

cells

� ../""' Neurosecretory �-:0:::':�:�:�rOthoracic gland

OCH3 Juvenile hormone

Ecdysone

---

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Corpora

JH receptor OHR) OH

si"Mol gnalt"ing

HO

("molting"

\ '7 ��

ProthO�iC� gland

V JH-JHR

of

-�� � n allata

o

"Differentiating signal"

o

20-Hydroxyecdysone (20E) hormone) OH HO :

Ecdysone

f

e /'

20E

OH HO HO o

FIGURE 1 5,16 Regulation of insect meta­ morphosis. (A) Structures of juvenile ho r­ mone (JH), ecdysone, and the ilctive molting hormone 20-hydroxyecdysone (20E), (B) Gen eral pathway of insect metamorphosis. 20E and I H toge the r cause molts that form the next larval instar. When the concentra­ tion of I H becomes low enough, Ihe 20E­ induced molt produces a pupa i nstead of an instar. When ecdysone acts in the absence of JH, the imaginal discs differentiate and the molt gives ri se to an adult (Imago). (After Gilbert and Goodman 1 981 .)

PrQtein """h,esi,

Protein

t

t

Protein synthesis

synthesis

Adult structures

Pupal structures Cuticle

Larva

isoform. Mutations in specific codons that are found in only some of the splicing isoforms indicate that the different forms of EcR play different roles in metamorphosis and that the different receptors activate dUferent sets of genes when

they bind 20E (Davis et a!. 2005).

BINDING OF 20-HYDROXYECDYSONE TO DNA During molt­

ing and metamorphosis, certain regions of the polytene

chromosomes of Drosophila puff out in the cells of certain organs at certain times (CLever 1966; Ashbumer 1972; Ash­ burne r and Berondes 1978). These chromosome p uffs are areas where DNA is being actively transc ribe d . Moreover,

P up

a

these organ-specific patterns of chromosome puffing can be reprod uced by c ul turing larval tissue and add ing hor­ mones to the medium, or by adding 20E to an c arl y-stafie larva. When 20E is ad d ed to larval salivary glands, certain puffs are produced and others regress (Figure 1 5.1 7) . The puffing is mediated by the binding of 20E at specific places on the chromosomes; fluorescent antibodies against 20E find this hormone localized to the regions of the genome that are sensitive to it (Groneme yer and Pongs 1980). At these sites, the ecdysone bound receptor complex recruits a hislone methyltransferase that me thylatos lysine 4 of his­ tone H3, thereby loosening the nuclcosornes in that area -

-

(Sedkov et aJ.

2003).

See VADE MECUM Chromosome squash

558

(AI

CHAPTER 1 5

I I"' I!" ,� , ��\"� il .,,: I

L

FIGURE 15.17 20E-induced puffs in cultured salivary gland cells of D. mel.nogaster. (A) Uninduced control. (S-E) 20E-stimulated chromosomes at (S) 25 minutes, (C) , hour, (D) 2 hours, and (E) 4 hours. (Courtesy of M. Ashburner.)

Figure

15.18A shows a simplified schematic for the

in Drosophila. 20E binds to the EcR/USP receptor complex. It activates the "earl\' response genes," including E74 and £75 (the puffs in Figure 15. 17), as well as Broad and the EcR gene itself. The tran­ scription factors encoded by these genes activate a second series of genes, sucl1 as £75, DHR4, and DHR3. The prod­ ucts of these genes are transcription factors that work together. First, they activate {3FTZ-Fl, a gene encoding a transcription factor that enables a new set of genes to respond to the second burst of 20E. Secondly, the products of these genes shut off the early genes so that they do not interfere with the second burst of 20E. Moreover, DHR. coordinates growth and behavior in the larva. It allows the larva to stop feeding once it reaches a certain weight and to begin searching for a place to glue itself to and form a pupa (Urness and Thummel 1995; Crossgrove et al. 1996; Klng-Jones et al. 20OS). The effects of these two 20E pulses can be extremely dif­ ferent. One example of this is the ecdysone-mediated changes in the larval salivary gland. The early pulse of 20E activates the Broad gene, whicl1 encodes a family of tran­ scription factors through differential RNA splicing. The targets of the Broad complex proteins include those genes that encode the salivary gland "glue proteins." The glue proteins allow the larva to adhere to a solid surface, where it becomes a pupa (Guay and Guild 1991). So the first 20E pulse stimulates the function of the larval salivary gland. However, the second pulse of 20E calls for the destruction of this larval organ (Buszczak and Segraves 2000; Jiang et a1. 2000). Here, 20E binds to the EcR-A form of the ecdysone receptor (Figure When complexed with USP, it activates the transcription of early response genes E74, E75, and Broad. But now a different set of targets is activated. These transcription factors activate the genes encoding the apoptosis-promoting proteins Hid and i p2 gene Reaper, as well as blocking the expression of the da (whim would otherwise repress apoptosis). Thus, the first 20E pulse activates the salivary gland, and the second 20E pulse destroys it. Like the ecdysone receptor gene, the Broad gene can gen­ erate several different transcription factor proteins through differentially initiated and spliced messages. Moreover, the variants of the ecdysone receptor may induce the synthesis of particular variants of the Broad proteins. Organs such as the larval salivary gland that are destined for death during metamorphOSiS express the 21 isofonn; in1aginal eliscs des­ tined for differentiation express the 'Z2 isoform; and the cen­ tral nervous system (whim undergoes marked remodeling during metamorphOSis) expresses all isoforms, with Z3 preframework of metamorphosis

(E)

20E-regulated chromosome puffing occurs during the late stages of tl,e third instar Drosophiln larva, as it prepares to form the pupa. The puffs can be divided into three cat­ egories: "early" puffs tl,at 20E induces rapidly; "intermolt" puffs that 20E causes to regress; and "late" puffs that are first seen several hours after 20E stiml).lation. For example, in the larval salivary gland, about six puffS emerge with­ in a few minutes of hydroxyecdysone trealment. No new protein has to be made in order for these early puffs to be induced. A much larger set of puffs is. induced later in development, and these late puffs do need protein synthe­ sis to become transcribed. Ashburner (1974, 1990) hypoth­ esized tl1at the "early pulf" genes make a protein product that is essential for the activation of the "late pulf" genes and that, moreover, this early regulatory protein itself twns off the transcription of the early genes ' These insights have been confirmed by molecular analyses. "The observation that 20E (ootroHed the transcriptional units of chromosomes was an extremely important and exciting discovery. This was our first real glimpse of gene regulation in eukaryotic organisms. At the time when this discovery was made, the only examples of transcriptional gene regulation were in bacteria.

15.18B).

POSTEMBRYONIC D EVELOPMENT

(A)

Pupatio , , , 20E + EcR/USP , , , ,,

t

n

Head eversion , , 20E + EcRfUSP ,, , , , , , , , , , , , , ,

j

EeR, E74, E75, Broad

�75 {3FTZ-Fl

L-

Third instar

Ir::atel :�

_ ,, , ,, ,

_ _

Mid prepupa

late prepupa

Pupa

+

- y

20 Ecd sone

(B)

t

USP + EcRA 20E

FIGURE 15 .18 20-Hydroxyecdysone initiates deve lopmenta l cascades. (A) Schematic of the major gene expression cascade in Drosophila metamorphosis. When 20E binds to the EeR/USP receptor complex, it activates the early response genes, including £74, £75, and Broad. Their products activate the "late genes." The activated EeR/USP complex (1lso acti­ vates a series of genes whose products are transcription factors and which activate the {3FTZ-F/ gene. The PFTZ-F I protein modifies the chromatin so that the next 20E pulse acti­ vates a different set of late genes. The products of these genes also inhibit the early­ expressed genes, including the EcR receptor. (B) Postulated cascade leading from ecdysone reception to death of the larval salivary gland. Ecdysone binds to the EcR-A isoform of the ecdysone receptor. After complexing with USP, the activated transcription factor complex stimulates transcription of the early response genes E74A, E758, and the Broad complex. These make transcription factors that pmmote apoptosis in the salivary gland cells. (A after King-Jones et ,l. 2005; B after Buszczak and Segraves 2000.)

559

560

CHAPTER 1 5

dominating (Emery et a1. 1994; Crossgrove et al. 1996). Juve­ nile hormone may act to prevent ecdysone-inducible gene expression by interfering with the Broad complex of pro­ teins (Riddiford 1972; Restifo and White 1991).

See WEBSITE 1 5.4 Precocenes and synthetic JH Like those of amphibian metamorphosis, the stories of insect metamor­ phosis involve complex interactions betvveen ligands and receptors. The "target tissues" are not mere passive recip­ ients of hormonal signals. Rather, they become responsive to hormones only at particular times. For example, when there is a pulse of 20E at the middle of the fourth instar of the tobacco hornworm moth Manduca, the epidermis is able to respond because this tissue is expressing ecdysone receptors. The wing discs, however, are unaffected by ecdysone until the prepupal stage, at which time they syn­ thesize ecdysone receptors, grow, and differentiate (Nijhout 1999). Thus, the timing of metamorphic events in insects can be controlled by the synthesis of receptors in the tar­ get tissues. Metamorphosis remains one of the most striking of developmental phenomena, yet we know only an outline of the molecular bases of metamorphosis, and only for a handful of species. COORDI NATION OF RECEPTOR AND LIGAND

REGENERATION Regeneration is the reactivation of development in postem­ bryonic life to restore missing tissues. The ability to regen­ erate amputated body parts or nonfunctioning organs is so "unhuman" that it has been a source of fascination to humans since the beginnings of biolOgical science. It is dif­ ficult to behold the phenomenon of limb regeneration in newts or starfish without wondering why we cannot grow back our own arms and legs. What gives salamanders this ability we so sorely lack? In fact, experimental biology was born of the efforts of eighteenth-century naturalists to answer this question. The regeneration e�periments of Trem­ blay' (hydra), Reaumur (crustaceans), and Spallanzani (sala­ manders) set the standard for experimental research and for the intelligent discussion of one's data (see Dinsmore 1991). More than two centuries later, we are qeginning to find answers to the great questions of regeneration, and at some point we may be able to alter the human body so as to per­ mit our own limbs, nerves, and organs to regenerate. Suc-

cess would mean that severed limbs could be restored, dis­ eased organs could be removed and then regrown, and nerve cells altered by age, disease, or trauma could once again function normally. Modem medical attempts to coax human bone and neural tissue to regenerate are discussed in Chapter 17, but to bring these treatments to humanity, we must first understand how regeneration occurs in those species that already have this ability.+ Our recently acquired knowledge of the roles of paracrine factors in organ formation, and our ability to clone the genes that produce those factors, have propelled what Susan Bryant (1999) has called "a regeneration renaissance. Since ren­ aissance literally means "rebirth/' and since regeneration can be seen as a return to the embryonic state, the term is apt in many ways. Regeneration does in fact take place in all species and can occur in four major ways: II

1. Stem-cell m edia ted regeneration. Stem cells allow an

organism to regrow certain organs or tissues that have been lost; examples include the regrowth of hair shafts from follicular stem cells in the hair bulge and the con­ tinual replacement of blood cells from the hematopoi­ etic stern cells in the bone marrow. 2. Epimorphosis. In some species, adult structures can undergo dedifferentiation to form a relatively undiffer­ entiated mass of cells that then redifferentiates to form the new structure. Such epimorphosis is characteristic of planarian flatworm regeneration and also of regen­ erating amphibian limbs. 3. Morphallaxis. Here, regeneration occurs through the repatterning of existing tissues, and there is little new growth. Such regeneration is seen in Hydra (a cnidari­ an). 4. Compensatory regeneration. Here, the differentiated cells divide but maintain their differentiated functions. The new cells do not come from stem cells, nor do they corne from the dedifferentiation of the adult cells. Each cell produces cells similar to itself; no mass of undiffer­ entiated tissue forms. This type of regeneration is char­ acteristic of the mammalian liver. Numerous examples of stern-cell mediated regeneration have been discussed throughout this book. In this chapter we will concentrate on epimorphosis in the salamander limb, morphallaxis in Hydra, and compensatory regenera­ tion in the mammalian liver.

+ Mammals do have a small amount of regenerational ability. In *Tremblay's advice to researchers who would enter this new field is

addition to regenerating body parts continuously through adult

pertinent even today: he advises us to go directly to nature and to

stem cells, rodents and humans can regenerate the tips of their dig­

avoid the prejudices that our education has given us. Moreover,

its if the animal is young enough. This ability has been correlated

"one should not become disheartened by want of success, but should try anew whatever has failed. It is even good to repeat suc­ cessful experiments a number of times. All that is possible to see is not discovered, and often cannot be discovered, the first time" (quoted in Dinsmore 1991).

2003; 2(05).

2004).

with the expression of the homeodomain transcription factor MSX1 fetal digit tips express MSXl in the migrating epidermis and subja­ (Han et al.

Kumar et al.

Apparently, amputated human

cent mesenchyme, just as regenerating amphibian limbs do (Allan

et

at.

POSTEMBRYO N I C DEVELOPMENT

Distal amputation

ProximaJ amputation

561

Formation of the apical ectodermal cap and regeneration blastema When a salamander limb is amputated, a p lasma clot

Original limb

forms; within 6-12 hours, epidermal cells from the remain­ i ng StunlP migrate to cover the wOlUld s urface, forming the wound epidermis. [n contrast to wound healing in

Amputation

mammals, no scar forms, and the dermis does not move

7d

nerves innervating the limb degenerate for a short distance

with the epidermis to cover the site of amputation. The

p roxim al to the plane of amputation (see Chernoff and Stocum 1995). During the next 4 days, the extracellular matrices of the tissues beneath the wound epidermis is degraded by pro­ teases, liberating Single cells that undergo dramatic dedif­ ferentiation: bone cells, cartilage cells, fibroblas ts, and myocytes all lose their differentiated characteristics. Genes that are expressed in differentiated tissues (SUdl as the mrf4

21d 2Sd 32d

and myfS genes expressed in muscle cells) are downreguJat­

42d

ed, while there is

genes such as

72d

a dramatic increase in the expression of

msxl

that are associated with the proliferat­

ing progress zone mesenchyme

of the embryonjc limb (Simon et a1. 1995). This cell mass is the regeneration blastema, and these are the cells that will continue to pro­

FIGURE 15.19

Regeneralion of a salamander forelimb. The amputation shown on the left was made below the elbow; the amputation shown on the right cut through the humerus. In both instances, the orrec t positional information was re-specifiecl and a no rma l limb was regenerated within 72 days. (From Goss 1 969, courlesy of R. J. Goss.) c

liferate, and which will eventually redifferentiate to form the new structures of the limb

(Figure 1 5.20; Butler 1935).

Moreover, during this time, the wound epidermiS thickens to form the apical epi dermal cap (AEC), which acts simi­ larly to the apical ectodermal ridge during normal Hmb development (Han et al. 2001). Thus, the previously well-structured limb region at the cut

edge of the stump forms a proliferating mass of indis­ cap. One of the major que stions of regeneration has been: do the cells keep a "memory" of what they had been? In other tinguishable cells just beneath the apical ectodermal

Epimorphic Regeneration of Salamander Limbs When an adult salamander limb is amputated, the remain­ ing limb cells are able to reconstruct a complete new limb, with all its differentiated cells arranged in the proper order. In other words, the new cells construct only the missing structures and no more. For example, when a w rist is amputated, the salanlander forms a .t:1ew wrist and not a new elbow (Figure 1 5. 1 9). In some way, the salamander limb "knows" where the proximal-distal axis has been sev­ ered and is able to regenerate from that

point on. Salaman­ ders accomplish epimorphic regeneration by cell dediHer­ entiation to form a regeneration blastema-an aggregation

words, do new muscles arise from old muscle celis, or can

any cell

of the blastema become a muscle? Kragl and col­ (2009) found that the blastema is not a collection of homogeneous, fully dedifferentiated cells. Ra ther in the regenerating limbs of the axolotl salamander, muscle cells arise only from old muscle cells, dermal cells come only

leagues

,

from old dermal cells, and cartilage can arise only from old cartilage or old dermal cells. Thus, the blastema is not a collection of unspecified multipotential progenitor

cells. the blastema

is a heterogeneous assortment of restricted progenitor cells. Kragl and olleag ues performed an experiment in which they transplanted limb tissue from a salamander whose cells expressed green fluorescent protein (GFP) into differ­ Rather, the cells retain their specification, and

c

1 5.21). U they transplanted the GFP-expressing limb cartilage into a salamander limb that did not contain the GFP transgene, the GFP-express­ ing cartilage would integrate normally into the limb skele­

of relatively dedifferentiated cells derived from the origi­

ent regions of limbs of normal salamanders that did not

nally differentiated tissue-which then proliferates and

have the GFP transgene (Figure

redifferentiates into the new limb parts (see Brockes and Kumar 2002; Gardiner et aJ.

