Form of the worm:

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C. elegans epidermal morphogenesis

Form of the worm: genetics of epidermal morphogenesis in C. elegans The development of the epidermis of the nematode worm Caenorhabditis elegans illustrates many common processes of epithelial morphogenesis. In the worm, these morphogenetic movements have been described with single-cell resolution, and the roles of individual cells have been probed in laser killing experiments. Genetic dissection is yielding insights into the molecular mechanisms of these complex morphogenetic processes. Ian D. Chin-Sang chinsang@ biology.ucsc.edu Andrew D. Chisholm chisholm@ biology.ucsc.edu Dept of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA.

orphogenesis, the development of shape and form, has been described as the ‘next frontier’ of developmental biology1. This is a reflection of our mature, although incomplete, understanding of other processes in development, such as pattern formation, cytodifferentiation, and cell proliferation. In contrast, the dynamic fourdimensional nature of morphogenetic movements poses a challenge to the molecular geneticist accustomed to linear pathways and binary switch genes. Genetic model organisms such as Caenorhabditis elegans and Drosophila provide simple examples of most morphogenetic movements, and

M

FIGURE 1. Topology of C. elegans epithelia (a)

Mouth

Pharynx Intestine

Anus

Gonad Vulva

Epidermis Mesoderm

(b)

Mouth Pharynx Intestine Vulva Gonad

Anus trends in Genetics

(a) The shape of an adult C. elegans hermaphrodite, with the major organs shown. (b) Schematic topology of the epithelia. Like most metazoans, C. elegans comprises an outer epithelial layer (epidermis, orange) enclosing an inner epithelial layer (endoderm, light brown), the two layers being separated by mesenchymal mesoderm. The epidermis is breached by three holes: the mouth, the anus, and (in adult hermaphrodites) the vulva. Each orifice is built from stacked toroidal epidermal cells that connect the epidermal and internal tubes. (Smaller openings in the epidermis, such as the excretory pore, are formed within single epidermal cells.) The gonads (yellow) form internal tubes, opening to the outside via the vulva in adult hermaphrodites or the cloaca/anus in adult males. 544

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the power of genetics is beginning to elucidate the mechanisms by which multicellular structures are molded. Many morphogenetic movements involve epithelia. Spreading, invagination, extension, and fusion or detachment of epithelia are accomplished by changes in cell shape, and the coordination of these changes involves interactions among the extracellular matrix, the cell surface, and the cytoskeleton. Here we highlight recent progress in understanding one aspect of C. elegans epithelial morphogenesis: the embryonic development of the epidermis. The epidermis, also known (for historical reasons) as the hypodermis, is the worm’s largest organ, and its structure largely defines the shape and size of the animal. Worm epidermal development involves several common epithelial movements, all of which are being subjected to genetic scrutiny. The morphogenesis of other epithelial organs, such as the gut2, gonads3, hermaphrodite vulva4, and male tail5, have recently been described in detail, and are already proving fertile ground for genetic approaches (e.g. Refs 6, 7). The vermiform body shape of C. elegans reflects the morphogenesis of the embryonic epidermis (Figs 1, 2). Epidermal cells are produced from dorsal ectodermal precursors, and form a layer that spreads to enclose the embryo, closing up at the ventral midline. This process of epidermal enclosure is an example of epiboly, a term that encompasses a variety of spreading movements of cell sheets (Table 1 for examples). Following enclosure, contractions within the epidermis cause elongation of the embryo. From 6 to 11 h after first cleavage, the embryo elongates from a ball of about 550 cells into the cylindrical worm. Mutants with defects in epidermal morphogenesis have been recovered by several genetic approaches (see Box 1 for a description of how ‘morphogenes’ have been found, and see Table 2 for a summary of genes for which the molecular identities are known). In this review, we focus on genes specifically required for the shaping of the embryonic epidermis. Many mutations with dramatic effects on body shape (phenotypes known as ‘dumpy’, ‘small’, ‘long’, ‘roller’, etc.) affect components of the collagenous cuticle8. Whilst these indicate the importance of the cuticle in maintaining the shape of the epidermis, in general such 0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(00)02143-0

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mutations do not affect the embryonic cell movements discussed here. For reasons of space, we also omit any discussion of genes such as vab-3 (a C. elegans Pax-6 homolog) and vab-7 (homologous to even-skipped) whose effects on epidermal morphogenesis probably reflect defects in epidermal cell-fate specification9,10.

