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Riddle DL, Blumenthal T, Meyer BJ, et al., editors. C. elegans II. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997.

Cover of C. elegans II

C. elegans II. 2nd edition.

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Section VSpatial Patterning by the C. elegans HOM-C Gene Cluster

A. C. elegans HOM-C Gene Cluster

The basic pattern of Pn.a neuroblast cell lineage varies over the length of the animal. For example, the VC neurons that innervate the vulval muscles are only generated by 6 of the 12 Pn.a neuroblast cell lineages that are located in the middle of the animal, P3.a to P8.a (Fig. 5). In the other Pn.a cell lineages, the cells at the position in the cell lineage homologous to the VC neuron undergo programmed cell death. Such modifications to the reiterated neuroblast cell lineages are spatially clustered and are under the control of homeotic genes.

As in Drosophila and vertebrates, a cluster of homeobox genes regulates spatial patterning of C. elegans. Six members of the ancient metazoan homeobox cluster (HOM-C) genes (Kenyon and Wang 1991; Bürglin and Ruvkun 1993; Wang et al. 1993) have been detected in C. elegans, and mutations have been identified in four of them. The C. elegans homologs of the Drosophila Dfd, Antp, and Abd-B genes, called lin-39 , mab-5 , and egl-5 , respectively, control spatial patterning of mesodermal and ectodermal cells in adjacent spatial domains. These genes mediate the spatial variations on the canonical Pn.a neuroblast cell lineages and thus are likely to modulate the activity of, or response to, the neural patterning genes described above. The C. elegans homolog of labial, ceh-13 , has not yet been analyzed genetically, although it is expressed in a spatial pattern consistent with the function of labial in Drosophila (C. Wittmann and F. Muller, pers. comm.). vab-7 , the C. elegans homolog of eve, which in vertebrates is part of the HOM-C gene complex, is expressed in the posterior region of early embryos and is necessary for normal patterning in the most posterior regions of the animal (Ahringer 1996).

mab-5

Mutations in mab-5 affect cell identities in a region just anterior to that affected by egl-5 and just posterior to the region affected by lin-39 (Fig. 5) (Kenyon 1986; Wang et al. 1993). A variety of cell types are regulated by mab-5 . For example, the fates of the VB descendants of neuroectoblasts P11 and P12, the production of rays or alae by V-cell descendants in the male, the fate of ventral Pn.p hypodermal cells, and the migration of the descendants of the Q and M cells are all controlled by mab-5 . mab-5 encodes a homeodomain protein most closely related to the Antp class (Costa et al. 1988). The finding that the spatial patterning function of mab-5 is mediated by a homolog of a Drosophila spatial patterning gene was the first indication that C. elegans might also bear a homeotic gene cluster.

MAB-5 protein is expressed in a simple spatial pattern that includes the cells affected by mab-5 mutations (Salser et al. 1993; Wang et al. 1993). Genetic mosaic analysis revealed autonomous mab-5 function in these patterning activities (Kenyon 1986). However, the mab-5 activities in patterning are much more complex than its simple expression pattern would indicate—the boundaries of its activity vary depending on cell type (Salser et al. 1993). For example, mab-5 is required for the death of the VB neurons in the P11 and P12 cell lineages only; in the absence of mab-5, these neurons survive, as is normally observed in P-cell lineages more anterior to P11 and P12. Thus, the boundary for mab-5 activity in the VB neurons is between P10 and P11. The boundary for mab-5 function in Pn.p specification is between P6 and P7. However, the expression pattern of MAB-5 spans the region from P7 to P12. The discordance between the simple mab-5 expression pattern and complex pattern of genetic activities suggests that other developmental factors modify mab-5 activity in each of these P-cell sublineages. Ectopic expression of MAB-5 from a heat shock promoter is not sufficient to confer apoptosis to VB neurons in other P-cell lineages, suggesting that some combinatorial partner to MAB-5 is essential to confer apoptosis in P11 and P12 (Salser et al. 1993). This partner has not been identified. However, it is known that egl-5 is not the partner for VB cell death. egl-5 , however, is a possible regulator of mab-5 expression, for all P11 and P12 descendants continue to express MAB-5 in an egl-5 mutant, unlike wild type. Thus, mab-5 modifies the canonical P-cell lineage in the Pn.p descendants of P7 and P8 and in the Pn.a descendants of P11 and P12.