2002). Bone, dermis, and carti­ lage j ust beneath the site of amputation contribute 10 the regeneration blastema, as do satellite cells from nearby muscles (Morrison el at. 2006).

ton. They later amputated the limb through the region con­ taining GFP-marked cartilage cells. The blastema was

562

CHAPTER I S

(A)

... ;.--..".,...,. ".--; (B) ,...-..,.".

(C)

(D)

(E)

(F)

FIGURE 1 5.20 Regeneration in the larval forelimb of the spotted salamander Ambystoma maculatum. (A) Longitudinal section of the upper arm, 2 days after amputation. The skin and muscle (M) have retracted from the tip of the humerus. (B) At 5 days after amputation, a thin accumulation of blastema cells is seen beneath the thickened epidermis, where the apical ectodermal cap (AEC) forms. (el At 7 days, a large population of mitotically active blastema cells lies distal to the humerus. (0) At 8 days, the blastema elongates by mitotic activity; much dedifferentiation has occurred. (El At 9 days, early redif­ ferentiation can be seen. Chondrogenesis has begun in the proximal part of the regenerating humerus, H. The letter A marks the apical mesenchyme of the blastema, and U and R are the precartilaginous condensations that will form the ulna and radius, respectively. P represents the stump where the ampu­ tation was made. (F) At 1 0 days after amputation, the precartilaginous condensations for the carpal bones (ankle, C) and the first two digits (D1, 02) can also be seen. (From Stocum 1 979, courtesy of D. L Stocum.)

POSTEMBRYONIC DEVELOPMENT

(A)

563

i.\age transplantc': d Cal:t

GFP-expressing limb (E)

Graft (e)

-

Wild-type limb

Ampl1tation

Regenerate

B

�

Fa[e? Positional identity?

Blastema

e

l ast m a

Regenerate

FIGURE 15.21 Blastem'a cells retain th ei r spec i fi ca­ tion, even though they dedifferentiate. (A,B) Schemat­ ic of the procedure, wherein a pa rti c ular tissue (in this case, carti lage) is t ra nsplanted from a salamander e p ess i ng green fluorescent protein (GFP) transgene into a wild-type salamander limb. Later, the limb is x r

a

amputated through the region of the limb containing GFP exprcssion, and a blastema is formed containing GFP-expressing cel ls that had been cartilage p recur­ sors. The regenerated limb th en studied to sec if GFP is found only in the regenerated cartilage tissues or in other tissues. (C) Longitudinal section of a regen­ erated limb 30 d ay after amputation. The muscle cells

is

s

are stained red, and nuclei are stained blue. The majority of GFP cells (green) were found in the regen­ erated ca rtilage; no GFP was seen in the muscle. (After Kragl et al. 2009, c u rtesy of E. Tanaka.)

o

found to contain CFP-expressing cells, and when the blastema differentiated, the only CFP-expressing cells found were in the limb cartilage. Similarly, CFP-marked

gradient protein (nAG). This protein can cause blastema

( F igure 15.22; 11 activated nAG genes are electopo­

muscle cells gave rise only to muscle, and GFP-marked

cells to proliferate in culture, and it permits nonmal regen­

epidermal cells only produced the epidermis of the regen­

eration in limbs that have been denervated

erated limb.

Kumar et al. 2007a).

rated into the dedifferentiating tissues of limbs that have

Proliferation of the blastema cells: The requirement for nerves and the AEC

been denervated, the limbs are able to regenerate.

If nAC is

not administered, the limbs remain as stumps. Moreover, nAC is only minimally expressed in normal limbs, but it

The growth of the regeneration blastema depends on the

is induced in the Schwann cells that surround the neurons

presence of both the apical ectodennal cap and nerves. The

within

AEC stimulates the growth of the blastema by secreting

5 days of amputation.

The creation of the amphibian regeneration blastema

ridge does in normal

may also depend on the maintenance of ion currents driv­

limb development), but the effect of the AEC is only pos­

en through the stump: if this electric field is suppressed,

FgfB (just as the apical ectodermal

sible if nerves are present (Mullen et aJ. 1996). Singer (1954)

the regenera tion blastema fails to form (Altizer et al. 2002).

demonstrated that a minimum number of nerve fibers

Such fields have been shown to be necessary for the regen­

must be present for regeneration to take place. The neu­

eration of tails in the frog

rons are also believed to release factors necessary for the

an,phibian). The

Xenopus laevis

(an anuran

Xenopus tadpole regenerates its tail, and

proliferation of the blastema cells (Singer and Caston 1972;

the notochord, muscles, and spinal cord each regenerate

Mescher and Tassava 1975). There have been many candi­

from the corresponding tissue n i the stump (Deuchar 1975;

dates for such a nerve-derived blastema mitogen, but the one that is probably the best candldate is newt anterior

Slack et aI. 2004). In this frog, the V-ATPase proton pump is

activated within 6 hours after tail amputation, changing

564

CHAPTER 1 5

Right limb denervated

(A)

,

Both limbs amputated

,

Electroporation with nAG

,

--'---' -'---...; . �

-

7 days

5 days

Regeneration

FIGURE 15.22 Regeneration of newt limbs depends on nAG Inormally supplied by the limb nerves). (A) Schematic of the pro· cedure. The limb is denervated and a week later is amputated. After 5 five days, nAG is electroporated into the li mb blastema. (B) Results show that in the denervated control {not given nAG), the amputated li mb (ye ll ow star) remains a stump. The limb that is given nAG regenerates tissues and proximal-

"

�

E E

� V>

�

Second year

�

"

�

'"

c

c ·"V>

§, � �

V>

"

'"

ro �

Third year

� c

'j

CHAPTER 1 6

604

"!

Germinal vesicle

t

Progesterone

Di plotene block (no active MPF)

c-mes

Active MPF

)g::

t .

Ca2+ flux � Ferlilization CSF Metaphase block

p 34

Cydin

Inactive MPF

Calmodu l m

jI

--

--

Meiotic arrest (First meiotic prophase)

First meiotic metaphase

Second meiotic metaphase

,

FIGURE 16.22 Schematic representation of Xenopus oocyte mat­ u ration sh owing the regu l ation of meiotic cell division by proges­ terone and fertil ization. Oocyte maturation is arrested at the

d i plotene stage of first meiotic prophase by the lack of active MPF. Progesterone activates the production of the (-mDS protein . This protein initiates a cascade of phosphorylation that eventual l y p hosp hory lates the p34 subunit of MPF, allowing the MPF to become active. The MPF drives the cell cycle through the first meiotic division, but further division is blocked by eSF, a com-

n,e mediator of the progesterone signal is the c-mos

protein. Progesterone reinitiates meiosis by causing the egg to polyadenylate the maternal c-mos mRNA that has been stored

n i

,

Calpain II ---'� CSF degraded Cam- PKlI

its cytoplasm (Sagata et al. 1988; Sheets et aL 1995;

Mendez e t aL 2000). This message is translated into a 39-

Fertilization. completion of meiosis II

pou nd conta ini ng c-mos, cyclin-dependent kinase 2, and Erp l . CSF inhibits the anaphase-promoting comp lex from degrading cyel in . Upon fertilization, calcium ions released into the cy to­ plasm are bound by calmodulin and are used to activate WO enzymes, calmod u l i n-dependen t prote in kinase I I and calpain I I , which inactivate a n d degrade (SF. Second meiosis i s comp leted , and the two haploid pronuclei can fuse. At this time, cyc li n B is resynthesized, allowing the first cel l cycl e of cleavage to begin.

the first meiotic division. The proteins o f the CSF complex

interact, eventually activating Erpl by phosphorylating it. the anaphase-promoting comp l ex

Phosphorylated Erpl blocks the degradation of cyclin by

(Figure 1 6.23).

This metaphase block is broken by fertilization. The cal­

kDa phosphoprotein. This c-mos protein is detectable only

cium ion flux attending fertilization activates the ca1cium­

fertilization. Yet during its brief lifetime, it plays a major cole n i releasing the egg from its dormancy. The c-mos pro­ tein activates a phosphoryla tion cascade that phosphory­ lates and activates the p34 subunit of MPF (Ferrell and

binding protein calmodulin, and calmodulin, in tum, can

during oocyte maturation and is destroyed quickly upon

Machleder 1998; Ferrell 1999). The active MPF allows the

c-mos is inhibited by injecting

activate two enzymes that inactivate CSF. These enzymes

II, a cal ci um-depend en t protease (Watanabe et aL 1989; Lorca et aL 1993). This action promotes cell division in two ways. First,

are calmodulin-dependent protein kinase II, which inac ti­ vates cdk2, and calpain that degrades c-mos

germinal vesicle to b rea k down and the chromosomes to

withoutCSF, cyclID can be degraded, and the meiotic divi­

c�mos an tisense mRNA into the

sion can be completed . Second, calcium-dependent pro­

divide. If the translation of

breakdown and the resump tion of oocyte maturation do oocyte, germinal vesicle

not occur.

However, oocyte maturation then encounters a second block. MPF can take the chromosomes only throu gh the division. The oocyte is arres ted once again in the

first meiotic division and prophase of the second meiotic

tein kinase II also allows the centrosome to duplicate, thus forming the poles of the meiotic spindle (Matsumoto and Maller 2002). ln 1911, Frank Lillie wrote, "The nature of the inhibition that causes the need for fertilization is a most

fundamental problem." The solution to tha t p roble m appears to be oocyte-derived CSF and the sperm-induced wave of calcium ions.

metaphase of the second meiotic division. This metaphase CSF is a complex of proteins that includes c-mos, cyclin­

block is caused by cytostatic factor (CSF; Ma tsui 1 974).

Gene transcription in amphibian oocytes

dependent kinase 2 (cdk2), MAP kinase, and ErpI (Gabriel­

The amph ib ian oocyte has certain periods of very active

Erp1

RNA synthesis. During the diplotene stage, certain chro­

is the active protein, and it is synthesized immediately after

mosomes stretell out large loops of DNA, causing them to

li et aL 1993; lnoue et aL 2007; Nishiyama et aL 2007) .

CSF c

om

T H E SAGA O F T H E GERM L I N E

plex

c- m as

FIGURE 16.23 The main pathway l ead i ng to metaphase arrest in the second meiotic division. The (SF protein complex consists of c-mos, three transducer kinases, and the effector protein Erp1. Activation of c-mos activates the kinases, which eventually phos­ phorylate Erp l . Phophorylated Erp! binds to and inhibits the anaphase-pro oting com­ plex, thus blocking the deg radation of cyclin B that would allow the cell to enter anaphase. (After Inoue et al. 2007.)

MEK

I MAPK I t ce90rs0 t

Anaphase­ promoti complexng Cyetin Cde2

m

are being transcribed (Figure 1 6.24A). Electron micrographs of gene transcripts from lampbrush chromosomes also enable one to see chains of mRNA coming off each gene as it is transcribed (Figure 16.24B; also see Hill iJl1d MacGregor

� 1

Metaphase arrest

B

Metaphase II

Anaphase 11

resemble a lampbrush (which was a handy instrument for cleaning test tubes in the days before microfuges). In situ hybridization reveals these lampbrush chromosomes to be sites of RNA synthesis. Oocyte chromosomes can be incubated with radioactive RNA probe and autoradiog­ raphy used to visualize the precise locations where genes

a

(A)

605

1980).

In addition to mRNA synthesis, ribosomal RNA and transfer RNA are also transcribed during oogenesis. Fig­ ure 1 6.25A shows the pattern of rRNA and tRNA synthe­ sis during Xenopus oogenesis. Transcription appears to begin in early (stage I, 25-40 �.m) oocytes, during the diplotene stage of meiosis. At this time, all the rRNAs and tRNAs needed for protein synthesis until tile mid-blastula stage are made, and all the maternal mRNAs needed for early development are transcribed. This stage lasts for months in Xenopus. The rate of rRNA production is prodi­ gious. The Xenopus oocyte genome has over 1800 genes encoding 185 and 285 rRNA (the two large RNAs that form the ribosomes), and these genes are selectively amplified such that there are Over 500,000 genes making rRNA in the oocyte (Figure 1 6.25B; Brown and Dawid 1968). When the mature (stage Vl) oocyte reaches a certain size, its chromo-­ somes conden�e, and the rRNA genes are no longer tran-

(B)

FIGURE 16.24 In amphibian oocytes, lampbrush chromosomes are active in the diplotene germinal vesicle during first meiotic p rop hase. (A) Autoradio­ graph of ch romosome I of the newt Tritu­ rus crislalUs after in situ hybridization with radioactive histone mRNA. A his­ tone gene (or set of histone genes) is being transcribed (arrow) on one o t e loops of this lampbrush (B) Lampbrush romos m ma nder NOlOphlhalmu5 viridescens. Extended DNA (w te) transcribed i to (A

fh chromosome. ch o e of the sala­ hi loops out and is n RNA (red). from Old et al. 1 977, courtesy of H . G. Callan; B co u rtesy of M. B. Roth and J. GaiL)

606

CHAPTER 1 6

In and moths) that undergo meroistic oogenesis, in which cytoplasmic connections remain between the cells pro­ duced by the oogonium. The oocytes of meroistic insects do not pass through a transcriptionally active stage, nor do they have lampbrush chromosomes. Rather, RNA synthesis is largely confined to the nurse cells, and the RNA made by those cells is actively transported into the oocyte cytoplasm (see Figure 6.7). Oogenesis takes place in only 12 days, so the nurse cells are metabolically very active during this time. Nurse cells are aided in their transcriptional efficiency by becom­ ing polytene-instead of having two copies of each chro­ mosome, they replicate their chromosomes until they have produced 512 copies. The 15 nurse cells pass ribosomal and messenger RNAs as well as proteins into the oocyte cyto­ plasm, and entire ribosomes may be transported as well. The mRNAs do not associate with polysomes, and they are not immediately active in protein synthesis (Paglia et a1. 1976; Telfer et a1. 1981). The meroistic ovary confronts us with some interesting problems. If all 16 cystocytes derived from the PGC are connected so that proteins and RNAs can shuttle freely among them, how do 15 cystocytes become RNA-produc­ ing nurse cells while one cell is fated to become the oocyte? Why is the flow of protein and RNA in one direction only? As the cystocytes divide, a large, spectrin-rich structure called the fusome forms and spans the ring canals between the cells (see Figure 16.4A). It is constructed asynunetrical­ Iy, as it always grows from the spindle pole that remains in one of the cells after the first division (Lin and Spradling 1995; de Cuevas and Spradling 1998). The cell that retains the greater part of the fusome during the first division becomes the oocyte. It is not yet known if the fusome con­ tains oogenic determinants, or i f it directs the traffic of materials into this particular cell.

scribed. This "mature oocyte" condition can also last for months. Upon hormonal stimulation, the oocyte completes its first meiotic division and is ovulated. The mRNAs stored by the oocyte now jOin with the ribosomes to initiate protein synthesis. Within hours, the second meiotic divi­ sion has begun, and the egg is fertilized in second meiot­ ic metaphase. The embryo's genes do not begin active tran­ scription until the mid-blastula transition (Newport and Kirschner 1982). As we saw in Chapter 2, the oocytes of several species make two classes of mRNAs-those for immediate use in the oocyte, and those that are stored for use during early development. In frogs, the translation of stored oocyte mes­ sages (maternal mRNAs) is initiated by progesterone as the egg is about to be ovulated. One of the results of the MPF activity induced by progesterone may be the phosphoryla­ tion of proteins on the 3' urn of stored oocyte mRNAs. The phosphorylation of these factors is associated with the lengthening of the polyA tails of the stored messages and their subsequent translation (Paris et a1. 1991).

Meroistic oogenesis in insects There are several types of oogeneSis in insectsi but most studies have focused on those insects (including Drosophi-

(A)

�

High

tRNA

�

.� � .c

Ribosomal RNAs (IB5 and 285)

� 0

DNA

.'l • -

.�

;; 0; '"

Low

55 rRNA

�!

:��

cations

tI

Accumulation of yolk starts

�L

grown oocyte

__ __ __

Transcription

(B)

==}�!.'...;,.':" -'

"\")' (VI� -';:� � -:'�f(� :,.,.. ' ,: t '; ;< .:H

.