FIGURE 2. C. elegans embryonic morphogenesis

0 Hours 20 °C

First cleavage (Time = 0)

Setting the stage: gastrulation To put the formation of the epidermis in context, a brief review of the preceding cell movements of gastrulation is necessary. C. elegans gastrulation involves the ingression of gut, germline, and mesoderm precursors from the ventral side of the early embryo (Fig. 2a). Despite this relative simplicity, we still know little of the mechanisms controlling cell movement and adhesion during gastrulation11. A depression created on the ventral side of the embryo by such movements, known as the ‘entry zone’ or ‘gastrulation cleft’, is then closed by short-range movement of ectoderm cells (mostly neuroblasts) that flank the cleft (Fig. 2b). The VAB-1 Eph receptor, tyrosine kinase, and its ephrin ligand VAB-2/EFN-1 are required for these neuroblast movements; Eph signaling mutants display ventral clefts characteristically broader, deeper and more persistent than those found in the wild type12,13. Eph signaling has been studied intensively in the context of vertebrate neural development, where it promotes axon growth cone collapse14. The primary defect in the worm Eph mutants is unclear, but, by analogy, could be excessive or inappropriate adhesion between neural precursors during gastrulation. Strikingly, several vertebrate Eph receptors and ephrin ligands are expressed during gastrulation in vertebrate embryos15, where they may function in mesodermal and neural morphogenesis16. Thus, the involvement of Eph signaling in gastrulation cell movements might be conserved in evolution. The correct movement of neuroblasts following gastrulation might be a prerequisite for the next major morphogenetic movement, i.e. ventral epidermal enclosure.

(a) Gastrulation 2

(b) Ventral cleft closure

(c) Dorsal 4

epidermal intercalation

(d) Ventral epidermal enclosure 6

(e) Elongation

10

Epiboly: epidermal enclosure of the embryo The epidermis begins as a dorsal sheet that spreads around the embryo until the edges meet at the ventral midline (Figs 2d and 3). Such spreading of a bounded epithelium over a substrate is known as epiboly. Epiboly movements are common in animal development; examples include the movement of the animal cap in Xenopus gastrulation and dorsal closure of the epidermis in Drosophila, the latter being the subject of intensive genetic analysis (reviewed in Ref. 17). An important motivation for understanding epiboly is its similarity to aspects of wound healing18. Time-lapse analysis of C. elegans epidermal enclosure has defined two cell populations that undergo partly independent movements19. The initial ventral migration is led by an anterior quartet of epidermal cells, known as leading cells, which extend actin-rich filopodia toward the ventral midline (Fig. 3a). Laser inactivation of all four leading cells blocks enclosure, as does treatment with cytochalasin D, which shows that these cells provide the driving force necessary to pull the epithelial sheet around the embryo. Epidermal cells posterior to the leading cells, known as ‘ventral pocket’ cells, do not have protrusive edges but have actin microfilaments along their free edges, forming a ring that runs around the edge of the pocket (Fig. 3b). Thus, enclosure of the ventral pocket is probably driven by a supracellular ‘purse-string’ mechanism analo-

trends in Genetics

Major morphogenetic movements are outlined; developmental time at 20°C is relative to first cleavage46 (note that in some papers, timing is erroneously given as post-fertilization). The Nomarski differential interference contrast (DIC) photomicrographs and schematic diagrams presented show lateral or ventral views, oriented with the anterior to the left. (a) Gastrulation (100–250 min). The first cells to move inwards from the ventral surface are gut precursors, followed by mesoderm and germline precursors. Gut and pharyngeal cells form a central column; cells in this column epithelialize to form the pharynx and gut2. (b) Closure of the ventral gastrulation cleft (230–290 min), by short-range movement of ventral ectoderm cells. (c) Formation of the epidermis and dorsal intercalation. Epidermal cells are born at ~240 min and form an epithelium soon afterwards. The two rows of dorsal epidermal cells intercalate into a single dorsal row (290–340 min), causing a slight lengthening of the dorsal epidermis relative to the ventral epidermis (dorsal view). Dorsal epidermal cells (represented here in pink), lateral epidermis (yellow) and ventral epidermis (red) are shown. (d) Epiboly: ventral epidermal enclosure (310–360 min). The epidermal layer spreads to enclose the embryo, sealing up at the ventral midline (ventral view). (e) Elongation (~360–600 min). Circumferential contraction within the epidermis causes elongation of the embryo, which increases in length approximately fourfold, and becomes approximately three times thinner. Synthesis of the larval cuticle begins at ~650 min. Post-embryonic epidermal growth increases the size of the worm but does not significantly alter its shape.