lin-39

lin-39 controls fates in the region anterior to and partially overlapping that controlled by mab-5 . The most obvious phenotype of lin-39 null mutant hermaphrodites is that the Pn.p cells that normally generate the vulva fuse with the hyp7 syncytium, rather than remaining unfused and competent to respond to signals from the anchor cell as they do in wild type (Fig. 6) (Clark et al. 1993; Wang et al. 1993). Thus, in lin-39 mutants, the vulval equivalence group is not specified, and P3.p to P8.p fuse with an adjacent syncytium, as their spatial homologs P1, P2, P9–12 do normally. From the pattern of cell fate changes, it appears that lin-39 acts in the P cells, because both anterior neural and posterior ectodermal derivatives of these P cells are affected. For example, in the hermaphrodite, P3.a to P8.a normally generate VC motor neurons, but in a lin-39 mutant, these cells die, like their homologs in the Pn.a cell lineages normally generated by P1, P2, P9–12. Other neurons generated by P3.a to P8.a in a lin-39 mutant migrate anteriorly, like the P1, P2a derived neurons in wild type, suggesting that P3 to P8 are transformed to a more anterior fate in a lin-39 mutant. In lin-39 mutant males, the Pn.aapp serotinergic neuron normally generated by P3 to P8 is not generated; instead, this cell in the P3 to P6 sublineages undergoes programmed cell death, more like P1 and P2, whereas P7 and P8 generate neuronal cell lineages more like P9 to P11. Hypodermal cells P7.p and P8.p are transformed in a lin-39 mutant to the fate more similar to those of posterior cells. Thus, in lin-39 mutants, cells located in the mid-body region are transformed to either more anterior or more posterior cell fates. Genetic mosaic analysis of lin-39 revealed cell-autonomous function in P-cell specification as well as in Q-cell migration (see below).

lin-39 encodes a homeodomain protein of the Dfd class, consistent with the spatial patterning functions of this gene class in other animals (Clark et al. 1993; Wang et al. 1993). A lin-39/lacZ fusion gene is expressed in P5 to P8 and to a lesser extent in P3 and P4, as well as weakly in the V cells and in the Q cells also located in this spatial domain (Fig. 7). Muscle cells in the region also express the lacZ fusion gene.

Figure 7. Embryonic expression pattern of the lin-39-lacZ, mab-5-lacZ, and egl5-lacZ fusion genes.

Figure 7

Embryonic expression pattern of the lin-39-lacZ, mab-5-lacZ, and egl5-lacZ fusion genes. The schematic drawing shows a mid-stage embryo (460 min after (more...)

The transformations of cell identity in a lin-39 mutant involve both anterior transformations and posterior transformations of cell fate. The simplest interpretation of this observation is that lin-39 modifies the activity of mab-5 in the posterior region of the lin-39 expression pattern but that in the anterior region of its expression pattern, lin-39 modifies the activity of an unknown other gene, perhaps the HOM-C labial homolog ceh-13 . There are not yet mutations in ceh-13 to test this notion.

egl-5

egl-5 corresponds to the posterior-most homeobox gene in the cluster and may be the homolog of Drosophila Abd-B. Mutations in egl-5 cause homeotic changes in cell fate in ectoblast cells and neurons that are clustered in the tail of the animal (Chisholm 1991). These cells become transformed toward the fates of more anterior homologs. An egl-5/lacZ fusion gene is expressed in the cells of the posterior region affected by egl-5 mutations (Fig. 7) (Wang et al. 1993).

B. The Molecular Basis of Homeosis

The cell fate transformations that result from mutations in HOM-C genes are homeotic because of their antagonistic regulation of other HOM-C genes. In Drosophila, Abd-A represses expression of Ubx in the posterior regions, so that in an Abd-A mutant, the Ubx expression domain is expanded posteriorly (Struhl and White 1985). This posterior expansion of the Ubx expression transforms posterior segment identity to that of anterior fates. In this case, homeosis results from cross-regulation of HOM-C cluster gene expression. In other cases, homeosis results not from cross-regulation of expression but from cross-regulation of activity. For example, in Drosophila, the expression of HOM-C genes from the posterior regions of the complex renders impotent the expression of other members of the complex (Mann and Hogness 1990). Examples of both these phenomena have been observed in the C. elegans HOM-C cluster genes.