�'-

l

Fertilization

� �� '�

Y -y Oocyte growth 3 months

L__ __ __ __

one

Fully

�,

16

FIGURE 16. 25 po ocyte . mph bian oogenesis u ng the as 3 t (8) T a cr i ti the large RNA p ec e e er, hap o d genome. (A aher O. L. M l er,

Ribosomal and transfer RNA r duction in Xeno­ o s (A) Relative rates of DNA, tRNA, and rRNA synthesis in a i d ri l t mon hs before ovula­ tion. r ns p on of r ursor of the 285, 1 85, and 5.85 ribosomal RNAs. These units ar tand mly linked togeth­ with some 450 per l i Gurdon 1 976; 8 courtesy of i l Jr.)

pus

Maturation months

\

Transcription of ribos mai RNA

Transcription

Nontranscribed

T H E SAGA OF T H E G ERM L I N E

607

TABLE 1 6.2 Sexual dimorphism in mammalian meioses

Female oogenesis

Male spermatogenesis

One gamete produced per meiosis

FOllr gametes produced per meiosis

Meiosis initiated once in a finite population of cells Completion of meiosis delayed for months or years

Meiosis initiated continuously in a mitoticall y dividing stem cell population Meiosis completed in days or weeks

Meiosis arrested at first meiotic prophase and

Meiosis and differentiation proceed continuously without ceU cycle arrest

Differentiation of gamete occurs while diploid, in first meiotic prophase

Differentiation of gamete occurs while haploid, after meiosis ends

All chromosomes exhibit equivalent transcription and recombination during meiotic prophase

Sex chromosomes excluded from recombination and transcription during first meiotic prophase

reinitiated in a smaller pOPlllation of cells

Source: Handel nnd Eppig 19Y8.

Once the patterns of transport are established, the

Gametogenesis i n Mammals

cy toskeleton becomes actively involved in transporting

As outlined in Table 1 6.2, there are profound differences

Tbeurkauf 1994). An array of microtubules

between spermatogenesis and oogenesis in mammals. One

mRNAs from the nurse cells into the oocyte cytoplasm (Cooley and

that extends through the ring canals (see Figure 16.4C) is

of the fundamental differences concerns the timing of

meiosis onset. In females, meiosis begins in the embryon­

critical for oocyte determination. In the nurse cells, the

ic gonads; in males, meiosis is not initiated until puberty.

Ex uperantia protein binds

This critical difference in timing is due to

bicoid message to the micro­

If the microtubular array is dis·

tubules and transports it to the anterior of the oocyte (Cha et a1. 2001; see Chapter 6).

rupted (either chemically or by mutations such as bicau·

dal-D Or egalitarian), the nurse cell gene products are trans­

retinoic acid (RA)

produced by the mesonephric kidneys (Figure 1 6.26). This

RA stimulates the germ cells to undergo a new round of DNA repl ic a ti on and initiate meiosis (Baltus et al. 2006;

Bowles et .1. 2006; Lin et al. 2008). In males, however, the

mitted in all directions and a11 1 6 cells d ifferentiate into

embryonic testes secrete the RA-degrading enzyme

nurse cells (Gutzeit 1986; Theurkauf e t a l . 1992, 1993;

Cyp26bl . This prevents RA from promoting meiosis. Later,

Spra dl ing 1993).

Nanos2 will be expressed in the male germ cells, and this

The Bicaudal·D and Egalitarian proteins are probably

will also prevent meiosis and ensure that the cells follow

core components of a dynein motor system that transports

the pa thway to become sperm (Koubova et al. 2006; Suzu·

mRNAs and proteins throughout Ule oocyte (Bullock and

ki and Saga 2008).

Ish·Horowicz 2001). It is possible that some compounds transpor ted from the nurse cells into the oocyte become

VADE MECUM Gametogenesis in mammals

associated with transport prote�ns such as dynein and kinesin, which would enable them to travel along the tracks of microtubules extending through the ring canals

oskar for instance, is linked to kinesin through the Bar· entsz protein, and kinesin can transport the oskar message to the posterior of the oocyte (van Eeden et a1. 2001; see

Spermatogenesis

(Theurkauf et a1. 1992; Sun a n d Wyman 1993). The

Once mammalian PGCs arrive a t the genital ridge of

message,

male embryo, they are called gonocytes and become incor·

Figure 6.7).

Actin may become important for maintaining the polar·

a

porated into the sex cords (Culty 2009). They remain there

until maturity, at which time the sex cords hollow out to form the

seminiferous tubules. The epithelium of the

tubules differentiates into the Serto1i cells that will nOUIish

ity of transport during later stages of oogenesis. Mutations

and protect the developing spenn cells. TIle gonocytes dif·

that prevent actin microfilaments from lining the 'ring

ferentiate into a population of stem ceUs that have recent·

canals prevent the transPOlt of mRNAs from the nurse cells

ly been named the

to the oocyte, and disruption of the actin microfilaments

nia (Yoshida e t a l . 2007). These cells can reestablish

Wa tson et al. 1993). Thus, the cyt oskeleton controls the

randomizes the distribution of mRNA (Cooley et a1. 1992;

sperma togenesis when transferred into mice whose sperm

movenlent o f organelles and RNAs between nurse cells

production was eliminated by toxic chemicals. TIley appear

only in the appropriate direction.

blood vessels.

and oocyte such

that developmental cues are exchanged

und

i ffe ren ti ate d

type

A sperm atogo·

to reside in stem cell niches created by the junction of Ser­ toli cells, interstitial (testosterone·producing) cells, and

608

CHAPTER 1 6

FIGURE 16.26 Retinoic acid (RA) (A) Female germ ceUs determines the ti m ing of meiosis and sexu al di fferenti ation of m am m alian Nanos2 added Normal germ cells. (A) In fem al e mouse MesoRA RA embryos, RA secreted from the mesonephros reaches the gonad and triggers meiotic initiation via the induction of Stra8 transcription fac­ tor in female germ cells (beige). StraS .....- Nanos2 Stra8 However, if activated Nanosl genes are added to female germ ce li s they suppress StraB expression, leading the germ ce lls i n to a mal e pathway (gray) . (B) In embryonic testes Meiosis Meiosis Cyp26bl blocks RA signal ing there­ Gonad by preventing male germ cells from Male fate initiating meiosis until embryonic day 1 3 .5 (l eft pa nel). After embryon(e) RA synthesized ic day 1 3 .5, when Cyp26bl expression is decreased, Nanos2 is expressed and pre­ vents meiotic initiation by blocking Stra8 expressi on. This induces male-type differen­ tiation in the germ cells (right panel) . (C,D) Day 1 2 mouse embryos stained for mRNAs encoding the RA-synth esi zing enzyme Aldhla2 (C) and the RA-degrading enzyme Cyp26bl (D). The RA-synthesizing enzyme is seen in the mesonephros of both the male and female; the RA-degrading enzyme is seen only in the male gonad. (A,S (rom Saga 2008; C,D from Bow les et al. 2006, co urtesy of P. Koopman.) ,

, ,

=C:mJ m l mnnnm

j

t t

Male

The decision to proliferate or differentiate may involve interactions between the Wnt and BMP pathways. Wnt sig­ naling appears to promote proliferation of stem ceils, and the spermatogonia appear to have receptors for both Wnts and BMPs (Golestaneh et aJ. 2009). The initiation of sper­ matogenesis during puberty is probably regulated by the synthesis of BMPs by the spermatogenic germ ceils, the spermatogonia. When BMP8b reaches a' critical concentra­ tion, the germ cells begin to differentiate. The differentiat­ ing ceils produce high levels of BMP8b, which can then further stimulate their differentiation. Mice lacking BMPSb do not initiate spermatogenesis at puberty (Zhao et aJ. 1996). The spermatogenic germ cells are bound to the Sertoli cells by N-cadherin molecules on the surfaces of both cell types, and by galactosyltransferase molecules on the sper­ matogenic cells that bind a carbohydrate receptor on the Sertoli cells (Newton et aJ. 1993; Pratt et aJ. 1993). Sper­ matogenesis-the developmental pathway from germ cell to mature sperm--occUIs in the recesses between the Ser­ tali ceils ( F i gu re 1 6.27). FORMING THE HAPLOID SPERMAT I D The undifferentiated

type Aj spermatogonia (sometimes called the dense type

Female

(B) Male germ ceUs Before day 13.5 Meso­ RA nephros Somatic Germ line

Gonad

After day 13.5 RA

f- eyp26bl

Stra8

t

>- eyp26bl

StraB.....- Nanos2

Me i osis

i

M

(D) RA degraded

j

Male fate

Male Female

A spermatogonia) are found adjacent to the outer basal lamina of the sex cords. They are stem cells, and upon reaching maturity are thought to divide to make another type Aj spermatogonium as well as a second, paler type of cell, the type A2 spermatogonia. The A2 spermatogonia divide to produce type A3 spermatogonia, which then beget type A4 spermatogonia. The A4 spermatogonia are thought to differentiate into the first committed stem cell type, the intermediate spermatogonia. Intermediate sper­ matogonia are committed to becoming spermatozoa, and they divide mitotically once to form type B spermatogo­ nia (see Figure 16.27). These cells are the precursors of the spermatocytes and are the last cells of the line that under­ go mitosis. They divide once to generate the primary sper­ matocytes-the cells that enter meiosis_ The transition between spermatogonia and spermato­ cytes appears to be mediated by the opposing influences of glial cell line-derived neurotrophic factor (GDNF) and stem ceil factor (SCF), both of which are secreted by the Sertoli cells. GDNF levels determine whether the dividing spermatogonia remain spermatogonia or enter the path­ way to become spermatocytes. Low levels of GDNF favor the differentiation of the spermatogonia, whereas high le,-­ els favor self-renewal of the stem cells (Meng et aJ. 2000).

T H E SAGA O F T H E GERM L I N E

Type B spermatogonium

Vas deferens

609

A, um spermatogoni Type

spermatogonium

spermatocyte

spermatocyte

seminiferous tubule

FIGURE 1 6.27

Section of the seminiferous tubule, showing the relationship between $ertol i cells and the developing sperm. As germ cells mature, they progress toward the lumen of the seminiferous tubule. (Mer Dym ' 9 77.)

SCF promotes the transition to spermatogenesis

(Rossi et upregulated by

still connected to one another through their cytoplasmic

al. 2000). Since both GDNF and SCF are

bridges. The spermatids that are connected in this manner

follicle-stimulating hormone (FSH), these two factors may

have haploid nuclei but are functionally diploid, since a

serve as a link between the Sertoli cells and the endocrine

gene product made in one cell can readily diffuse into the

the testes to produce more sperm (Tadokoro et al. 2002).

During the divisions from type A, spermatogonia to

Keeping the stem cells in equilibrium-producing neither

spermatids, the cells move farther and farther away from

too many undifferentiated cells nor too many differentiat­

the basal larnina

system, and they provide a mechanism for FSH to instruct

ed cells-is not easy. Mice with the Iuxoid mutation are ster­ ile because they lack a transcription factor that regulates

cytoplasm of its neighbors (Braun et a!. 1989).

of the seminiferous tubule and closer to Figure 16.27; Siu and Cheng 2004). Thus, each type of cell can be found in a particular layer of the its lumen (see

this division. All their spermatogonia become sperm at

tubule. The spermatids are located at the border of the

once, leaving the testes

lumen, aJ1d here they lose their cytoplasruic connections

devoid of stem cells (Buaas et aJ.

2004; Costoya et al. 2004). Looking

and differentiate into spermatozoa.

at Figure 1 6.28, we find that during the sper­

matogonial divisions, cytokinesis is nC?t complete. Rather, the cells form a syncytium in which each cell communi­

In humans, the pro­

gression from spermatogonial stem cell to mature sperma­ tozoa takes 65 days (Dyrn 1994). The processes of spermatogenesis require a very

spe­

cates with the others via cytoplasmic bridges about 1 �.m

cialized network of gene expression (Sassone-Corsi 2002).

in diameter (Dym and Fawcett 1971). The successive divi­

Not only are histones substantially

sions produce clones of interconnected cells, and because

b y sperm-specific

ions and molecules readily pass through these cytoplas­

RNA polymerase n transcription factors are exchanged for

mic bridges, each cohort matures

synchronously. During

this time, the spermatocyte nucleus often transcribes genes whose

products will be used later to form the axoneme and

acrosome.

remodeled and replaced variants (see below), but even the basal

sperm-specific variants. The TFIID complex, which con­

tains the TATA-binding protein and 14 TAFs, functions in the recognition of RNA polymerase.

One of these TAFs,

TAF4b, is a sperm-specific TAF required for mouse sper­

Each primary spermatocyte undergoes the first meiot­

matogenesis (Falender et a!. 2005). Without this factor, the

ic division to yield a pair of secondary spermatocytes,

spermatogonial stem cells faU to make Ret (the receptor for

which complete the second division of meiosis. The hap­

GDNF) or the

loid cells thus formed are called spermatids, and they are

genesis fails to occur.

luxoid

transcription

factor, and spermato­

610

CHAPTER 1 6

SPERMIOGENESIS: DIFFERENTIATION OF THE SPERM The mammalian haploid spermatid is a round, unflagellated cell that looks nothing like the mature vertebrate sperm. The next step in sperm maturation, then, is spermiogenesis (or spermateliosis), the differentiation of the sperm cell. For fertilization to OCCUI, the sperm

has to meet and bind with an egg, and spermiogenesis prepares the

sperm for these functions of motility and interaction. The process of mam­

Type Aj

spermatogon ia

Type A2 spermatogonia

or �

� More type A I � spermatogonia

Type A3

spermatogonia Type A, spermatogonia Intermediate spermatogonia

Type B spermatogo nia

malian sperm differentiation was shown in Figure 4.2. The first step is the construction of the acrosomal vesicle from the Golgi apparatus. The

Secondary spermatocytes (2nd meiotic divisio n )

acrosome forms a cap that covers the sperm nucleus. As the acrosomal cap is formed, the nucleus rotates so that the cap will be facing the basal lam­ ina of the seminiferous tubule. This rotation is necessary because the fla­ gellum, which is beginning to form from the centriole on the other side of the nucleus, will extend into the

Spermatids

lumen. During the last stage of spermiogenesis, the nucleus flattens and condenses, the remaining cyto­ plasm (the residual body, or "cyto­ plasmic droplet") is jettisoned, and the mitochondria form a ring arowld the base 01 the flagellum. During spermiogenesis, the his-

-���

••••••••••o•••••

tones of the spermatogonia are often replaced by histone

variants, and widespread nucleosome dissociation takes place. This remodeling of nucleosomes might also be the

point at which the PGC pattern of methylation is removed and the maJe genome-specific pattern of methylation is

��

sperm cellS

FIGURE 16.28 Format i on of syncytial clones of human male germ cells. (After Bloom and Fawcett 1 975.)

established on the sperm DNA (see Wilkins 2005). As spermiogenesis ends, the histones of the haplOid nucleus are eventually replaced by protamines.' This replacement

In the mouse, development from stem cell to sperma­

results in the complete shutdown 01 transcription in the

tozoon takes 34.5 days: the spermatogonial stages last 8

nucleus and facilitates the nucleus a5suJ!ling an almost

days, meiosis lasts 13 days, and spermiogenesis takes

crystalline structure (Govin et aJ. 2004). The resulting

another 13.5 days. Hwnan sperm development takes near­

sperm then enter the lumen of the seminiferous tubule.

ly twice as long. Because the original type A spermatogonia

See WEBSITE 1 6.5 The Nebenkern

are stern cells, spermatogenesis can occur continuously. Each day, some 100 million sperm are made in each hwnan testicle, and each ejaculation releases 200 million spenn.

·Protamines are relatively small proteins that are over 60%, argi­ nine. Transcription of the genes for protamines is seen in the early haploid spermatids, although translation is delayed for several days (Peschon et al. 1987). The replacement, however, is not com­ plete,

Normal blood cells

�

Leukemia progenitor

Leukemia cells

FIGURE 17.18 Model of cancer stem cell production, using leukemia (a white blood cell tumor) as an exampl e. (A) A hematopoietic stem cel l (HSC) usually gives rise to normal blood progenitor cells that can become mature white blood oells. Microenvironment (8) If the HSC undergoes mutations or epigenetic changes involving gene activation it can become a cancer stem cell (esC) that can divide to produce more of itself plus other relatively differentiated (leukemic) cells. As in normal blood devel op­ ment, the CSC reta in s the abil ity for self-renewal and thereby becomes the malig­ nant portion of the cancer. IC) The CSC may also be produced by changes in the microenvironment, which allows cert.ain cells to display a stem cel l phenotype that they would not otherwise possess. (After Rosen and Jordan 2009.) ,

MEDICAL ASPECTS O F DEVELOPMENTAL BIOLOGY

dividing stem cell population that gives rise to more can­ cer stem cells and to populations of relatively slowly divid­

ing differentiated cells (Lapidot et aI.