gous to that in Drosophila dorsal closure20. The ventral cells form adherens junctions with their contralateral partners to complete ventral enclosure. When epidermal cells are killed by laser ablation to create a hole in the epidermis, pressure due to later movements of elongation causes internal cells to ooze out of the TIG December 2000, volume 16, No. 12

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TABLE 1. Some processes of epithelial morphogenesis Process

C. elegans example

Other examples

Epiboly: Spreading of the edges of an epithelial sheet over a substratum

Ventral enclosure of epidermis

Dorsal closure of epidermis (Drosophila) Blastodisc and enveloping layer (zebrafish) Animal cap in Xenopus gastrulation Wound healing

Ingression: Movement of individual cells into interior of embryo

Gastrulation

Formation of primary mesenchyme in sea urchin

Convergent extension: Extension of tissue along one axis and its narrowing along a perpendicular axis

Dorsal epidermal intercalation

Dorsal marginal zone in Xenopus gastrulation Archenteron extension in sea urchins

Contraction-driven elongation: Expansion of a tube along one axis by contractile forces developed around its circumference

Epidermal elongation

?

hole in the epidermis, and the embryo dies21. This distinctive phenotype allows genetic screening for enclosure mutants, although other, incompletely penetrant, enclosure mutants (such as vab-1) were identified on the basis

BOX 1. How the genes involved in morphogenesis have been isolated Genes with roles in epidermal morphogenesis have been recovered from a variety of forward genetic screens.

Genome-wide screens for viable mutants These have been the source of most mutants with dramatic epidermal defects47. Many cuticle mutants (gene classes such as ‘dumpy’, ‘roller’, ‘small’) were identified in hunts for viable mutants. Such screens might be thought unlikely to recover mutations affecting essential embryonic processes such as epidermal enclosure. However, mutations causing incompletely penetrant and variably expressed defects in epidermal morphogenesis (such as those affecting the VAB-1 Eph receptor) could be recovered. For several vab genes, the null phenotype is variable, perhaps reflecting a high degree of redundancy in the genetic control of morphogenesis.

Screens for essential genes To recover lethal mutations, more labor-intensive clonal screens have been performed, usually for specific embryonic phenotypes such as defects in epidermal enclosure or in elongation (e.g. hmr-1, see Ref. 22). Several such loci were also initially found in screens designed to saturate certain genomic regions for lethal mutations (e.g. let-502). Screens for mutations affecting specific post-embryonic morphogenetic processes such as vulval invagination7 or gonadal morphogenesis have also been productive. As mentioned in the text, pat mutants were first found in a screen for mutations eliminating MYO-3, and were later screened for directly. An elegant screen using genetic mosaics to identify cell type-specific lethals48 yielded the mup-4 locus. Some mutants, such as hmp-1, are partly rescued by a maternal wild-type contribution, raising the possibility that mutations in other genes with roles in early morphogenesis will show similar rescue, and thus might not be found in conventional F2 or deficiency screens. To identify such genes, direct screens for maternal-effect morphogenetic mutants can be performed; alternatively, early zygotic functions of such genes can be probed using germline clones.

Chromosomal deficiency screens Both Nomarski microscopy and antibody or green-fluorescent protein (GFP) markers have been used to screen collections of multigene deficiencies (representing approximately 70% of the genome) for zygotic loci required for epidermal morphogenesis49,50, leading to the identification of let-413.

Reverse genetics Targeted screens for knockouts of specific genes, or RNA-mediated interference, provide the only way of probing the functions of genes with highly redundant roles; large multigene families such as the cadherins are excellent candidates for such approaches.

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of later epidermal defects (Box 1). Enclosure involves the movement of epidermal cells over a neuronal substrate. Thus, in analysing the defects of enclosure mutants it is important to define the focus of the defect, i.e. is the gene product required in epidermal cells, in substrate cells, or in both?