The C. elegans HOM-C genes lin-39 and mab-5 do not regulate each other's expression. Rather, in this case, the homeosis results from combinatorial control of cell fates by mab-5 and lin-39 . For example, the fates of P7 and P8 descendants are controlled by the combination of lin-39 and mab-5 gene activities (see Fig. 5). In the absence of lin-39 gene product, the fates of P7 and P8 descendants are governed solely by MAB-5 and vice versa. Since the expression of lin-39 is generally more anterior to mab-5 and only overlaps in the region of P7 and P8, in the absence of lin-39 gene activity, P7 and P8 descendants take on the fates normally associated with cells in the mab-5 expression domain that do not express lin-39 , normally located more posteriorly. Similarly, in the absence of mab-5 gene activity, P7 and P8 descendants take on the fates normally associated with cells in the lin-39 expression domain that do not express mab-5 , normally located more anteriorly. This lin-39 / mab-5 interaction in the P7/P8 spatial domain is not due to cross-regulation of mab-5 or lin-39 gene expression; MAB-5 protein is expressed in the same low level in P7 and P8 even in a lin-39 mutant, and lin-39 /lacZ fusion gene expression is not altered in a mab-5 mutant (Wang et al. 1993). Even more compelling is the observation that expression of mab-5 in the lin-39 expression domain caused all P cells in the lin-39 domain to become transformed to the P7 and P8 fates, which, for example, include cell fusion of P7.p and P8.p in males (Salser et al. 1993). This fate depends on both lin-39 and mab-5 gene activities. Thus, expression of MAB-5 does not repress or neutralize LIN-39 expression; rather, it acts combinatorially with LIN-39 to respecify cells.

In the anterior regions, lin-39 may conspire with ceh-13 to specify P1 to P6 fates. The transformations in cell fate that accompany mutations in these genes are not automatically posterior or anterior; they rather reflect which HOM-C gene products combinatorially interact in particular regions. In the case of the P11 and P12 cell lineages, egl-5 appears to repress the expression of mab-5. mab-5 expression in P11 and P12 descendants continues in an egl-5 mutant, unlike wild type.

C. ceh-20 Encodes a Combinatorial Partner to HOM-C Proteins

Mutations in ceh-20 cause defects in spatial patterning in the lin-39 and mab-5 spatial domains, including defects in Pn.p specification and defects in M-cell migrations (E. Chen and M. Stern, pers. comm.). ceh-20 encodes a homeodomain protein of the PBC type, which bears a divergent homeodomain as well as an upstream conserved domain that has been found in mammalian and Drosophila homologs (Bürglin and Ruvkun 1992). CEH-20 is expressed in a variety of mesodermal and ectodermal cells, including the Pn.a and Pn.p cells (T. Bürglin and G. Ruvkun, pers. comm.). The Drosophila homolog of CEH-20 is Exd, which has been shown to act as a combinatorial partner to Ubx and Antp (Rauskolb et al. 1993; Rauskolb and Wieschaus 1994). The probable bipartite DNA-binding domain of PBC proteins and their synergistic binding with HOM-C cluster homeodomain proteins probably increase the specificity of HOM-C gene action. For example, in isolation, HOM-C homeodomain proteins are rather promiscuous in their DNA-binding specificity, to the point that it has proven difficult to detect downstream genes biochemically using isolated HOM-C homeodomain proteins. But in combination with Exd, the Ubx and Antp proteins bind synergistically to downstream gene sequences (Chan et al. 1994; Lu et al. 1995; Popperl et al. 1995). The P-cell fate defects in the ceh-20 mutant suggest that like Exd in Drosophila, ceh-20 functions as a combinatorial partner for C. elegans HOM-C genes. The HOM-C proteins in combination with CEH-20 are likely to modify the activity of genes analogous to unc-4 and unc-30 (see below).