1994; Bonnet and Dick 1997; Singh et a1. 2004; Schatton 2008). Whether the tumor

is initiated by an adult stem cell "gone bad" or by a more differentiated cell that has regained stem cell abilities is a

645

ed. Thus, one might get cancer if faulty methylation either

inappropriately methylated the tumor suppressor genes (turning them off) or inappropriately demethylated the

oncogenes (turning them on;

Figure 1 7.19).

Some genes may be oncogenes in one set of cells and

tumor suppressor genes in another set of cells. In the

2009; Schatton et aI. 2009). In some tumors, such as prostate

breast, for instance, estrogen receptors can act as oncogenes

stem cell that has escaped the control of its niche (Wang e t

receptors function as tumor-suppressor genes. Issa and col­

matter of controversy (Gupta et a1. 2009; Rosen and Jordan

cancer, the origin of the tumor is most Ukely a normal adult

for estrogen-dependent breast cancer. In the colon, how­

ever, estrogen stops the proliferation of cells, and estrogen

aI. 2oo9).

leagues

(1994) showed that in addition to the age-associ­

Cancer as a return to embryonic invasiveness: Migration reactivated

genes in colon cancers. Even the smallest colon cancers had

Another crucial point in considering cancers as diseases of

of the estrogen receptor gene.

of the malignant cell into other tissues. Like embryonic

genetic cause. Indeed, several studies indicate that these

ated methylation of estrogen receptors, there was a much

disrupted development involves metastasisi the invasion

cells, tumor cells do not usually stay put-they migrate and form colonies. Chapter 3 discussed the roles of cad­

herin proteins in the sorting-out of cells to form tissues dur­ ing development, and how cells form boundaries and seg­

regate into tissues by al tering the strengths of their

higher level of DNA methylation nearly

in the estrogen receptor

100% methylation of the cytosines in the promoter

The epigenetic causation of cancer does not exclude a

mechanisms augment one another. Numerous mutations

occur in each cancer celi, and recent evidence suggests that

as many as

14 Significant tumor-promoting mutations are 2006). Jacinco and Esteller (2007) have presented evidence that the large num­

found in each cancer cell (Sjoblom et aJ.

In cancer metastasis, this property is lost: cad­

ber of mutations that accwnuJate in cancer cells may have

ment to the extracellular matrix and other types of cells

itself from mutations. One is the editing subunits on DNA

attachments.

herin levels are downregulated, and the strength of attach­

becomes greater than the cohesive force binding the tissue together. As a result, the cells become able to spread into

other tissues (Foty and Steinberg

1997, 2004).

Another phase of metastasis involves the digestion of

extracellular matrices by metalloproteinases. These

enzymes are used by migrating embryonic cells to digest a path to their destination. They are commonly secreted by

an epigenetic cause. DNA has several means of protecting

polymerase; these "proofreaders" get rid of mismatched

bases and insert the correct ones. Another mechanism is the

set of enzymes that repair DNA when the DNA has been

damaged by light or by cellular compounds that are prod­ ucts of metabolism. In cancer cells, the genes encoding these DNA repair enzymes appear to be susceptible to inactiva­

tion by methylation. Once DNA repair enzymes have been

trophoblast cells, axon growth cones, sperm cells, and

dowmegulated, the number of mutations increases.

malignant cancer cells, allowing the cancer to invade other

linked by the common denominator of aberrant DNA

somitic cells. Metalloproteinases can be reactivated

in

tissues. The presence of these enzymes is a marker that the

tumor is particularly dangerous (see Gu et a1.

2005).

Cancer and epigenetic gene regulation In Chapter

15, we saw evidence that the methylation pat­

terns of mammalian genes change with age. We specifical­

It is therefore possible that aging and cancer may be

methylation. If metabolically or structurally important

genes (such as the estrogen receptors) become heavily

methylated, they don't produce enough receptor proteins,

and our body function suffers. If tumor suppressor genes

or the genes encoding DNA repair enzymes are heavily methylated, tumors can arise.

Tumors can be generated by a combination of genetic

ly looked at genes that might cause elements of the aging

and epigenetic means. Changes in DNA methylation can

dependent patterns of gene methylation altered the tran­

thereby initiating tumor formation. Conversely, oncogenes

Two types of genes control cell division. The first are

which also aids tumorigenesis. Moreover, the tissue envi­

sion, and prevent cell death. These are the genes that can

processes. The complexities of tumors, including their mul­

phenotype. But what would happen if the random, age­ scription of the genes regulating cell division?

oncogenes, which promote cell division, reduce cell adhe­

activate oncogenes and repress tumor-suppressor genes,

can cause the methylation of tumor suppressor genes,

ronment of the cell may be critical in regulating these

promote tumor formation and metastasis. The second set

tiple somatic mutations and their resistance to agents that

genes usually put the brakes on cell division and increase

combination of genetic and epigenetic factors rather than

of regulatory genes are the tumor suppressor genes. These

induce apoptotic cell death, may best be explained by a

the adhesion between cells; they can also induce apopto­

just by the basis of mutations. Knowledge of the epigenet­

and tumor suppressor genes has to be very finely regulat-

of cancer therapy.

sis of rapidly dividing ceUs. The interplay of oncogenes

ic causes of cancer can provide the basis for new methods

CHAPTER ·1 7

646

(A)

(B)

NORMAL CELL

NORil4AL CELL

/

Methylated

Unmethylaled CpG

\

()

Tumor-suppressor gene: Open chromatin conformation

CANCER CELL

c

J

)

pG

I It ) )

1 1 I )) )

II II I It I )

t:)) 1 )

11n

Oncogene:

Repressed chromatin conformation

CANCER CELL Hypomethylated

J

)

I

I

CpG

\ G:I)

1 1 1 Ci )

I

) 1

Repressed chromatin conformation on tumor-suppressor gene

Open chromatin conformation on oncogene

CeU cycle entry

CeU cycle entry

Blocking of apoplosis

Inappropriate gene expression

DNA repair deficits

Loss of cell adhesion

_ _ _ _ .. . L�

I

Tumor formation

I

Loss of dosage regulat ion

FIGURE 1 7.19 Cancer can arise (A) i( tumor-suppressor genes are inappropriately turned off by DNA methylation or (8) if onco­ genes are inappropriatel y demethylated (and thereby activated). (After Esteller 2007.)

SIDEL IG H TS SPECULATIONS

The Embryonic Origins of Adu lt-Onset Illnesses

T

eratogenesis is usually associated with congenital disease ( i .e., a condition appearing at birth) and is also associated with disruptions of organogenesis during the embryonic period. However, D. J. P. Ba rker and

colleagues (1 994a,b) have offered evi­ dence that certain adult-onset diseases may also result from conditions i n the uterus prior to birth. Based on epi­ demiological evidence, they hypothe­ size that there are critical periods of development during which certai n physiological insults or stimuli can cause specific changes in the body.

The "Barker hypothesis" pos tulates that .certain anatomical and physiological parameters get "programmed" during embryonic and fetal development, and that deficits in nutrition during this time can produce permanent changes in the pattern of metabolic activity­ changes that can predispose the adult to particular diseases. Speciiically, Barker and colleagues showed that i nfants whose mother experienced protein deprivation (because of wars, famines, or migra­ tions) during certa i n months of preg­ nancy were at high risk for having cer-

tain d iseases as adults. Undernutrition during a fetus's first trimester could lead to hypertension and strokes i n adult life, while those (etuses experi­ enCing undernutrition during the sec­ ond trimester had a high risk of devel­ oping heart disease and diabetes as adults. Those fetuses experiencing undernutrition rluring the third trimester were prone to blood clotting defects as adults. Recent studies have tried to deter­ mine whether there are physiological or anatomical reasons for these corre­ lations (Gluckman and Hanson 2004,

MED ICAL ASPECTS O F DEVELOPMEN TA L BIOLOGY

647

SIDELIGHTS & SPECULATIONS (Co nti n ued) Figure 17.20 Anatomical changes associated with hypertension. (A) In age-m atched individuals, the kidneys of men with hypertension had about half the number of nephrons as the kidneys of men with normal blood pressure. (B) The glomerul i of the nephrons in hypertensive kid­ neys were much larger than the glomerul i in control subjects. (After Keller et al. 2003, photographs courtesy of G. Keller.) (A) 2,250,000 2,000,000 '" � �

1,750,000

v E

1,500,000

Z

1,000,000

.M�r--:- 1,429,200

0

'0

Oh 1,250,000 " v .D

E "

750,000

'"

2> ;; � v

2005; Lau and Rogers 2005). Anatom­ ically, undernutrition can change the number of cells produced during a critical time of organ formation. When pregnant rats are fed low-protein diets at certain times during their pregnan­ cy, the resulti ng offspring are at high risk for hypertension as adult. The poor diet appears to cause low nephron numbers i n the adult kidney (see Moritz et a l . 2003). In humans, the n u mber of nephrons present i n the kidneys of men with hypertension was only about half the n u mber found i n men without hypertension (Figure 1 7.20A; Keller et a l . 2003). In addition, the glomeru l i (the blood-fi ltering unit of the nephron ) of hypertensive men were larger than those in control sub­ jects (Figure 1 7.20B). Similar trends have been reported for non-insu l i n dependent (Type II) d iabetes and glucose i n tolerance (Hales et a l . '1 991 ; Hales and Barker 1 992). Here, poor nutrition reduces the number of p cells in the pancreas and hence the ability to synthesi ze insulin. Moreover, the panc reas isn't the o n ly organ involved. Undernutri­ tion in rats changes the h i stological

Matched controls

5

2

� Patients with hypertension

6

E

�

250,000

7

4

0 "

702,379

8

E

� �

500,000

0

�E E "1

3

0

Patients with hypertension

architecture in the liver as well. A low­ protein d iet duri ng gestation appeared to increase the amount of periportal cells that produce the glucose-synthe­ sizi n g enzyme phosphoenolpyruvate carboxykinase while decreasing the number of perivenous cells that syn­ thesize the glucose-degrading enzyme glucokinase in the offpsring (Burns et a l . 1 997). These changes may be coor­ di nated by glucocorticoid hormones that are stimulated by mal nutrition and which act to conserve resources, even though such actions might make the person prone to hypertension later in life (see Fowden and Forhead 2004). (Since, historical ly, most humans died before age 50, this would not be a detrimental evolution­ ary trade-off.) Hales and Barker (200 1 ) have pro­ posed a "thrifty phenotype" hypothe­ sis wherein the malnourished fetus is "programmed" to expect an energy­ defic ien t environment. The developing

Matched cont rols

fetus sets its biochemical parameters to conserve energy and store fat. * Resulting adults who do indeed meet with the expected poor environment are ready for it and can survive better than individuals whose metabolisms were set to utilize energy and not store it as efficiently. However, if such a "deprivationally developed" person l ives in an energy- and protein-rich environment, their cells store more fats and their heart and kidneys have developed to survive more stringent conditions. Both these developments put the person at risk for several later­ onset diseases. How can conditions experienced in the uterus create anatomical and bio­ chemical conditions that will be main­ tained throughout adulthood? One place to look is DNA methylation. Lil­ Iycrop and colleagues (2005) have shown that rats born to mothers having a low-protein diet had a different pat­ tern of liver gene methylation than did

*In other words, the embryo has phenotyp i c pl as tic i ty-t he ability to modulate its phe­ notype dependi ng on the environment; this plasticity w i l l be discussed further i n Chapter 1 8.

648

CHAPTER I 7

SIDELIGHTS & SPECULATIONS (Continued)

t he offsp r i ng of mothers fed a normal diet. These differences in methylation changed the metabol ic profi Ie of the rats' livers. For instance, t he methy la­ tion of the promoter region of th e PPARa gene (which is critical in the regulation of carbohyd rate and lipid me ta bo l i s m) is 20% l ower i n the off­ spring of protei n-res tri cted rats, a n d the ge n e 's tra n sc ri pti ona l activity is tenfold greater (Figure 1 7.21). More­ over, the difference between these met hylat i on patterns can be abo l ished by includ ing folic ac i d in the protein­ restricted diet. Thus, the difference in methylati on probab l y results from changes in folate meta bo lis m caused by the l imited amount of protein avail­ able to the fetus.

I t does appear that prenatal nutri­ tion can induce long-lasting, gene­ spec i fic a l terati on s i n tra nscr i p ti ona l activity and metabolism. The preven­ tion of ad u l t di sease th rou gh p ren ata l diet could thus become a p u b l i c health issue i n the co m i n g decades.

(A)

(B) 110

." � • �

= -

8� 3 '0 '';:: 1:::: � g � v � 8 = « z Q

1200 '"

�

1000

� Q,

E

100

8-

c ??- 800

.2 :::; � o

90

8 o

0 0 v 0

0 v «

12

80

E

70

0 -

-

Control

Protein� Proteinrestricted restricted + folate

600 400 200 0

Control

Pr otei n - Proteinrestricted restricted + folate

Figure 17.21 Activity of the l iver gene for peroxisomal prol iferator-activated receptor (PPARa) is susceptible to dietary differences. (A) DNA methylation pattern of the PPARa pro­ moter region, showing highly methylated control promoters compared with poorly methylat­ ed promoters from the livers of mice whose mothers had protein-restricted diets (p < 0.00'1). Adding folate to the protein-res tricted diet abolished Ihis di fference. (8) Levels of mRNA for the PPARa gene were much higher in the mice fed the protein-restricted diel (p < 0.0001). (After lillycrop et al . 200S.)

DEVELOPMENTAL THERAPIES

can contribu te to cancer therapies is the inhibition of angio·

Know ledge gained from research in the field of deve lop­

The c ri tical p oi n t at which a node of cancerous cells becomes a rapidly growi ng tumor occurs when the node

mental biology is now

genesis (blood vessel forma tion) .

being focused on several diseases.

The abil ity to

b lock p aracri ne factors, to use stem cells to the body to become a pluripotenti.1 stem cell may enable us to block the spread of cancer, repair bodily inj uries, and

becomes vascularized. A microtumor can expand to 16,000

regenera te b ody parts, and to induce nearly any cell in

times its original volwne in the 2 weeks following vascular·

even to ameliorate genetic disease.

stances called tumor angi ogene si s factors, which often

ization (Folkman 1974; Ausprunk and Folkman 1977). To achieve vascularization, the microtumor secretes sub­ include the same factors that engender blood vessel growth in the embryo-VEGFs, Fgf2, placenta-like growth factor.

Anti-Angiogenesis

and others. Tilinor angiogenesis factors stimulate mitosis

Judah Folkman (1974) has estimated that as many as 350

in endothelial cells and direct the cell differentiation intu

billion mitoses occur i n the human

blood vessels in the direction of the tumor.

b ody every day.

With

each cell division comes the chance that the resulting cells will be

ma lignant .

Indeed , a u topsies have shown that

every person over 50 years old has

m icroscopic tumors in

their thyroid glands, although less than 1 in 1000 persons

Tu mor angiogen esis can be demonstrated by implan ti­ ng a piece of tumor tissue withi.n the layers of a rabbit or mouse cornea. The cornea itself is not vascularized, but it is surrounded by a vascu lar border, or limb us. The tumor

have thyroid cancer (Fol kman and Kalluri 2004). Folkman

tissue induces blood vessels to form and grow toward the

suggested that cells capab le of forming tumors develop at

tumor

a certain frequency, but that most never form observable

1979). Once the blood vessels enter the tumor, the tumor

rumors. The reason is that a solid tumor, like any other rap­

ceUs W'ldergo explosive growth, eventually bu rsti n g

idly div i ding tissue, needs oxygen and nutrients to sur­ vive. Withou t a b lood supply, potential tumors eW,er die or remain as dormant "microtumors," stable cell popula­ tions wherein dying cells are replaced by new cells. Thus one important area in which knowledge of developlnent

(Figure 1 7.22; Mu thu kkaruppan and A uerba ch

the eye. Other adult solid tissues do not induce blood vessels to form. It might therefore be pOSSible to block tumor devel opmen t by blocking a ngiogenesis . Numerous chem­ icals are bei ng tested as natura l and artificial angiogenesis inhibitors. These compounds act by preventing endothe-

MEDICAL ASPECTS OF DEVELOPMENTAL BIOLOGY

(A)

649

(B)

Vein

Growing tumor

Artery

/

Tumor

2 Days ---�.� 6 Days

o .. . .