Role of the epidermal cells: actin-based motility Elegant genetic analysis has shown that a cadherin/catenin complex containing the HMP-1/a-catenin, HMP-2/ b-catenin and HMR-1/cadherin proteins, localized to epidermal adherens junctions, is critical for enclosure movements22. In the absence of any of these proteins, the leading cells do not migrate fully to the ventral midline (in the case of the HMP catenins, this occurs only when both the maternal and the zygotic contributions are removed). The HMP/HMR complex anchors the actin filaments at the adherens junctions and may transmit actin contractions into cell-shape changes. Once the leading cells have met at the ventral midline they rapidly form a seal. This sealing of epidermal contacts involves cadherin/HMR-1-dependent recruitment of a-catenin/HMP-1 from cytoplasmic stores to filopodial contacts at the ventral midline23. Mammalian cells have recently been shown to exhibit a strikingly similar cadherin-based filopodial adhesion process24. Only the four leading cells may require this ‘priming’, as sealing of the pocket cells is not affected in the absence of these proteins. Such specific functions of cadherin/catenin complexes in epidermal cell movements are in contrast to the more widespread defects in epithelial polarity and cell adhesion observed in cadherin mutants in vertebrates or Drosophila25, and suggest that other cell-signaling pathways may be responsible for general cell adhesion in the worm embryo. Genetic analysis of Drosophila dorsal closure has identified several signaling pathways [including the Jun N-terminal kinase (JNK) pathway and transforming growth factor b (TGFb) signaling pathways] that function in movements of the leading edge17. The worm homologs of such genes do not appear to be essential for epidermal enclosure, although they could play redundant or subtle roles. Genes specifically involved in enclosure of the ventral pocket have not yet been found; since this step resembles the ‘purse-string’-mediated dorsal closure in Drosophila, perhaps molecular similarities will emerge here.

Role of the neuronal substrate: permissive substrate or active signals? The migration of the leading cells over substrate neurons raises the question of whether substrate cells play an active role in guiding leading-cell migrations. None of the genes implicated in enclosure has been indicative of chemoattraction

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of leading cells to the ventral midline. Laser killing experiments have been inconclusive: ablation of small groups of neurons had no effect on enclosure; although larger-scale kills disrupted enclosure, this effect was attributed to cellular debris resulting from the kills19. Thus, if there is a neuronal cue for epidermal cells it must be provided redundantly by several cells. Although it is unclear whether neuroblasts provide a specific cue for attracting the leading cells, it is evident that the neuronal cells are an indispensable substrate for epidermal movements. Evidence of a role for the neuronal substrate in enclosure has come from the Eph signaling mutants. As mentioned above, mutations in the VAB-1 Eph receptor and the ephrin ligand, VAB-2/EFN-1, cause defects in neuroblast movements during gastrulation-cleft closure. However, striking defects in the Eph mutants are seen in epidermal morphogenesis; some embryos lacking the VAB-1 receptor completely fail to enclose the epidermis, and arrest, wheras other embryos enclose and then rupture at later stages. Despite these epidermal defects, expression patterns and genetic mosaic analysis strongly suggest that, in embryos, signaling involving VAB-1 and its ligands occurs mostly in the developing nervous system12. The nature of this non-autonomous effect of neurons on epidermal cells remains elusive. The epidermal defects may result from the earlier defects in gastrulationcleft closure; failure to close the cleft might result in a substrate that is not permissive for epidermal movements. Alternatively, Eph signaling might set up a specific cue for epidermal guidance; it is notable that the leading cells migrate close to the interface between VAB-1- and VAB-2/EFN-1-expressing cells (Fig. 3a, right panel). Recent work on Drosophila has also implied an active role for the amnioserosal substrate in dorsal epidermal enclosure20; whether this reflects a passive mechanical interaction or an instructive signaling event between substrate and epidermis remains to be determined. Another signaling pathway, previously known largely from its function in axon guidance, has roles in epidermal enclosure. Mutants lacking the secreted semaphorin-2a MAB-20 display enclosure defects (albeit different from those seen in Eph mutants) reflecting inappropriate contacts between epidermal cells26. Semaphorin signaling might regulate the formation or stabilization of contacts between epithelial cells; as MAB-20 is expressed ubiquitously, further insights will require the identification of receptors for MAB-20. Although semaphorin signaling has been studied mainly in the context of axon guidance, some vertebrate semaphorin mutants have been shown to have defects in the morphogenesis of non-neural tissues (e.g. Ref. 27), suggesting that this may be a conserved aspect of semaphorin function.