D. Control of Cell Migration

mab-5 and lin-39 are also necessary for the normal migration of Q-cell descendants. In the wild type, QL and its descendants migrate posteriorly, whereas QR and its descendants migrate anteriorly (Fig. 8). In a mab-5 mutant, QL descendants migrate anteriorly rather than posteriorly, but QR migrates normally. lin-39 mutants disrupt anterior migrations of QR descendants but do not affect posterior migrations of QL descendants. In a lin-39 mutant, QR and its descendants begin their anterior migration normally but do not continue migrating into the head region. In genetic mosaics, a Q-cell migration defect is always associated with a mab-5 or lin-39 mutant genotype in the Q cell (Kenyon 1986; Clark et al. 1993). The cell autonomy of mab-5 and lin-39 suggests that both activate the expression of signaling pathways that sense migration cues, rather than regulating the production of migration cues themselves.

Figure 8. Dorsal view of the migration patterns of the Q neuroblast descendants.

Figure 8

Dorsal view of the migration patterns of the Q neuroblast descendants. QL and QR (open circles) are initially located directly opposite (more...)

mab-5 is expressed in QL descendants but is not expressed in QR descendants (Salser and Kenyon 1992). Thus, the changes in QL migration that are observed in the mab-5 mutant can be understood as a homeotic transformation from QL to QR fates. mab-5 begins to be expressed as soon as the migrating Q cell moves into the posterior region of the animal, where mab-5 is expressed by other cells. Thus, a system that detects spatial cues in the migrating Q cell may activate mab-5. The mab-5 gene activity in this migrating cell may modify this detection system or the mechanism by which detection of these cues is translated to directed migration to allow new cues or new migratory behaviors to be induced.

A mab-5 gain-of-function mutation results in the opposite transformation: Both QR and QL descendants migrate posteriorly. mab-5 may be expressed in both QR and QL in this mutant. Interestingly, expression of MAB-5 from a heat shock promoter at any point in the anterior migration of Q.a is sufficient to cause its neuroblast daughter Q.ap to migrate posteriorly (Fig. 8) (Salser and Kenyon 1992). Thus, if migratory cues are detected by a MAB-5-regulated gene, they must be distributed globally. The lag between heat shock and migration direction change is 4 hours, suggesting that a cascade of gene regulation could be necessary for the change in migration imposed by MAB-5 expression (Salser and Kenyon 1992). Within a few hours of MAB-5-induced posterior migration, the neuroblasts and neurons begin to migrate again anteriorly, but further heat shock induction of MAB-5 again causes these cells to reverse direction (Salser and Kenyon 1992). These observations show that mab-5 does not cause an irreversible switch in migratory behavior.

The factors that asymmetrically activate mab-5 expression in QL are not known. The mab-5(gf) mutation is a partial duplication of the entire gene that truncates the promoter of one of the duplicate mab-5 genes at –4 kb (Salser and Kenyon 1992). The Mab-5 gain-of-function phenotype is not suppressed in trans to a deficiency, suggesting that it is not a twofold increase in mab-5 that creates the phenotype. Rather, it is more likely that the truncated mab-5 promoter either removes a negative regulatory element or generates a novel positive regulatory element for QR to symmetrize mab-5 expression. This suggests that repression of mab-5 in QR is the basis of asymmetric expression.

The distinct activities of the closely related lin-39 and mab-5 genes in, for example, QL where both are expressed suggest that these genes regulate distinct sets of genes. lin-39 may regulate the expression of genes that respond to anterior migration cues, whereas mab-5 may regulate the expression of genes that respond to posterior migration cues. Precedent for how such related genes can have such distinct activities comes again from Drosophila. For example, ectopic expression of Ubx or Antp proteins cause distinct segmental transformations, and this specificity maps to particular residues in the homeodomain that are distinct between Antp and Ubx (Mann and Hogness 1990). These differences mediate the distinct biological activities of related homeobox genes presumably by controlling which combinatorial partners interact with each subtype.