I�ii>r

�

8 Days -----�.� 12 Days

FIGURE 17.22 New blood vessel growth to the site of a mam­ nlary tUnlor transplanted into the cornea of an albino mouse. (A) Sequence of events leading to vascularization of the tumor on days 2, 6, 8, and 1 2 . Both the veins and the arteries in the limbus surrounding the cornea supply blood vessels to the tumor. (B) Photograph of living cornea of an albino mouse, with new blood vessels from the limbus approaching the tumor graft. (From Muthukkaruppan and Auerbach 1 979; B courtesy of R. Auerbach.)

lial cells from responding to the angiogenetic signal of the tumor.' One of the advantages of these compounds is that the tumor cells are unlikely to evolve resistance to them, since the tumor cell itself is not the target of these agents (Boehm et al. 1997; Kerbel and Polkman 2002). In one set of clinical trials, an antibody against VEGP­ A was fOWld to be successful against colon cancer but not against breast cancer. This is probably because colon cancer is more dependent on VEGP-induced angiogenesis than are mammary tumors (Whisenant and Bergsland 2005). Antibodies against a placental form of VEGP were able to inhibit the growth of tumors without affecting healthy blood vessels (Fischer et al. 2007). Blocking the VEGP receptor VEGFR3 prevents the angiogenic sprouting need­ ed for new blood vessels (Tammela et al. 2008) and stops tumors from getting blood-borne nutrients and oxygen. Interestingly, thalidomide, the tenitogen responsible for birth defects in the 1960s, is now being used to block l1.unor-induced blood vessels. Thalidomide has been found to be a potent anti-angiogenesis factor tha t can reduce the growth of cancers in rats and mice (0' Amato et aJ. 1994; Dredge et a l. 2002). Cancer and congenital maJiorrnations are opposite sides of the san1e coin. Both involve disruptions of normal devel­ opment. Thus, as we have seen, agents that have been known to cause congenital malformations-thalidomide, retinoic acid, and cyclopamine-can be used as drugs to *This is the flip side of differentiation therapy discussed above. In differentiation therapy, paracrine factors or hormones are added to promote differentiation. Here, the substances being administered block paracrine signals in order to prevent tissue (Le., blood vessel) formation.

prevent cancers. Just as they disrupt normal development, these substances can disrupt the caricature of development that is caused by tumor cells. When angiogenesis is blocked, the tumor cells can be starved.

Stem Cells and Tissue Regeneration Embryonic stem cells As we discussed in Chapter 8, the inner cell mass of the mammalian blastocyst generates the entire embryo. 1den­ tical twins and chimeric individuals show that each of the cells of the inner cell mass are pluripotent. When cultured, the cells of the inner cell mass blastomeres can become embryonic stem (ES) cells, which remain pluripotent. Cur­ rently, pluripotent stem cells are obtained by two major techniques (Figure 17.23A). They can be derived from the inner cell mass of blastocysts, such as those left over from in vitro fertilization (Thomson et al. 1998), and they can also be generated from germ cells derived from sponta­ neously aborted fetuses. The latter are generally referred to as embryonic germ (EG) cells (Gearhart 1998). Some experimental evidence (Stre1chenko et aJ. 2004) suggests that it may also be possible to derive embryonic stem cells from late morulae, befoTe they form blastocysts. While adult stem cells are rare and do not usually remain undif­ ferentiated in culture, embryonic stem cells can be readily harvested and retain their undifferentiated state for years of culturing. The IDlportance of pluripotent stem cells in medicine is potentially enormous. The hope is that human ES cells can be used to produce new neurons for people with degener­ ative brain disorders (such as Alzheimer and Parkinson dis­ ease) or spinal cord injmies, and new pancreatic � cells for people with diabetes. People with deteriorating hearts might be able to have damaged tissu e repla ced with new heart cells, and those suffering from immune deficiencies might be able to replenish their failing immune systems. Such ther­ apies have already worked in mice. Murine ES cells have been cultured under conditions causing them to form insulin-secreting cells, muscle stern cells, glial stem cells, and

CHAPTER 1 7

650

.

(A)

Q� @ -:. . •

.

•

.

�

G:::l �

••

. . .. . . .

B lastocyst-stage em b ryo

Cells from inner cell mass

Fetus

Embryonic stem cells;

I

Primordial germ mass

cultured pluripotent stem cells

Lineage-specific stem ceUs

FIGURE 17.23 E mb ryo n i c stem ce l l ther­ a pe u ti cs IA) ES cel l s can be obtained from the inner cell mass of the blastocyst or from primordial germ cells and cul­ tured in different ways to produce lineage· spec ifi c stem cells. These cells (or the pre­ cursor cells derived from them) can then be tran sp lan ted into a host. (B) Differentia· tion of mo u se ES cells into l ineage-restrict­ ed (neuronal and glial) stem cells can be accomplished by al tering the media in which the ES cells grow. (A after Gearhart 1 998; B, photographs from Brustle et al. 1 999 and Wi cke l gren 1 999, courtesy of O. Brustle and J . W. McDonald.)

neural stem cells ( Figure McDonald et al.

1999).

1 7.236; Brustle et aJ. 1999; Dopaminergic neurons

derived from ES cells have been shown to signifi· cantly reduce the symptoms of Parkinson disease

Heart cell precursor

•

(B)

• ES cell

. .

-tI

�

:::::--...

.-

Neural precursor

I

in rodents (Bjorklund et al.

2002; Kim et al. 2002).

Wheu neural stem cells derived from germ cell· derived ES cells were transplanted into the injured

� \ "--; ----- /---' ,-------,

Transplantation therapy

brains of mice, the neural stem cells replaced neu­ rol15 and

gUal ceUs

in the forebrains of the newborn 2005). However, in most cases

mice (Mueller et al.

when human ES cells have been transplanted into the brains of immunosuppressed animals, the results have been less encouraging. Typically, trans­ plantation or thousands of ES cell-generated neu­

�:�����:;�r

Medium . . contammg basic fibroblast growth factor and platelet-derived growth factor Glial stem cells

I ansp!antation t �mto mICe

rons into animals results in very few surviving

Medium containing retinoic acid

• t _

dopaminergic neurons and a high frequency of tumors (Li et aJ. 2006). For such transp!antatiol15 to work, one must be able to find the neural stem cells made by the ES cells and to prevent undifferentiat­ ed ES cells from being transplanted. Human embryonic stem cells differ from their murine counterparts in their growth requirements.

In most ways, however, they are very similar, and have a similar, if not identical, pluripotency. Like mouse ES cells, human ES cells can be directed

Neural stem cells

Kaufman and his colleagues

down specific developmental paths. For example,

(2001) directed human

ES cells to become blood-forming stem ceUs by plac­ ing them on mouse bone marrow or endothelial cells. These ES-derived hematopoietic stem cells could further differentiate into numerous types of blood cells (Figure

1 7.24) . Human embryonic germ in

cells were able to cure virus-induced paraplegia

rats. These stern cells appear to do thls both by dif­ ferentiating into new neurons and by producing paracrine factors (BDNF and TGF-a) that prevent the death of the existing neurOI15 (Kerr et aJ.

Functional glial cells

2003).

Similarly, ES cells from monkey blastocysts have

Functional neurons

been able to CUIe a Parkinson-like condition in adult

MEDICA L ASPECTS OF DEVELOPMENTAL BIOLOGY

FIGURE 17.24 Differentiated blood cells developing from human ES cel l s cultured on mouse bone marrow. (Courtesy of The University of Wisconsin.)

monkeys whose dopaminergic neurons had been destroyed (Tagaki et al. 2005). Research is now being done to find ways of directing the differen tia tion of ES cells by using small molecules (rather than paracrine factors). Chen and colleagues (2009), for instance, have found tha t indolactam V molecules can induce Prix1 expression in ES cells, di.recting them into the pancreatic lineage. Thus, ES cells may be able to provide a reusable and readily available source of cells to heal dam­ aged tissue in adult men and worn,en. See WEBSITE 1 7.4 Therapeutic cloning

651

The newfound knowledge of the transcription factors needed to maintain pluripotency has illuminated a star­ tlingly easy way to generate embryonic stem cells that have the exact genotype of the patient.' In 2006, Kazutoshi Taka­ hashi and Shinya Yamanaka of Kyoto University demon­ strated that by inserting activated copies of four genes that encoded some of these critical transcription factors, nearly any cell in the adult mouse body could be made into a cell with the pluripotency of embryonic stem cells. Such cells are called induced pluripotent stem (iPS) cells. Using a strategy very similar to the one used to identi­ fy the active components of Spemann's organizer (see Chapter 7), Takahashi and Yamanaka obtained mRNA from mouse ES cells and made these into cDNAs, which they placed onto active viral promoters. They transfected sets of 24 of these recombinant viruses into cultured fibrob­ lasts that had a neomycin-resistance gene placed onto an Fbx15 regulatory region. The Fbx15 gene is usually turned on in ES cells, so if the transfected genes activated the Fbx15 gene, then the neomycin-resistance gene would be activat­ ed and the cells would survive in neomycin-mntaining cul­ ture medium (Figure 1 7.25A). U a cohort of 24 active genes was found to activate the Fbx15 promoter (as shown by the cells' growing in neomycin-containing medium), then the group of cloned genes was further split. Eventually, Taka­ hashi and Yamanaka discovered that only four active genes were needed to tum on the Fbx15 gene. These genes encod­ ed the transcription factors Oct4, Sox2, .Klf4, and c-Myc (Figure 1 7.256). When the researchers cultured those cells whose Fbx15 promoter was activated, they found that in many cases the cells had become pluripotent, as demon­ strated in a series of tests:

• When the cells were aggregated together, they formed a

teratoma-a tumorlike amalgam containing cell types of all three germ layers.

Induced pluripotent stem cells

The mammalian inner cell mass is known to be character­ ized by certain transcription fadors, including Nanog, Oct4, and Sox2. Knocking out these. genes in mice abolish­ es the pluripotency and self-renewal of the inner mass blas­ tomeres, eventually leading to the demise of the embryo (see Boyer et aJ. 2006; Niwa 2007). Sox2 and Oct4 can dimerize to form a transcription factor complex tha t acti­ vates thei.r own genes (Oe14 and Sox2) as well as the Nnnog gene. It appears that Oct4 not only promotes the synthesis of Sox2 and Klf4, but it also blocks the production of the miR145 microRNA that would otherwise prevent the trans­ lation of Oct4, Sox2, and KIf4 messages (Chivukula and Mendell 2009; Xu et al. 2009). These transcription factors (and microRNAs) initiate a transcription network in the inner cell mass and ES cells that is essential for pluripoten­ cy (see Welstead et al. 2008). This fi.rst set of transcription factors probably acts by activating other transcription fac­ tors, such as Nanog and FbxlS, which are critical in main­ taining the pluripotent and dividing state.

•

•

When injected onto the blastocyst of a normal mouse, the induced cells showed that they could contribute to the production of cells in each of the three germ layers (Figure 1 7.25C; Maherali et a1. 2007; Okita et a1. 2007; Wernig et a!. 2007). 'When the inner cell masses of mouse embryos were C0111pletely compo;;ed of induced pluripotential cells, normal mice were generated (Boland et al. 2009; Zhao et al. 2009).

• The transcription and DNA methylation pattern of the induced pluripotential cells was found to be almost iden­ tical to that of actual mouse embryonic stem cells. The genes for Nanos, Sox2, and other ES-cell transcription factors were hypomethylated, as they are in ES cells. Interestingly, the methylation problems that plague

:tIn order to be a successful therapeutic agent embryonic stem cells have to be the same genotype as the patient, SO that their differenti­ ated products will not be rejected. In fact, however they are not quite exact, because the viruses used to induce pluripotency are integrated into the chromosomes, as we'll see next. ,

CHAPTER 1 7

652

(A) Pbx 15

Ii

enhan�er X� F I

enhancer �

" ' &) " N � � _ Fibroblast iPS cell _

Pbx 15

II

(B)

C=I ,

•

"

N,

1Retroviral infection and neomycin selection 5' LTR II Viral II I 3'LTR promoter

N

Control

(C)

eomyc -resistant in

eDNA

FIGURE 1 7.25 Produ ction of i nduced pluripotent stem cells by expression of four transc ri pti on factors in adult mouse tail fibro­ blasts. (A) A mouse was produ ced wherein the 5' regul a to ry region of the Fbx7 5 gene was attached to a neomycin-resistance (N eo RI gene. When Fbx l5 gene is activated (as it is in ES cellsl, th e n eomycin-res stan ce gene is a ctivated and the cells are able to grow in medium containing neomycin. Various combinations of genes encoding mRNAs found in ES cells were added to the fibroblasts, and the fibroblasts grown in neomycin to see if a ny combination of genes activated the Fbx 15 promo ter. (8) Some neomyc in-resistant colonies survived . These resistant colonies were found to have genes encoding Sox2, Oct4, Klf4, and c-Myc. (C) The pluripotency of these cells was shown by their i nsertion into the inner cell mass of a mouse blastocyst. GFP-Iabeled iPS cells were subsequ e tly found in all organs of the embryo. (After Takahashi and Yamanaka 2006.1 -

i

n

normal cells. However, Nakagawa and colleagues (2007) showed that by altering the culturing techniques, one could circumvent the need for c-Myc. In other words, one should be able to obtain induced pluripotent stem cells by adding merely three active genes to a patient's cell. Another way of "getting rid of c-Myc" was to place the genes for the key ES transcription factors onto episomal viral vectors (Kaji et a1. 2009; Yu et a1. 2009). Episomal vectors are derived from viruses (such as the Epstein-Barr virus that causes mononucleosis) that do not insert themselves into host DNA. rn this manner, the genes from the vector generated the transcription factors that converted human fibroblasts into iPS cells. Once the iPS cells were produced, the cul­ ture media could be changed so that the vector would be eliminated. This technique generated human iPS cells that did not have a v:irus inserted in it. Thus, not only was the

cloned animals do not appear to be a problem for embry­ on.ic stem cells derived by somatic cell nuclear transfer. It appears that the nuclei in the small population of ES cells that survive in culture have had their methylation pat­ terns erased, thus enabling them to redifferentiate (Jaenisch 2004; Rugg-Gwm et a1. 2005).

problem of c-Myc circumvented, but so was the problem of the viral insertion capable of causing a gene mutation. Recently, adult stem cells have been found that are already "part of the way" to becoming pluripotent and do not need as many factors to make them so. Giorgetti and colleagues (2009) showed that blood stem cells from the human umbilical cord can be converted into pluripoten­ tial stem cells merely by adding activated OCT4 and SOX2

This technique does nothing less than transform any cell

genes, and Kim and colleagues (2009) found that they

in the body into an embryonic stem cell. By 2007, the

could transform human neural stem cells into iPS cells by

Yamanaka laboratory (Takahashi et a1. 2007) had used the

adding only a single activated gene, OCT4.

same set of four transcription factors to induce pluripoten­

The therapeutic potential of iPS cells was demonstrat­

tiality in adult human fibroblast cells. These induced cells

ed by the ability of iPS-derived hematopoietic stem cells

formed teratomas containing cells of all three germ layers,

to correct a sickle-cell anemia phenotype in mice

and they had the same transcription profile as the human

(Figure 17.26; Hanna et a1. 2007). Here, tail-tip fibroblasts from a

ES cells.

mouse with sickle-cell hemoglobin were made into iPS cells

Yamanaka (2007) proposed that c-Myc helped "immor­

b y being infected in culture by viruses containing Oct4,

talize" the cell, keeping it in an undifferentiated state of

Sox2, Klf4, and c-Myc. The iPS cells were selected, then

proliferation, and that Klf4 prevented apoptosis and senes­

electroporated with DNA containing wild-type globin

cence. But this situation would have created a tumor, were

genes. These genetically corrected iPS cells were cultured in

it not for Oct4 and Sox2, which redirected growth and gave

media that promoted the production of hematopoietic stem

the cell properties similar to those of germ cells. The need

cells, and the hematopoietic stem cells were injected back

for c-Myc was disconcer ting, however, since this is a well­

into the mice with sickle-cell anemia. By

known oncogene, capable of initiating tumor formation in

mia had been cured.

2 months, the ane­

MEDICAL ASPECTS OF DEVELOPMENTAL BIOLOGY

The combination of iPS cells and genetic engineering may

be able to cure certain genetic diseases. Raya and colleagues

(2009)

have cultured dermal fibroblast cells from patients

653

paracrine factors that appear to activate the heart's own stem cells to repair the damage (Cho et al.