Directed intercalation in the dorsal epidermis At the same time as the epidermis is extending ventrally, two rows of dorsal epidermal cells undergo a type of convergent extension movement that may initially drive extension of the epidermis along the body axis. Convergent extension involves the directed intercalation of epithelial cells towards the axis of extension28 and is a common mechanism for driving the elongation of a tissue in one direction and the narrowing of the tissue in an orthogonal direction. In C. elegans, the dorsal epidermal cells extend processes circumferentially and intercalate to form a single row along the dorsal midline (Fig. 2c). The

FIGURE 3. Epidermal enclosure (a)

VAB-1

VAB-2/EFN-1

Leading cells Leading-edge cells Ventral pocket

(b)

Pocket cells

(c)

Ventral midline

Actin microfilaments

Dorsal epidermis

VAB-1-expressing neurons

Lateral epidermis

VAB-2-expressing neurons

Ventral epidermis trends in Genetics

Cartoons of ventral views are paired with immunofluorescence micrographs of comparable stages; the red staining is the MH27 antigen, recognizing a component of adherens junctions. (a) Epidermal leading cells move approximately 15 mm (from the equator to the ventral midline) in ~30 min. The filopodia of the leading cells appear to migrate between neuronal precursor cells underneath them. The immunofluorescence shows the expression of Eph receptor VAB-1 (blue) and ligand VAB-2 (green) in substrate neurons during enclosure. For simplicity, we refer to the underlying cells as neurons, although at the time of enclosure they include neuroblasts, post-mitotic neurons, and neuronal support cells or their precursors. (b) Enclosure of the ventral pocket; an actin-rich ring forms around the ventral pocket cells. Anterior leading cells have already formed a seal at the ventral midline. (c) Completion of epidermal enclosure and beginning of elongation. It is unclear how the head region anterior to the leading cells is enclosed; here, epidermal cells might be born in their final positions and form a sheet without long-range movements.

intercalation movements are generated cell-autonomously and require both actin microfilaments and microtubules29. Mutations in the die-1 gene block dorsal intercalation and elongation, but not epidermal enclosure29. Although die-1 might function independently in intercalation and elongation, it is possible that elongation (which requires oriented cytoskeletal elements) is dependent on the cell and cytoskeletal rearrangements accomplished by intercalation.

Elongation of the epidermis Perhaps the most obvious morphogenetic change in the C. elegans embryo is elongation, whereby the embryo changes from a shape resembling a lima bean to the long, TIG December 2000, volume 16, No. 12

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TABLE 2. C. elegans morphogenetic processes and cloned genes Process

Gene producta

Mutant defect

Refs

Closure of gastrulation cleft

VAB-1 Eph receptor VAB-2/EFN-1 Ephrin

Defective movements of neuroblasts

12,13

Epidermal enclosure

HMP-1 a-catenin HMP-2 b-catenin HMR-1 Cadherin

Blocked leading cell movements and sealing of contacts

22,23

VAB-1 Eph RTK VAB-2/EFN-1 Ephrin

Aberrant leading cell movements

12,13

MAB-20/Semaphorin-2a

Ectopic epithelial cell contacts

26

LET-502 Rho-kinase MEL-11 Phosphatase MLC-4 Myosin light chain kinase

Elongation block (twofold arrest)

30–32

HMP-1 HMP-2 HMR-1

Detachment of actin bundles from cell membrane; constrictions in dorsal epidermis

22

Epidermal elongation

SQT-3 cuticle collagen

Elongation then retraction

21

SMA-1 bH-Spectrin SPC-1 a-Spectrin

Slow elongation

35,36

LET-413/CeScribble

Twofold arrest

33

NHR-25 Nuclear hormone receptor

Twofold arrest

34

MYO-3 Muscle myosin PAT-2 a-integrin PAT-3 b-integrin LET-2/EMB-9 Type IV basement membrane collagens UNC-52 Perlecan DEB-1 Vinculin EGL-19/Calcium channel (1 many other genes)

Pat (paralyzed, arrest at twofold)