mab-5 and lin-39 also regulate the migration of the P-cell descendants. Normally, just the P1 and P2 descendants migrate anteriorly, whereas in a lin-39 mutant, P1 to P6 descendants all migrate anteriorly, and in a lin-39 ; mab-5 double mutant, P1 to P11 descendants migrate anteriorly (Clark et al. 1993). Thus, in these cases, expression of lin-39 or mab-5 inhibits anterior migration, whereas in the case of the Q cell, expression of lin-39 activates anterior migration and expression of mab-5 activates posterior migration. Other factors may modify the activity of lin-39 and mab-5 in P descendants versus Q descendants. On the other hand, differential splicing of mab-5 or lin-39 in the P cells and Q cells, analogous to the differential splicing observed in Drosophila HOM-C genes (Weinzierl et al. 1987), could modify the sets of downstream genes regulated, and thus the migration cues detected by P- and Q-cell descendants. For example, alternative microexons in Ubx confer differential ability of the protein to specify PNS neural fates, suggesting that these Ubx microvariants may cooperate with distinct sets of other transcription factors to activate PNS-specific genes (Mann and Hogness 1990).

egl-5 also regulates cell migration (Desai et al. 1988; Chisholm 1991). The HSN neuron is born in the posterior egl-5 spatial domain, and its migration and development depend on egl-5 function. The HSN expresses an egl-5 lacZ fusion gene, even after this cell migrates away from the posterior regions. If this represents continued egl-5 expression rather than β-galactosidase perdurance, it suggests that extracellular regional-specific cues are not necessary for maintenance of egl-5 expression once it has been initiated. A similar perdurance of mab-5/lacZ expression has been noted in the migrating M cell (Wang et al. 1993).

Mammalian HOM-C genes are expressed in migrating neural crest cells and in migrating cells during gastrulation (Lufkin et al. 1991; Dush and Martin 1992). Together with the regulation of neuroblast migration by C. elegans HOM-C genes, this suggests that the regulation of migration by HOM-C genes may be general. Interestingly, mammalian evx genes have been implicated in control of gastrulation, and vab-7 , the C. elegans evx homolog, appears to act in an analogous region of the C. elegans embryo (Ahringer 1996). During such migrations, cell movements into the domains of signaling molecules may induce expression of particular HOM-C genes, as has been observed in the case of mab-5 , and the expression of these genes may in turn cause changes in adhesive properties and the migratory properties of these cells.

The regulation of migration by such HOM-C genes may be mediated by membrane receptors whose expression is regulated by these transcription factors. In Drosophila, the gene for the surface adhesion protein Connectin was isolated based on binding to Ubx protein and regulation by Ubx (Gould and White 1992). This adhesion molecule may mediate recognition of particular muscle cells by motor neurons; expression of Connectin in both muscle and motor neurons may be regulated by HOM-C genes (Gould and White 1992; Nose et al. 1994). This precedent suggests that there is not a deep regulatory hierarchy downstream from HOM-C genes but that these transcription factors directly regulate, in some cases, molecules that mediate morphogenetic movements and adhesions.

Across phylogeny, HOM-C cluster genes continue to be expressed in neurons (Krumlauf et al. 1993). Because of the function of HOM-C genes in migration of neuroblasts, they may also function in neural pathfinding as well. It is possible that similar cues guide migrating cells and outgrowing neurons so that the same regulatory pathways from HOM-C genes to receptors for guidance cues control cell migration and neural pathfinding (see Antebi et al., this volume).

E. Modification of Reiterated Pattern Elements

The HOM-C genes generate spatial diversity along the A-P axis of animals across phylogeny. Mutations in genes that specify particular spatial domains reveal underlying reiterated spatial domains in the A-P dimension that express similar patterns of cell identity. Thus, the HOM-C genes can be understood as evolutionary adaptations to diversify reiterated basic units of development, the segment in Drosophila, or the P-cell-based segmental unit in C. elegans. The HOM-C genes may modify the activity of other pattern formation genes that specify these more ancient pattern elements.