2007; Mirotsou

2007).

et at.

with the genetic disease Fanconi illlemia and have added to them a good copy of the defective gene. These cells were

BONE REGENERATION Several stem cell therapies can be

es bearing the activated forms of OCT4, SOX2, KLF4, and c­

seen

then converted into iPS cells by the addition of retrovirus­

MYC. Moreover, these iPS cells could be directed to become

can become normal parts of

in an incredibly important area of regenerative med­

icine: forming new adult bone. VVhile fractured bones can heal, bone cells

in adults usually do not regrow to bridge

the blood stem cells needed by the patients. However, we

wide gaps. The finding that the same paracrine and

still do not know if these cells

endocrine factors involved in endochondral ossification

the patients and not produce tumors themselves.

are also involved in fracture repair (Vortkamp et al.

1998)

raises the pOSSibility that new bone could grow if the prop­ er paracrine factors and extracelJular environment were

Adult stem cells and regeneration therapy

provided. Several methods are now being tried to devel­

In the introduction to Part 1II, we mentioned the potential

op new functional bone in patients with severely fractured

these new therapies.

Bonadio and his colleagues

medical uses of adult stem cells, especially the mesenchy­

mal stem cells. We will now provide details about some of

or broken bones. One solution to the problem of delivery was devised by

(1999), who developed a

col­

lagen gel containing plasmids carrying the human parathy­

CARDIAC REGENERATION When mesenchymal stem cells

roid hormone gene. The plasmid-impregnated gel was

from the bone marrow of heart attack patients are injected

placed in the gap be tween the ends of a broken dog tibia

into their own hearts, these cells can differentiate into heart

or femur. As cells migrated into the collagen matrix, they

and vessel cells and can significantly m i prove the patients'

incorporated the plaSmid and made parathyroid hormone.

outcomes (Yousef et al.

2009).

Interestingly, in many

A dose-dependent increase in new bone formation was

1 7.27). This type of treat­

instances the stem ceUs do not create new structures them­

seen in about a month (Figure

selves to circumvent the blockage. Rather, they secrete

ment has the potential to help people with large bone frac­ tures as well as those with osteoporosis.

Humanized sickle ceU

(HbSIHbS)

anemia mouse model

Uo

,

o Transplant corrected

hematopoetic progenitors back into irradiated mice

t

� "'"

_ _ _

I

\

e Differentiate into embryoid bodjes

t;-y HbAlHbS

iPS clones

,

in

IPS cells by speCific

gene targeting

E;�y\

FIGURE 1 7.26 Protocol for curing a "human" disease in a mouse, using iPS cells fibroblas t plus recombinant genetiCS. (1 ) Tail-tip fibroblasts are taken from a mouse whose genome contains the human alleles for f) Infect with Oct4, sickle cell anemia (HbS) and no mouse Sox2, Klf4 and genes for this protein. (2) The cells are cultured and infected with viruses con­ taining the four transcription factors known to induce pluripotentiality. (3) The iPS cells are identified by their distinctive shapes and are given DNA containing the wild-type al lel e of human g l ob in (HbA). (4) The embryos are allowed to differenti­ ate in culture . They form Ilernbryoid bod­ ies" that contain blood-forming stem ce lls. (5) Hematopoietic progenitor and stem cells from these embryoid bodies are injected into ./ mouse-derived the original mouse and cure its sickl e-ce ll aneiPS dones mia. (After Hanna et aJ. 2007.)

�

o �.rrect sick1e-ceU �utation

· Harvest tail tIp fibroblasts

I

@

654

CHAPTER 1 7

b

(e) Whole on e, 53 wk

(A) Treated fracture

FIGURE 17.27 Bone formalion from collagen matrix containing plasm ids bearing the human parathyroid hormone. (A) A 1 .6-cm gap was made in a dog femur and stabilized with screws. Plasmid-containing gel was placed on the edges of the break. Radiographs of Ihe area at 2, 8, 1 2, 1 6, and 1 8 weeks afler the sur­ gery show the formalion of bone bridging the gap at 1 8 weeks. (B) Control fracture (no plasmid in Ihe gel) al 24 weeks (C) Whole bone a year after surgery, showing repaired region. (From Bonadio el aJ. 1 999, courtesy of J. Bonadio.) .

and mechanical engi neering, is called tissue engineering. Li and collea gue s (2005) made scaffolds of ma terial that resembles normal extracellular matrix and which can be molded to form the shape of bone needed. The bone

(B)

Untreat.ed fracture, 24

wk

Another approach is to find the right mixture of paracrine factors to recruit stem cells and produce normal bone. Peng and colleagues (2002) genetically modified muscle stem cells to secrete BMP4 or VEGF-A. These cells were placed in gel matrix discs, which were implanted in wowlds made in mouse skulls. The researchers found that certain ratios of BMP4 and VEGFs were able to heal the wounds by making new bone. Similarly, BMP2 has been used to heal large fractures of primate mandibles and rab­ bit femurs (Li et a1. 2002; Marukawa et a1. 2002). A third approach is to make sca ffolds that resemble those of the bone and seed them with bone nlarrow stem cells. This approach, combining developmental biology

marrow stem cells can be placed in these scaf· folds and plac ed into the existing bone. These stem cells are told how to differentiate by the local conditions, and histological and gene expression studies have shown that these cells form bone with the appropriate amounts of osteocytes and chondrocytes. Similarly, when cells are removed from the tracheae of recent cadavers, they can be reseeded with bone marrow stem cells of patients with damaged tracheae. The stem cells dif­ ferentiate into chondrocytes, and the newly reseeded tra­ cheae can be substituted for ille damaged tracheae and pro­ vide a normal airway (M acchi arini et a1. 2008). A fourth ap proach has been to let the body do the work. While bones can't regenerate if wide gaps appear, many bones can undergo normal healing of small wounds. Here, cells in the periosteum (the sheath of cells that surrounds the bone) will differentiate into new cartilage, bone, and ligaments to fill the crack. Stevens and colleagues (2005) have used this normal healing process to make new bones. They injected saline solutions between the rabbit tibial bone and its periosteum, mimicking a fracture, and kept this space open by adding a gel containing calcium (to push the per iosteal cells to differentiate into bone rather than cartilage). Within a few w eeks, these cav ities filled with new bone, which could be tra nspl a nted into sites where b one had been damaged. This technique might pro­ vide a r ela tively painless way to produce new bone for fus­ ing vertebrae and other surgical techniques. NEURONAL REGENERATION While the central nervous sys­ tem is characterized by its ability to change and make new connections, it has very little regenerative capacity. The motor neurons of the peripheral nervous system, however, have Significant regenerative powers, even in adult mam· mals. The regeneration of motor neurons involves the

MEDICAL ASPECTS OF D EVELOPMENTAL B I OLOGY

J£ the cell body of a motor neu­

655

regrowth of a severed axon, not the replacement of a miss­

myelination, and these transcription factors appear to be

ing or diseased cell body.

repressed by high levels of Wnt signaling (Arnett et a1.

ron is destroyed, it cannot be replaced.

2004; Fancy et al. 2009). Therefore, the use ofWnt inhibitors

The myelin sheath that covers the axon of a motor neu­

(such as Frzb and Dickkopf) may be a mechanism for treat­

ron is necessary for its regeneration. This sheath is made

ing this disease. Research into CNS axon regeneration may

by the Schwann cells, a type of glial cell in the peripheral

become one of the most important contributions of devel­

nervous system (see Chapter

opmental biology to medicine.

9). When an axon is severed,

the Schwann cells divide to form a pathway along which the axon can regrow from the proximal stump . This pro­ liferation of the Schwann cells is critical for directing the

Direct transdifferentiation

regenerating axon to the original Schwann cell basal lam­

One of the newest developmental therapies involves using

ina. If the regrowing axon can find that basal lamina, it can

transcription factors to convert one cell type into another

be guided to its target and restore the original connection.

without the intermediary of stem cells, a procedure known

In turn, the regenerating neuron secretes mitogens that

as transdifferentiation. In Chapter

allow the Schwann cells to divide. Some of these utitogens

transdifferentiation of exocrine pancreatic cells into insulin­

2, we

discussed the

are specific to the developing or regenerating nervous sys­

secreting pancreatic p cells by three transcription factors

tem (Livesey et a1.

(Ngn3, Mafal, and Pdxl). More recently; Kajiyama and col­

1997).

The neurons of the central nervous system cannot regenerate their axons under normal conditions. Thus,

leagues

(2010) have transfected .one of these factors, Pdxl,

into mouse adipose-derived stem cells and converted the

spinal cord injuries can cause permanent paralysis. One

stem cells into insulin-producing pancreatic cells. These

strategy to get around this block is to find ways of enlarg­

induced pancreatic cells were able to reduce the hyper­

ing the population of adult neural stem cells and to direct

glycemia in diabetic mice.

their development in ways that circumvent the lesions

Vierbuchen and colleagues (2010) found that ti,e viral

caused by disease or trauma. The neural stem cells found

insertion of three active transcription factor genes (Ascll,

.in adult mammals may be very similar to embryonic neu­

Bm2,

ral stem cells and may respond to the same growth factors

dermal fibroblasts into ftmctional neurons in vitro. These

Oohe et al.

1996; Johansson et a1. 1999;

Kerr et a1.

2003).

Another strategy for CNS neural regeneration is to cre­

and My tIl) sufficed to effiCiently convert mouse

induced neuronal cells expressed several neuron-specif­ ic proteins, were able to generate action potentials, and

ate environments that encourage axonal growth. Unlike

formed functional synapses (resembling excitatory neu­

the Schwann cells of the peripheral nervous system, the

rons of the forebrain). It remains to be seen if this trans­

myelinating glial cells of the central nervous system, the

differentiation is stable and whether it will ameliorate

oligodendrocytes, produce substances that inhibit axon

disease in vivo. However, the ability to generate pancre­

regeneration (Schwab and Caroni

atic � cells and neural cells that are genetically identical

1988).

Schwann cells

transplanted from the peripheral nervous system into a

to a patient holds the promise of significant therapies for

CNS lesion are able to encourage the growth of CNS axons

patients with diabetes and degenerative neural diseases.

to their targets (Keirstead et a1.

1999; Weidner et a1. 1999).

Three substances that inhibit axonal outgrowth have been isolated from oligodendrocyte myelin: myelin-associated glycoprotein, Nogo-l, and oligodendrocyte-myelin glyco­

1994; (hen et a1. 2000; Grand­ 2002). Each of these substances

protein (Mukhopadyay et a1. Pre et al.

2000; Wang et al.

Coda Developmental biology is gaining increasing importance in modern medicine. First, preventive medicine, pubHc health, and conservation biology demand that we learn

binds to the Nogo receptor (NgR). Thus, NgR may be the

more about the mechanisms by which industrial chemi­

critical target for therapies allowing regeneration. Chen

cals and drugs can damage embryos. The ability to effec­

and colleagues

(2009) lISed RNA interference to block

tlIe

tively and inexpensively assay compounds for potential

synthesis of NgR and found that this helped rat optic neu­

harm is critical. Second, developmental biology is pro­

rons to regenerate. The axons regenerated even better

viding us new ways of preventing and curing cancers.

when this therapy was combined with nutrients and pos­

Third, developmental biology is providing the explana­

itive growth factors.

tions for how mutated genes and aneuploidies cause their

As mentioned in the case with bone, there is a dose rela­

aberrant phenotypes. Fourth, the field of regenerative

tionship between wound healing and regeneration.

medicine is using developmental biology to provide

Patients with multiple sclerosis suffer from the demyeli­

induced pluripotent stem cells as well as the knowledge

nation of axons

of paracrine factors needed to form new cells, tissues, and

in the brain and spinal cord.

Several tran­

scription factors are needed to reinitiate the pathway to

organs in adults.

656

CHAPTER 1 7

Snapshot Summary: Medical Aspects of Developmental Biology 1. Pleiotropy occurs when several different effects are

produced by a single gene, In mosaic pleiotropy, each effect is caused independently by the expres­ sion of the same gene in different tissues . In relation­ al pleiotropy, abnormal gene expression

in one tissue

influences other tissues, even though those oilier tis­ sues do not express that gene,

2.

type,

in different individuals,

can produce different defects (or differing severities 4, Preimplantation genetics involves testing for genetic

abnormalities in early embryos in vitro, and implanting only those embryos that may develop normally. Selection for sex is also possible using preimplantation genetics,

5.

Teratogenic agents include certain chemicals such as alcohol and retinoic acid, as well as heavy metals, certain pathogens, and ionizing radiation. These agents adversely affect normal development, yield­ ing malformations and functional deficits.

6. Fetal alcohol syndrome is completely preventable. There may be multiple effects of alcohol on cells and tissues that result in this syndrome of cognitive and physical abnormalities.

7,

10, Cancer can be seen as a disease of altered develop­ ment. Some tumors revert to non-malignancy when placed in environments that fail to support rap id cell

11. Cancers can arise from errors in cell-cell communica­ tion. These errors include alterations of paracrine factor syn thesis.

Phenotypic heterogeneity arises when the same gene of the same defect)

by turning genes all,

methylation can alter metabolism and development

clivision.

Genetic heterogeneity occurs when mutations in more than one gene can produce the same pheno­

3,

inherited from one generation to the next. Such

Endocrine disruptors can bind to or block hormone receptors or block the synthesis, transport, or excre­ tion of hormones. DES is a powerful endocrine dis­ ruptor, Presently, bisphenol A and other compounds are being considered as possible agents of low sperm counts in men and a predisposition to breast cancer in women.

8. Environmental estrogens can caus� reproductive system anomalies by suppressing Hox gene expres­ sion and Wnt pathways,

9. In some instances, endocrine disruptors methylate DNA, and these patterns

01 methylation can be

12. Cancers metastasize in manners similar to embryon­ ic cell movement.

13. In many instances, tumors have a rapidly dividing as well as more quiescent and differentiated cells.

stem cell population which produces more stem cells

14, The methylation pattern 01 cancer cells is often aber­ rant� and these methylation differences can cause cancer by inappropriately inactivating tumor sup­ pressor genes or activating oncogenes.

15, Disrup ting tumor-induced angiogenesis may become an important means of stopping tumor progression.

16, Skin fibroblasts, and perhaps any normal adult cell, can be induced to form pluripotent stem cells by the incorporation of activated genes encoding certain transcription factors. These pluripotent stem cells would not be rejected by the patient from whom they were formed,

17, By altering conditions to resemble those in the embryo and by providing surfaces on which adult stem cells might grow, some adult stem cells might be directed to form numerous cell types.

19. Neurons in the central nervous system can be aided in regenerating by blocking the glial-derived

paracrine factors that stabilize neurons and prevent their growth.

20. Transdifferentiation is the viral insertion of tran­ scription factors (or transcription factor activation by small molecules), which can convert one stable cell

type into another,

For Further Reading Complete bib liographical citations for all literature cited in this chapter can be found at the free-access website www.devbio.com Anway, M, D" A. S. Cupp, M. Uzumcu and M, K. Skipper. 2005, Epigene tic transgeneration effects of endocrine dis­ ruptors and male fertility, Science 308: 146&-1469.

Baksh, D" L Song and R. S, Tuan. 2004, Adult mesenchymal stem cells: Charac­ terization� differentiation, and applica­ tion in cell and gene therapy, j, Cell Mol, Med. 8: 301-316,

Bissell, M, j" D. C. Rarlisky, A, Rizki, V, M, Weaver and 0, W. Petersen, 2002,

The organizing principle: Microenviron­ mental influences in the normal and malignant breast. Differentiation 70:

537-546.

MEDICAL ASPECTS OF DEVELOPMENTAL BIOLOGY

Foty, R. A. and M. s. Steinberg. 1997. Measurement of tumor cell cohesion and suppression of invasion by E- and P-cadherin. Cancer Res. 57: 5033-5036. Gilbert, S. F. and Epel. D. 2009. Ecologi­ cal Developmental Biology: Integratirlg Epigenetics, Medicine, and Evolution. Sin­ auer Associates. Sunderland, MA. Gluckman, P. D. and M. A. Hanson.

2004. Living with the past: Evolution, development, and patterns of disease. Science 305: 1733-1739. Gupta, P. S., C. L. Chaffer and R. A. Weinberg. 2009. Cancer stem cells: Mirage or reality?

15: 1010-1012.