38,39,41,42

LET-805/myotactin

Nonparalyzed twofold arrest

43

MUP-4 Epidermal transmembrane receptor

Variable defects in epidermal enclosure and muscle cell position, attachment

44

a Definition of gene names: hmp, humpback (contracted dorsal epidermis); hmr, hammerhead (deformed head epidermis); let, lethal; mab, male abnormal; mel, maternal-effect lethal; mup, muscle positioning defective; pat, paralyzed arrest at twofold; sma, small body size; vab, variable abnormal morphology.

thin shape of the larva (Figs 2e and 4). Elongation of the embryo reflects the elongation of individual epidermal cells along the body axis. These cell-shape changes are driven cell-autonomously by contractions of circumferentially oriented actin microfilament bundles (CFBs) in the epidermal cells21. As a result of these epidermal contractions, the interior of the embryo develops a hydrostatic pressure that may itself drive the elongation of internal cells. Disruption of microtubules or of an embryonic extracellular matrix layer that surrounds the epidermis (the ‘embryonic sheath’) causes deformations in the epidermis, suggesting that microtubules and the embryonic sheath distribute, uniformly, the contractile forces generated in elongation. Unlike other examples of directed elongation based on differential growth, in this case the epidermal cells do not change in size and change only in shape.

The driving force: circumferential actin contraction Analysis of elongation-defective mutants has identified several genes that probably function in the regulation of the actin-based contractions of elongation. Maternaleffect mutations in the LET-502 Rho-binding kinase and the MEL-11 myosin phosphatase both cause defective elongation30. Elongation-defective alleles of let-502 act as recessive gain-of-function alleles and appear to be antimorphs. Loss-of-function mutations in mel-11, isolated as suppressors of the let-502 lethality, result in over-contraction of epidermal cells, suggesting that LET-502 and MEL-11 548


play antagonistic roles in elongation31. A plausible model (Fig. 4) is that LET-502 promotes contraction of the CFBs whereas MEL-11 mediates relaxation; in the lateral epidermis, where LET-502 levels are higher, contractile forces predominate and the cells change shape. A likely target of the MEL-11 phosphatase is the non-muscle myosin regulatory light chain MLC-4. MLC-4 is expressed in the lateral epidermis, and these cells fail to change shape in mlc-4 null mutants (identified by reverse genetics)32. The regulators of LET-502 and MEL-11 have not been definitively identified, but, on the basis of genetic interaction data, they are likely to be members of the Rac/Rho GTPase family. The contractile force of the actin CFBs in lateral epidermal cells is transmitted to the rest of the epidermis via adherens junctions. The HMP-1/HMP-2/HMR-1 catenin/ cadherin complex is critical for this process22. In Hmp mutants, the CFBs detach from adherens junctions, resulting in deformation of the epidermis and severe elongation defects (hence the gene name, Hmp, for ‘humpback’). The basal boundary of adherens junctions in epidermal cells appears to be defined by the basolateral PDZ-domain containing protein LET-413 (the worm homolog of Drosophila Scribble). In let-413 mutants, adherens junctions are not formed properly and elongation is blocked33. Additional players in elongation include the nuclear hormone receptor NHR-25, loss of function in which causes defective elongation. The requirement for NHR-25

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FIGURE 4. Embryonic elongation (a)

High MEL-11

CFB contraction

High LET-502

HMR/HMP complex

Actin filaments (CFB)

(b)

rho

let-502 mlc-4

rac (mig-2)

Contraction of CFB (elongation)

mel-11 trends in Genetics

(a) Cell-shape changes in lateral epidermal cells are shown by monoclonal antibody MH27 staining for adherens junctions at the comma stage (left) and the threefold stage (right). During elongation, the dorsal and ventral epidermal cells do not show as dramatic a change in shape as the lateral cells, and might play a passive role. Consistent with lateral cells having more contractile force is the finding that LET-502 is expressed at higher levels in the lateral cells whereas MEL-11 is expressed at higher levels in the dorsal cells. (b) Models for the regulation of actin-based contractions by LET-502/MEL-11/MLC-4 (based on Refs 30, 31) are shown. LET-502 might negatively regulate MEL-11 activity; alternatively, LET-502 and MEL-11 might act in parallel antagonistic pathways. The Rho GTPase putatively regulating LET-502 has not been identified yet; genetic interactions with the Rac-like GTPase mig-2 suggest that it could regulate mel-11.