The modifications of the basic P-cell sublineages by the HOM-C genes suggest that these genes may modulate either the expression or the activity of genes that specify neural fates in these sublineages. The HOM-C genes probably do not modulate the expression or activity of unc-4 and unc-30 , which are expressed in all P-cell lineages. But, for example, the VC neurons that innervate the vulval muscles are only generated by 6 of the 12 Pn.a neuroblast cell lineages that are located in the middle of the animal, P3 to P8 (see Fig. 5). In the other Pn.a cell lineages, the cells at the position in the cell lineage homologous to the VC neuron undergo programmed cell death. The production of VC neurons is dependent on lin-39 . In the absence of lin-39 , these neurons undergo programmed cell death, like the homologous cells in the other P-cell lineages. On the other hand, in a ced-3 mutant, where programmed cell death is inactivated, six additional VC neurons are generated from the cell lineages where a programmed cell death normally occurs (Schinkmann and Li 1992). Thus, the expression of lin-39 in P3 to P8 may inactivate a cell death pathway directly or may act to control another transcription factor analogous to unc-4 that controls the generation of VC neurons in these cell lineages.

In this sense, one can view HOM-C proteins as another tier of gene regulation that modulates an earlier-acting (and phylogenetically more ancient) system of gene regulation for patterning reiterated elements. It is at the promoters for genes analogous to unc-4 and unc-30 or at the promoters of the genes which they regulate that we should look for mechanistic answers to how HOM-C genes pattern spatial domains. And it is at these promoters that the slight sequence differences between HOM-C proteins such as LIN-39 and MAB-5 will induce distinct cellular responses.

F. Regulation of Cellular Communication

One of the initial responses to mab-5 expression in Q is the detection of migration cues, whereas the initial response to mab-5 expression in P7 and P8 is the fusion of these cells with their neighbors. Both of these responses involve cell-cell communication. Interestingly, expression of lin-39 defines the fates of the P3.p to P8.p hypodermal cells that constitute the vulval equivalence group. These cells respond to anchor cell signaling via a let-23 signaling system (Aroian et al. 1990; see Greenwald, this volume). In the absence of lin-39 gene activity, P3.p to P8.p fuse with adjacent hypodermis rather than become competent to respond to the anchor cell signal. Expression in these Pn.p cells of the LET-23 receptor for the anchor cell signal, LIN-3, may thus be under the control of lin-39 . It will be interesting to see if let-23 and other genes in the signaling pathway downstream from let-23 are regulated directly by lin-39 .

It is provocative that downstream from Drosophila segmentation genes, many of which encode homeobox proteins or other transcription factors, are the cell-cell signaling genes such as dpp and hedgehog that may define segmental boundaries (Basler and Struhl 1994). These boundaries may correspond to the Drosophila polyclonal compartments that may constitute cell migration barriers (Blair et al. 1994). Such migratory barriers could be due to surface adhesion molecule expression, which could seal off a region from other migrating cells and signals from other compartments. It is tantalizing that the vulval equivalence group, P3 to P8, is also defined by HOM-C genes and may define a similar signaling domain.

G. What Regulates the HOM-C Genes?

Upstream of the HOM-C genes in Drosophila are the gap and pair rule genes that subdivide the embryo into domains. The pathway upstream of these genes in C. elegans is much less well characterized. However, candidate earlier-acting genes that may directly regulate the HOM-C genes have been identified.

The pal-1 / nob-2 gene is a candidate for a patterning gene that regulates HOM-C genes. Null mutations in pal-1 cause the posterior regions of the embryo to be malformed (L. Edgar et al., pers. comm.). The pal-1 mRNA is found in all blastomeres of the early embryo, but production of PAL-1 protein is repressed in particular nonposterior blastomeres by the maternal-effect patterning gene mex-3 (C. Hunter et al., pers. comm.). PAL-1 encodes a homeodomain protein related to Drosophila Caudal and mouse Cdx-1 (Waring and Kenyon 1990; Gamer and Wright 1993). These genes have been shown to regulate expression of HOM-C genes in the posterior region of the embryo. Thus, pal-1 may regulate HOM-C gene expression, as in distantly related organisms, but may be activated by cell lineage cues that are more parochial to C. elegans. This early blastomere regulation of C. elegans development is distinct from the syncytial mechanism of early Drosophila development. But flies, worms, and vertebrates clearly use the same HOM-C developmental mechanism after these distinct beginnings.

Copyright © 1997, Cold Spring Harbor Laboratory Press.
Bookshelf ID: NBK20116

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