Natw'e Medicine

Howdeshell, K L., A. K Hotchkiss, K. A. Thayer, ). G. Vandenbergh and F. S. vom Saal. 1999. Plastic bisphenol A speeds growth and puberty. Nature 401:

762-764.

Huang, G. T., S. Gronthos and S. Shi.

2009. Mesenchymal stem cells derived

from dental tissues vs. those from other sources: Their biology and role in regenerative medicine. J. Dental Res.

88:792-806.

The stroma as a crucial target in mam­ mary gland carcinogenesis. j. Cell Sci.

117: 1495-1502.

Nakayama, A., M. T. Nguyen C. C. Chen, K Opdecamp, C. A. Hodgkinson and H. Arnheiter. 1998. Mutations in microphthalmia, the mouse homolog of the human deafness gene MITF, affect neuroepithelial and neural crest­ derived melanocytes differently. Mech ,

Keller, G., G. Zimmer, G. Mall, E. Ritz and K Amann. 2003. Nephron number in patients with primary hypertension. New Engl j. Med. 348: 101-108. .

Lammer, E. ). and 11 others. 1985. Retinoic acid embryopa thy. N. Eng!.

Med. 313: 837-ll4 1.

I.

Lillycrop, KA., E. S. Phillips, A. A. Jack­ son, M. A. Hanson and G. C. Burdge. 2005. Dietary protein restriction of preg­ nant rats induces and folic acid supple­ mentation prevents epigenetic modifi­ cation of hepatic gene expression in the offspring. f. Nutrition 135: 1382-1386.

MacchiarinL P. and 14 others. 2008. Clinical transplantation of a tissue-engi­ neered airway. umcet 372: 2023-2030. Maffini, M. v., A. M. Soto, I. M. Calabro, A. A. Ucci and C. Sonnenschein. 2004.

Oev. 70: 155-166.

Sulik, K K 2005. Genesis of alcohol­ induced craniofacial dysmorphism. Exp. Bioi. Med. 230: 366-375.

Ta kahashi, K and S. Yamanaka. 2006. induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:

663-676. Tammela, T. and 20 others. 2008. Block­

ing VEGFR-3 suppresses angiogenic sprouting and vascular network forma­ tion. Nat"re 454: 656-660.

Outside Sites

Go Online WEBSITE 17.1

Human embryology and genetics. This

links to other websites that will connect you to tutorials in human development, as well as to the Online Mendelian Inheritance in Man (OMlM), which details all human genetic conditions.

WEBSITE 17.2

Association of Reproductive Health Professionals:

malfonned arms and legs, and it provided the first major

resources for health care providers and their Endocrine disruptor exchange:

Reproductive Toxicology website: www.reprotox.org has summaries of over 5000

agents, exposure levels, and their effects on development and

evidence that drugs could induce congenital anomalies. The mechanism of i ts action is still hotly debated.

reproduction.

WEBSITE 17.3

The NIH website on stem cell education.

Our stolen future. nus website monitors

the environmental effects of endocrine disruptors. It is a political and consumer action site as well as a scientific clearinghouse for

endocrine disruption. Run by the authors of the book Our Stolen Future, it also provides links to the websites of people who disagree with them.

t echniqu e, thera peu tic the immune rejection

Therapeutic cloning. Prior to induced

pluripotent stem ceils, another

cloning, was seen as a way around

of stem cell-derived differentiated cells.

Vade Mecum Somites and thalidomide.

These movies are from the lab­

oratory of Jay Lash, whose insightful work on cartilage for­ mation resulted in some of our first insights into the mechansisms b y which the drug thalidomide halts limb growth.

clients.

www.arhp,orgltopics/enviro-repro-health contains

www.endocrinedisruption.com

Thalidomide as a teratogen. The drug

thalidomide caused thousands of babies to be born with

WEBSITE 17.4

657

Stern cell basics: http://stemcells.nih.gov/info/basics "All things stem cell." An informative stem cell blog: http://www. allthingsstemcell.com For information on fetal alcohol syndrome: http://www.cdc,gov/ncbddd/fasdidata.htmi . NlH, FASD: http://www.niaaa.nih.gov/AboutNIAAAllnteragency/ AboutFAS.htm

Substance Abuse and M ental Health Services Administration (SAMHSA):

http://www.fascenter.samhsa.gov

Developm ental Plasticity and Sym biosis

iT WAS LONG THOUGHT THAT THE ENVIRONMENT played only a minor role in

development. Nearly all developmental phenomena were believed to be a "read­ out" of nuclear genes, and those organisms whose development was significant­

ly controlled by the environment were considered interesting odd ities. When

environmental agents played roles in development, they appeared to be destruc­

tive, such as the roles played by teratogens and endocrine disruptors (see Chap­ ter 17). However, recent s tudies have shown that the environmental context plays significant roles in the

narmaJ d evelopment of almost aU species, and that ani­

mal genomes have evolved to respond to environmental conditions. Moreover, symbiotic associations, wherein the genes of one organism are regulated by the products 01 another organism, appear to be the rule rather than the exception. One reason developmental biologists have largely ignored the environment's eflects is that a criterion for selecting wh.ich animals to study has been their abil­

C. elegans, Drosapl.i/fl, zebra fish, Xenopus,

ity to develop in the laboratory (Bolker 1995). Given adequate nutrition and tem­

perature, all "model organisms

"-

ch.icks, and laboratory mice-develop independently 01 their particular environ­ ment, leaving us with tl,e erroneous impression that everythlng needed to form

the embryo is wW1in the fertilized egg. Today, with new concerns about the loss of organismal diversity and the effects of environmenta l pollutants, there is renewed interest in the regulation of development by the environment (see van der Weele 1999; Gilbert and EpeI 2009).

The Environment as a Normal Agent in Producing Phenotypes Although the nucleus and cytoplasm of the zygote contribute a majority of phe­ notypic instructions, everything needed for producing a pa rticula r phenotype is not pre-packaged in the fertilized egg. Rather, crucial parts of phenotypiC deter­

mination are regulated by environmental facton; outside the organism.

Phenotyp­

ic plasticity is the ability 01 an organism to react to an env ironmental input with

seen in embryonic or larval stages of animals or p la nts , this ability to change a change n i form, state, movement, or rate of activity (West-Eberhard 2003). When phenotype is often called

developmental plasticity.

We have already enco untered several exampl es of developmental plasticity in this book. When we discussed environmental sex determination in turtles,

fish, and ech.iuroid worms (see Chapter 14), we were aware that the sexual phe­ notype was being instructed not by the genome but by the environment. When we discussed in Chapter

12 the ability of shear stress to activate gene expression

in capillary, heart, and bone tissue, we Similarly were studying the effect of an

We may now tum to consider adapw­ dons towards the extenlOl environment; and fimly the direct adaptations . . . in which an animal, during irs deuelop­ mem, becomes mod ified by exremal

factors in such a way as to increase its effiCiency

in dea li ng

Wilh them.

c. H. WADDINGTON

( 1 957)

)IAN xu AND )EFFREY I. GORDON

(2003)

HOllor thy sYl1lbionts.

660

CHA PTER 1 8

(A)

(6)

IC)

(D)

Spring morph among catkins

Summer morph on twig

environmental agent on phenotype . While studies of phe­ notypic plasticity play a central role in plant developmen­

(E)

lal biology, the mechanisms of plasticity have only recent­ ly been studied in animals. These studies are showing that

the integra tion of animals into ecological communities is

accomplished largely through developmental interactions. autonomous enti ties but rather that we are co-construct­ Indeed, these studies show that we d o not develop as ,

ed by other organisms and that we respond to abiotic agents within our loea1 communities.

FIGURE 18.1 Devel opmen ta l plasti ci ty in i nsects . (A,B) Density­ induced polyphenisrn in the desert (or " plague") locu st Schistocer­ ca gregaria. (AI The low-density m orph has green pigmentation and miniature wings. (B) The high-density morph has deep pig­ mentation and wings and legs suitable for migration. (C,O) Nemo­ ria arizonaria caterpillars. (0 Caterpill ars that hatch in the spring eat young oak leaves and develop a c utic l e thai resembles the oak's flowers (catkins). (D) Caterpi llars thai hatch in Ihe summer, after the catkins are gone, eat mature oak leaves and develop a cuticle that resembles a young twig. (E) Gyne (reproductive queen) and wo rker of Ihe ant Pheidologeton. This picture shows the remarkable dimorphism between the la rge, fertile queen and the small, sterile worker (seen near the queen's antennae). The differ­ ence between these two sisters is the result of larval feed ing (A,B from Tawfik et al. 1 999, courtesy of S. Tanaka; C,D courtesy of E. Greene; E ro Mark W. Moffett/National Geographic Society.) .

recognized: reac tion norms and polyphenisms (Woitereck Two main types of phenotypiC plasticity are currently

1909; Schmalhausen 1949; Stearns et al. 1991). In a reaction norm, the genome encodes the potential for a

range of potential phenotypes, and

continuoIls

the environment the

the most adaptive one). For instance, our muscle pheno­

individual encounters determines the phenotype (usually

type is determined by the amount of exercise our body is exposed to (even though there is a genelically defined limit

to how much muscular hypertrophy is possible) Similar­ .

ly, the length of a male's horn in some dung beetle species is determined by the quantity and quality of food (Le., the

dung) the larva eats before metamorphosis (see the uext section) The upper and lower limits of a reaction norm are .

feren t phenotypes produced by environmental conditions are called morphs (or, occaSionally, ecomorphs). The second type of phenotypic plasticity, po(yphenism,

also a property of the genome that can be selected. The dif­

refers to discontinuous ("either/or") phenotypes elicited by

(Al

Hornless male

Horned male

DEVELOPMENTAL PLASTICITY AND SYMBIOSIS

661

FIGURE 18.2 Diet and Onthophagus horn p henoty pe (AI Horned and hornless males of the dung beetle Onthophagus acuminatu5 (horns have been artificially colored). Whether a male is horned or hornless is determined by the liter of juvenile hor· mone at the last molt, which in turn depends on the size of the larva. (B) There is a sharp threshold of body size under which horns fail to form and above which horn growth is linear with the size of th e beetle. This threshold effect p roduces males with no horns and males with l arge horns, but very few with horns of intermediate size. (Aher Emlen 2000; photographs courtesy of D. Emlen.) .

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the environment. One obvious example is sex determina­

phenotypes in genetically identical organisms. Diet is also largely responsible for the formation of fertile "queens" n i ant, wasp, and bee colonies. Among these insects, each larva has the genetic potential to become either a worker or a queen; only those larvae fed adeguately become queens ( Figure

1 8.1 E).

In honeybees, we know that extra

nutrition results in the demethylation of particular genes associated with ovary growth and general metabolic rate (Maleszka

2008; Elango et al. 2009; Foret et al . 2009) .

See WEBSITE 1 8.1 Inducible caste determination in ant colonies WHEN DUNG REAllY MAnERS

For the male dung beetle

(Ol1thaphngus), what really matters in life is the amount and

quality of the dw'g he eats as a larva. The hornless femaLe

tion in turtles, where one range of temperatures will induce

dung beetle digs tunneLs, then gathers balls of dung and

female development in the embryo, and another set of tem­

buries them in these tunnels. She then lays a Single egg on

peratures will induce male development. Between these

each dung ball; when the larvae hatch, they eat the dung.

sets of temperatures is a small band of temperatures that

Metamorphosis occurs when the dung bail is finished, and

will produce different proportions of males and females,

the anatomical and behavioral phenotypes of the

but they do not induce intersexual animals. Another

dung beetle are determined by the quality and q uantity

important example of polyphenism is the migratory locust

this maternally provided food (Emlen

Schistocerca gregaria. These grasshoppers exist either as a

Emlen

2000).

male of

1997; Moczek and

The amount of food determines the size of

short-winged, green, solitary morph or as a long-winged,

the larva at metanwrphosis; the size of the larva at meta­

Cues in the

morphosis determines the titre of juveni le hormone dur­

environment determine which morphology a larva will

ing its last molt; and the titre of juvenile hormone regulates

develop upon molting (Rogers et al.

the growth of the imaginal discs that make the horns ( Fig­

brown, gregarious morph ( Figure

Sword

1 8.1A,B).

2003; Simpson and

ure

2008).

1 8.2A; EmLen and Nijhout 1999; Moczek 2005). If juve­

nile hormone is added to tiny

Diet-induced po/yphenisms The effects of diet in development can be seen in the cater­ pillar of

Nentoria arizonaria.

When it hatches on oak trees

in the spring, it has a form that blend, remarkably with the young oak flowers

O. taunts males during the

sensitive period of their last moLt, the cuticle in their heads expands to produce horns. Thus, whether a male is horned

or hornless depends not on the male's genes but on the food his mother left for him.

Horns do not grow until the male beetle reaches a cer­

(catkil1s). But those larvae hatching from

tain size. After this threshold body size, horn growth is

their eggs in the sununer would be very obvious if they

very rapid.' Thus, although body size has a normal distri­

still looked like oak flowers. Instead, they resemble newly

bution, there is a bimodal distribunon of horn sizes: about

formed twigs. Here, it is the diet (young versus old oak

half the males have no horns, while the other half have

leaves) that determines the phenotype ( Fig ure

horns of considerable length ( Figure

Greene

1 8 . 1 C, D;

1 8.2B).

1989).

Diets having different amounts of proteins or different concentrations of methyl donors have also been fOW1d to cause different genes to be expressed in mammalian embryos. Different diets can lead to remarkably distinct

*lnterestingly, the threshold size at which the phenotype changes from hornless to homed is genetically transmitted and can change when conditions favor one morph over the other (Emlen 1996, 2000).

662

CHAPTER 1 8

that gives mice yellowish hajr color; i t also affects lipid metabolism such that the mice become fatter. The

viable-yel­ low allele of Agouti has a transposable element inserted into its cis-regulatory regions. These transposon insertion sites

are

very interesting for regulation: whereas most regions of

the adult genome have hardly any intraspecies variation in CpG methylation, there are large DNA methylation differ­ ences between individuals at the sites of transposon inser­ tion. Such CpG methylation can block gene transcription. When the promoter of the Agouti gene is methylated, the gene is not transcribed. The mouse's fur remains black, and lipid metabolism is not altered. Waterland and Jirtle fed pregnant

viable-yellow Agouti

mice methyl donor supplements, including folate, choline, and betain. They found that the more methyl supplemen­ tation, the greater the methylation of the transposon inser­ tion site in the fetus' genome, and the darker the pigmen­ tation of the offspring. Although the mice in Figure

1 8.4

are genetically identical, their mothers were fed different diets during pregnancy. The mouse whose mother did not

FIGURE 18.3 The presence or absence of horns determines the male reproducti ve strategy in some du ng beet l e speci es . Females dig tunnels in the soil beneath a pile of dung and bring dung frag­ ments i n to the lunnels. These will be the food supply of the larvae. Horned males guard the tunnel entrances and mate repeated ly with the fema les. They fight to prevent other males from entering the tunnels, and thee males with long horns usually win such contests. Sma l l er, hornless males do not guard tunnels, but dig their own to connect with those of females. They can then mate and exit, u nCha l l enged by the guard i ng male. (After Emlen 2000.)

receive methyl donor sup plementation is fat and yellow­ the Agouti gene promoter was unmethylated, and the gene was active. The mouse born to the mother that was given supplements is sleek and dark; the methylated Agouti gene was not transcribed. As we saw in Chapter 17, such dlifferential gene methyla­ tion has been linked to human health problems. Dietary restrictions during a woman's pregnancy may show up as heart or kidney problems in her adult children. Moreover,

studlies in rats showed that dlifferences in protein and methyl donor concentration in the mother 's prenatal diet affected metabolism in the pup's livers (Lillycrop et a!.

2005).

The size of the hams detennines a male's behavior and chances for reproductive success. Horned males guard the females' twmels and use their horns

to prevent other males

from mating with the female; the male with the biggest

horns wins such contests. But what about the males with no horns' Hornless males do not fight with the horned males for mates. Since they, like the fe.males, lack horns, they are able to dig their own tunnels. These "sneaker males" dig tunnels that intersect those of the females and mate with the females while the horned male stands guard at the tunnel entrance (Figure and Emlen

2000).

1 8.3; Emlen 2000; Moczek

Indeed, about half the fertilized eggs in

most populations are from hornless males. The ability to produce a hom is inherited; but whether to produce a hom and how big to make it is regulated by the environment.