may reflect defects in extracellular matrix synthesis, as escapers were found to have problems in molting34. How is the rate of contraction regulated? A spectrin network in the epidermal cells may play a crucial role. sma-1 mutants are defective in the apical cytoskeletal component bH-spectrin, and elongate up to five times slower than the wild type; the mutants are consequently small (hence ‘sma’), but viable35. Inhibition of the SPC-1 a-spectrin (by RNA interference) causes elongation defects similar to those of sma-1 mutants36, suggesting that a SMA-1/SPC-1 complex in the apical cytoskeleton could cross-link actin microfilaments and link them to the cell membrane. The UNC-70 b-spectrin appears to play no role in this aspect of spectrin function37. Slower elongation in sma-1 mutants could reflect either a reduction in the contractile force itself, or a defect in coupling of the forces to changes in cell shape. As sma-1 mutants start and stop elongation at about the same time as wild-type embryos, currently unknown signals must function to trigger and terminate the elongation process.

Underlying muscle cells are needed for complete elongation Although cell-ablation experiments suggested that elongation is driven autonomously by the epidermis, studies of muscle-defective mutants show that complete elongation requires underlying muscle cells. This interaction was discovered serendipitously during screening for mutants lacking the MYO-3 body-muscle-specific myosin heavy chain38. myo-3 mutants were not only completely paralysed, but also failed to elongate beyond the twofold stage, a phenotype termed Pat (for ‘paralysed, arrest at twofold’). The Pat phenotype provides an efficient means of identifying new muscle mutants39, but its basis remains obscure. The requirement for muscle cells in epidermal elongation exemplifies the need to coordinate the morphogenesis of two adjacent tissues. Body-wall muscles lie directly beneath epidermal cells (separated by basement membrane), and transmit their contractions through the epidermal cells to the cuticle exoskeleton via complexes of hemidesmosomes TIG December 2000, volume 16, No. 12

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and intermediate filaments that span the epidermal cells, connecting the basement membrane to the cuticle (the ‘fibrous organelles’, FOs). Prior to elongation, muscle cells induce the organization of FOs in overlying epidermis40. Mutants with Pat phenotypes include those lacking muscle structural proteins, basement-membrane components41, or integrin subunits. The common theme in such mutants is a failure to assemble the contractile apparatus in muscle, or a failure to connect the contractile apparatus to the FOs in the epidermis. Some Pat mutants, such as those lacking the calcium channel subunit EGL-19, have normal muscle ultrastructure but defective contraction42, which suggests that muscle contraction itself is necessary for the effects on the epidermis. Mutations affecting ‘muscle-specific’ proteins such as myosin seem unlikely to cause cell-autonomous defects in epidermal cells, although in most cases this has not been rigorously demonstrated. If the effects of such Pat mutants are indeed cell-nonautonomous, this raises the question of whether muscle cells send specific signals to epidermal cells, or whether the effects are due to (for example) mechanical hindrance of epidermal cells by defective muscles. An epidermal receptor for specific muscle signals has not been identified to date; naively, one might predict mutants in such a re-ceptor to have a distinctive phenotype of non-paralysed twofold arrest. Mutations in the epidermal protein myotactin/LET-805 cause such phenotypes43, but myotactin appears to function to maintain the correct distribution of FOs after they have been initially positioned by the muscle signal. A simple model for the effects of muscle mutants on epidermis is based on the fact that the FOs somehow function in epidermal elongation past the twofold stage; failure in the coupling of muscle cells to epidermal FOs may account for the elongation defects in Pat mutants with normal FO ultrastructure. The need for reciprocal interactions between developing muscle and epidermis is underscored by the gene mup-4, which is required in epidermal cells for normal muscle positioning44. mup-4 mutants also display variable defects in epidermal morphogenesis. MUP-4 encodes a transmembrane protein localized to epidermal hemidesmosomes and thus could couple muscle attachments to FOs (E. A. Bucher, pers. commun.). The epidermal defects in mup-4 mutants should provide insights into the roles of FOs and their intermediate filaments in epidermal morphogenesis.

The cuticle holds epidermal cells in their final shape During elongation, the shape of epidermal cells is actively maintained by actin microfilaments, as shown by the rapid and reversible effects of cytochalasin D treatment21. After

References 1 Fraser, S. and Harland, R. (2000) The molecular metamorphosis of experimental embryology. Cell 100, 41–55 2 Leung, B. et al. (1999) Organogenesis of the Caenorhabditis elegans intestine. Dev. Biol. 216, 114–134 3 Newman, A.P. et al. (1996) Morphogenesis of the C. elegans hermaphrodite uterus. Development 122, 3617–3626 4 Sharma-Kishore, R. et al. (1999) Formation of the vulva in Caenorhabditis elegans: a paradigm for organogenesis. Development 126, 691–699 5 Nguyen, C.Q. et al. (1999) Morphogenesis of the Caenorhabditis elegans male tail tip. Dev. Biol. 207, 86–106 6 Blelloch, R. et al. (1999) The gon-1 gene is required for gonadal morphogenesis in Caenorhabditis elegans. Dev. Biol. 216, 382–393 7 Herman, T. et al. (1999) sqv mutants of Caenorhabditis

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elongation is complete, epidermal cells make the extracellular cuticle, which maintains the final shape of the epidermal cells. Animals carrying mutations in the cuticle collagen SQT-3 elongate normally but are unable to maintain the elongated shape, and retract21. Other mutants with abnormal cuticle structure show profoundly affected body shape, although most of them appear to have defects in post-embryonic growth rather than embryonic morphogenesis.

Conclusions and questions Genetics is beginning to scratch the surface of epithelial morphogenesis, and large-scale screens for morphogenetic mutants are likely to open up our understanding of these processes at the cellular and tissue levels. Many, perhaps most, key aspects of epidermal morphogenesis remain unclear. It is not known what drives epidermal leading cells to migrate during epidermal enclosure, nor is it clear as to whether they require specific cues from their substrate. The mechanical forces driving epidermal elongation have been defined (and candidate regulatory molecules are being identified), yet there is still no clear understanding as to what triggers elongation and how it comes to be so strikingly reproducible between animals. A final key area for future study is the coordination of morphogenesis of different tissues, exemplified by the complex reciprocal interactions between muscle and epidermis during elongation. Of course, whilst genetic screens can yield components of morphogenetic processes, results from genetic analysis must be integrated with information on the dynamics and mechanics of cells and tissues to clarify how cells and tissues move. Analysis of seemingly similar morphogenetic processes in different organisms has yet to uncover clear molecular conservation. For example, dorsal closure in Drosophila and ventral enclosure in C. elegans, as yet, appear to share no molecular pathways. This might reflect fundamental differences in the temporal and spatial scales of the two processes. However, given the limited data, such conclusions are premature – for example, sex determination in flies and worms had appeared to be completely different until the recent emergence of molecular similarities45.

Acknowledgements We are very grateful to L. Hinck, Y. Jin, M. Labouesse, M. Metzstein, the anonymous reviewers and members of the Jin and Chisholm laboratories for helpful comments. We thank E. Bucher and M. Labouesse for communicating results before publication. A.D.C. is an Alfred P. Sloan Fellow in the Neurosciences.

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Microsatellite mutations in the germline: implications for evolutionary inference Microsatellite DNA sequences mutate at rates several orders of magnitude higher than that of the bulk of DNA. Such high rates mean that spontaneous mutations that form new-length variants can realistically be seen in pedigree analysis. Data on observed mutation events from various organisms are now accumulating, allowing inferences on DNA sequence evolution to be made through an unusually direct approach. Here I discuss and integrate microsatellite mutation data in an evolutionary context. A striking feature of the mutation process is that it seems highly heterogeneous, with distinct differences between species, repeat types, loci and alleles. Age and sex also affect the mutation rate. Within genomes at equilibrium, the microsatellite-length distribution is a delicate balance between biased mutation processes and point mutations acting towards the decay of repetitive DNA. Indeed, simple repeats do not evolve simply. volutionary distance models make certain assumptions about the underlying mutation process. It is imperative that assumptions built into such models are as realistic as possible to optimize the models. However, as mutations are generally rare, it often proves

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most difficult to get detailed insight into the patterns and characteristics of mutation events. Therefore, mutation processes typically need to be studied through indirect approaches, which can be ambiguous and limited. TIG December 2000, volume 16, No. 12

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