DIET AND DNA METHYLATION Diet can also directly influ­ ence the DNA. As mentioned earlier, honeybee caste (queen or worker) is determined by dliet-induced changes in DNA methylation patterns. Dietary alterations can also produce changes in mammalian DNA methylation, and these methylation changes can affect the phenotype. Waterland and Jirtle

(2003) demonstrated this by

the viable-yellow aliele of Agouti.

using mice contalning

Agouti is a dominant gene

FIGURE 18.4 Maternal diet can affect p henotype. These two mice a re geneti ca l ly identical; both contain the viable-yellow all el e of the Agouti gene, whose protein product converts brown pigment to yellow and accelerates fat stora ge . The obese ye l low mouse is the offspring of a mother whose diet was not supple­

mented with methyl donors (e.g., folic acid) during her pregnancy. The embryo'S Agouti gene was not methyla ted, and Agouti protein was made. The sl eek b rown mouse was born of a mother whose pre natal diet was supplemented with methyl donors. The Agouti gene was turned off, and no Agouti protein was made. (After Waterland and J i rtl e 2003, photograph courtesy of R. L. Jirtle.)

DEVELOPMENTAL PLASTICITY A N D SYMBIOSIS

soluble filtrate from waler surrounding the predator is able to induce the changes . Chemicals that are released by a predator and can induce defenses in its prey are called kairomones Several roilier species will alter their morphology when they develop in pond water in which their predators were cultured (Dodson 1989; Adler and Harvell 1990). The predatory rotifer Asplnnchna releases a soluble compound that induces the eggs of a prey roilier species, Keralello slac­ ki, to develop into individuals with slightly larger bodies and anterior spines 130% longer than th ey otherwise would be, making the prey more difficult to eat When exposed to the effluent of the crab species that preys on it, the snail Thais lamellosa develops a thickened shell and a "tooth" in its aperture. [n a mixed snail population, crabs will not attack the thicker-shelled snails until more than half of the typicaJ-morph snails are devoured (Palmer 1985).

Predator-induced polyphenisms

Imagine an animal who is frequently confronted by a par­ tkular predator. One could then imagine an individual who could recognize soluble molecules secreted by that predator and could use those molecules to activate the development of structures that would make this individ­ ual less palatable to the predator. This ability to modulate development in the presence of predators is called preda­ tor-induced defense, or predator-induced pol ypheni sm. To demonstrate predator-induced polyphenism, one has to show that the phenotypic modification is caused by the presence of the predator, and that the modification increas­ es the fitness of its bearers when the predator is present (Adler and Harvell 1990; Tollrian and Harvell 1999). Fig­ ure 18.5A shows both the typical and predator-induced morphs for several species. In each case, the induced morph is more successful at su rviving the predator, and

(Keratella) Rotifer

pe

A

rtu re

�� t8/59

Mollusc

Barnacle (Chthama/us)

t t/43

(Thai,)

Thickened, "toothed"

r n

Preda to prese t

30/100

Predator

FIGURE 18.5 Predator-induced defenses. (A) Typical (upper row) and predator-induced (lower row) morp s of various organisms. The numbers beneath each column represent the percentages of organisms surviving predati on when both induced and uninduced individuals were presented wi th predators (in various assays). (8) Scanning electron micrograph s show predator induced (left) and typical ( right) morphs of genetically identical individuals of the water flea Daphnia. In the prese n ce of chemical signa l s from a -

/

Expanded body depth

(D)

h

(B)

(Carassius)

No predation until 50% of typical inorphs devoured

(C) Predator present

.

Carp

(A)

663

Predator absent

absent

in

predator Daphnia grows a protective "helmet." (e,D) Tadpole phenotypes. (e) Tadpoles of the tree frog Nyla chrysosce/is devel­ oping th e presence of cues from a predator's larvae develop strong trunk muscles and a red coloration (0) When predator cues are absent, the tadpoles grow sleeker, which helps them compete for food. fA aher Adler a nd Harvell 1 990 and references cited therein; B courtesy of A. A. Agrawal; C,O courtesy of T. Johnson/USGS.) ,

.

664

CHAPTER 1 8

One of the more in teres ting mechanisms of predator­

induced polyphenism is that of certain echinoderm larvae. When exposed

to the mucus of their fish pred a tor, sand budding off small groups of cells that quickly become larvae themselves. The small plutei are below the visual detection of the fish, and there­ by escape being eaten (Vaughn and Strathmann 2008; Vaughn 2009). dollar plutei clone themselves,

DAPHNIA AND THEIR KIN The predator-ind uced poly­ phenism of the parthenogenetic water flea Daphnia is ben­ eficial not only to itself but also to its offspring. When D. c"cul/atn encoun ter the predatory larvae of the fly Chaeoborus, their "helmets" grow to twice the normal size (Figure 18.5B). This increase lessens the chances th at Daph­ nia will be eaten by the fly larvae. nus same helmet induc­ tion occurs if the Daphnia are exposed to extracts of water in which the fly larvae had been swinuning. Agrawal and colleagues (1999) have shown that the offspring of such an induced Daphnia are born with this same altered head mor­ phology. It is possible that the OweabortlS kairomone reg­ ulates gene expression both in the adult and in the devel­ oping embryo. We still do not know the iden ti ty of the kairomone, the identity of its receptor, or the mechanisms by which the b indi ng of the kairomone to U,e receptor ini­

tiates the adaptive morphological changes. There are trade­ offs, however; the induced into making protective (Tollrian

Daphnia, having put resources structures, produce fewer eggs

1995).

AMPHIBIAN PHENOTYPES INDUCED BY PREDATORS Predator­

induced polyphenism is not limited to invertebrates," Among amphibians, tadpoles found in ponds or reared in the presence of other species may differ Significantl y from tadpoles reared by themselves in aquaria. For in sta nce, newly hatched wood frog tadpoles (Rona sylvetica) reared in tanks con taining the predatory la r va l dragonfly Anax (confined in mesh cages so U,ey cannot eat the tadpoles) grow smaller than those reared in similar tanks without predators. Moreover� their tail muscuJat\J-re deepens, allow­

ing faster turning and swinuning speeds (Van Buskirk and Relyea 1998). The addition of more preda tors to the tank causes a continuously deeper tail fin and tail musculature, and in fact what initially appeared to be a polyphenism may be a reaction norm that can assess the number (and type) of predators. McCollum and Van Buskirk (1996) have shown that in the presence of its predators, the tail fin of the tadpole of the tree frog Hyla chrysoscelis grows larger and becomes bright red (Figure 18.5C,D). nus phenotype allows the tad­ p ole to swim away faster and to deflect predator strikes *Indeed, when viewed biologically rather than medicilUy, the verte­ brate immune system is a wonderful example of predator-induced polyphenisru. Here, our immune cells utilize chemicilis from our

predators (viruses and bacteria) to change our phenotype so that we CCln better resist them (see Frost 1999).

toward the tail regi on . The trade-off is that noninduced tadpoles grow more slowly and survive better in preda­ tor-free environments. In some species, phenotypic plas­ ticity is reversible, and removing the predators can restore the non-induced phenotype (Relyea 2003a). The metabolism of predator-induced morphs may differ significantly from that of the uninduced morphs, and this has important consequences. Relyea (2003b, 2004) has found tha t in the presence of the chemical cues emitted by predators, the toxicity of pes ticides such as carbaryl (Sevin®j can become up to 46 times more lethal than it is without the predator cues. Bullfrog and green frog tadpoles were espeCially sensitive to carbaryl when exposed to pred­ ator chemicals. Relyea has related these findings to the global decline of amphibian populations, saying that gov­ ernments should test the toxicity of the chemicals under more natural conditions, including that of predator stress. He concludes (Relyea 2003b) that "ignoring the relevant ecology can cause incorrect estimates of a pesticide's lethal­ ity in nature, yet it is the lethality of pesticides under nat­ ural condi tions that is of utmost interest. The accumulat­ ed evidence strongly suggests that pesticides in na ture could be playing a role in the decline of amphibians ."

VIBRATIONAL CUES ALTER DEVELOPMENTAL TIMING The phe­ notypiC changes induced by environmental cues are not confined to structure. They can also include the timing of developmental processes. The embryos of the Costa Rican red-eyed treefrog (Agnlychnis callidryas) use vibrations trans­ mitted through their egg masses to escape egg-eating snakes. These egg masses (shown on this book's cover) are laid on leaves that overhang ponds. Usually, the embryos develop into tadpoles within 7 days, and these tadpoles wiggle out of the egg mass and fall into the pond wa ter. However, when snakes feed on the eggs, the vibrations they produce cue the remaining embryos inside the egg mass to begin the twitcl' ing movements that initiate their hatching (within seconds!) and dropping into the pond. The embryos are competent to begin these hatching movements a t day 5 (Figu re 1 8.6). Interes tingly, the embryos have evolved to respond this way only to vibrations given at a certain fre­ quency and interval (Warkentin et al. 2005, 2006). Up to 80% of the remaining embryos can escape snake predation in this way, and research has shown that these vibrations alone (and not smell or sight) cue these hatching movements in the embryos. There is a trade-off here, too. Although these embryos have escaped their snake predators, they are now a t greater risk from waterborne

predators than are fully of the early

developed embryos, because the musculature

hatchers has not developed fully.

Temperature as an environmental agent TEMPERATURE AND SEX There are many species in which

temperature does control whether testes or ovaries devel­ op. Indeed, among the cold-blooded vertebrates such as fish, tur tles, and all igators there are many species in which

DEVELOPME NTAL PLASTICITY A N D SYMBIOSIS

(A)

665

(B)

(e)

FIGURE 18.6 Preda tor- i nd uced polyphenism in the red-eyed tree frog Agalychnis callidryas. (A) When a snake eats a elutch of Aga/ychnis eggs, most of the remaining embryos inside the egg mass respond to the vibrations by hatching prematurely (arrow) and falling into the water. (8) I mmature tadpole, induced to hatch at day S. (C) Normal tadpol es hatch at day 7 and have better-developed musculature. (Cou rtesy of K. Warkentin.)

the environment determines whether an individual is male or female (Crews and Bull 2009). This type of environmen­ tal sex determination has advantages and disadvantages. One advantage is that it probably gives the species the ben­ efits of sexual reproduction without tying the species to a 1 : 1 sex ratio. In crococliles, in which extreme temperatures produce females while moderate temperatures produce males, the sex ratio may be as great as 10 females to each male (Woodward and Murray" 1993). 1n such instances, where the number of females limits the population size; this ratio is better for survival than the 1:1 ratio demand­ ed by genotypic sex determination. The major disadvantage of temperature-dependent sex determination may be its narrowing of the temperature lim­ its within which a species can persist. Thus thermal poUu­ tion (either locally or due to global warming) could con­ ceivably eliminate a species in a given area Uanzen and Paukstis 1991). Researchers (Ferguson and Joanen 1982; Miller et al. 2004) have speculated that dinosaurs may have had temperature�dependent sex determination and that their sudden demise may have been caused by a slight dlange in temperature creating conditions wherein only males or only females hatched. (Unlike many turtle species, whose members have long reproductive lives, can hiber­ nate for years, and whose females can store sperm, dinosaurs may have had a relatively narrow time to repro­ duce and no ability to hibernate through prolonged bad times.) See WEBSITE 1 8.2 Volvox: When heat brings out sex

Charnov and Bull (1977) argued that environmental sex determination would be adaptive in those habitats ellar­ acterized b y patchiness-that is, a habitat having some regions where it is more advantageous to be male and other regions where it is more advantageous to be female. Conover and Heins ( 1987) provided evidence for this hypothesis. In certain fish species, females benefit from being larger, since larger size translates into higher fecun­ dity. If you are a female Atlantic silverside fish (Menidia menidia), it is advantageous to be born early in the breed­ ing season, because you have a longer feeding season and thus can grow larger. (The size of males in this species doesn't influence mating success or outcomes.) In the southern range of Menidia, females are .indeed born early in the breeding season, and temperature appears to play a major role in this pattern. However, :in the northern reach­ es of its range, the species shows no environmental sex determination. Rather, a 1:1 sex ratio is generated at all tempera tures. Conover and Heins speculated tha t the more north.em populations have a very short feeding season, so there is no advantage for females in being born earlier. Thus, this fish has environmental sex determination in those regions where it is adaptive and genotypic sex deter­ mination in those regions where it is not. BUTTERFLY WINGS 1n tropical parts of the world, there is often a hot wet season and a cooler dry season. In Africa, a polyphenism of the dimorphic Malawian butterfly (Biclj­ elliS anljnafla) is adaptive to seasonal changes. The dry (cool) season morph is a mottled brown butterfly that sur­ vives by hiding in dead leaves on the forest floor. In con­ trast, the wet (hot) season morph, which routinely flies, has prominent ventral eyespots that deflect attacks from predatory birds and lizards (Figure 1 8.7).

666

CHAPTER 1 8

Dry-seas on form

Decreased amount of20-hydroxyecdysone OH HO '

� 24·C

Larva

OH

o

Increased amount of 20-hydroxyecdysone OH HO '

o

Distal-less expression

in imaginal disc OH

OH HO '

Wet-season form

OH _

o

FIGURE 18.7 Phenotypi c plasticity in Bicyclus anynana is regu­ lated by temperature during pupation. High temperature (either i n the wild o r in c ontroll ed laboratory conditions) allows t h e accu­ mulation of 20 hydrox yecdysone (20E), a hormone that is able to sustain Distal-less expression i n the pupal imaginal disc. The region of Distal-less expression becomes the focus of each eye­ spot. In cooler weather, 20E is not formed, Distal-Jess expression in the imaginal disc begins but is n ot sustained, and eyespots fa il to form. (Courtesy of S. Ca rrol l and P. Brakefield.) -

The importance of hormones such as 20E for mediating env ironmental signals controlling wing phenotypes has been docmnented in the Araschnin butterfly (Figure 1 8.8). Araschnia develops aIternative phenoty pes depending on whether the fourth and fifth instars experience a photope­ riod (hours of daylight) that is longer or shorter than a par­ ticular critical day length. Below tnis critical day length, ecdysone levels are low and the butterfly has the orange wings characteristic of spring butterflies. Above the criti­

The factor determining the seasonal pigmentation of B. nnynal1a is not diet, but the temperature d uring pupation. Low temperatures produce the dry-season morph; higher temperatures produce the wet-season morph (Brakefield and Reitsma 1991). The mechanism by which temperature regulates the Bicyclus phenotype is becoming known. In the late larval stages, transcription of the distal-less gene in the wing imagin al discs is restri cted to a set of cells that will become the signaling center of each eyespot. In the early pupa, higher temperatures elevate the formation of

20-hydroxyecdysone (20E; see Chap ter )5). This hormone sustains and expands the expression of distal-less in those regions of the wing imaginal disc, result.ing in prominent eyespots. In dry season, the cooler temperatures prevent the accumula tion of 20E in the pupa, and the foci of Dis­

tal-less signaling are not sus tained . In the absence of the

Distal-less signal, th e eyespots do not form (Brakefield et at.

1996; Koch et at. 1996). Distal-less protein is believed to

be the activating signal that determines the size of the eye­ spot (see Figure

18.7).

FIGURE 18.8 Environmentally induced morphs ofthe European map butterfly (Araschnia levana). Th e orange morph (bottom) forms i n the spri ng when levels of ecdysone in the la rva are low. The dark morph with a white stripe (top) forms i n summer, when h igher temperatures and longer photoperiods induce greater ecdysone p roduction in the larva. Linnaeus classified the two morph s as different species. (Courtesy of H. F. Nijhout.) ,

cal point, ecdysone is made and the sum mer pigmenta tion forms. The summer form can be ind uced in spring pupae by injecting 20E into the pupae. Moreover, by altering tne timing of 20E injections, one can generate a series of inter­

mediate forms not seen in the wild (Kodl and BUckmann

1987; Nijhout 2003).

DEVELOPMENTAL PLASTICITY A N D SYMBIOSIS

Environmental I nduction of Behavioral Phenotypes

667

� • Low maternal care

• Ell Plentiful maternal care

In many instances, the morphological phenotype is accom­ panied by a behavioral phenotype. This is obvious in the

l

envirorunental determination of sex, where an individual's sexual behavior generally matches the gonads and geni­

100 90

"

80

70

.g � 60 >-

talia. This is also seen in the cases of butterfly wings (fliers vs. crawlers) and dung beetle horns (fighters vs. "sneak­

-5 50 � E 40 � .S 30 � 8- 20 10 o '---u, -- .t ----.t - .-'-'--

ers"). Sometimes, however, the behavior is the major devel­ opmental phenotype induced by the environment.

Adult anxiety and environmen tally regulated DNA methylation

G