Abstract: Cells in the mid-body region of the nematode C. elegans develop differently from their anterior or posterior homologs. The gene lin-39 is required for mid-body region-specific development. In lin-39 mutants, mid-body cells express fates characteristic of more anterior or posterior homologs, and the migration of a neuroblast through the mid-body is defective. lin-39 acts cell autonomously in these mid-body cells and in the migrating neuroblast. lin-39 encodes a protein with an Antennapedia class homeodomain, most similar to those of the Drosophila homeotic genes Deformed and Sex combs reduced, and is located in a homeotic gene cluster with two other regional homeotic genes, mab-5 and egl-5. lin-39 and mab-5 function combinatorially in 2 ectodermal cells and have redundant functions in gonad development.
Intricate patterns of overlapping HOM-C gene expression along the A/P axis have been observed in many organisms; however, the significance of these patterns in establishing the ultimate fates of individual cells is not well understood. We have examined the expression of the Caenorhabditis elegans Antennapedia homolog mab-5 and its role in specifying cell fates in the posterior of the ventral nerve cord. We find that the pattern of fates specified by mab-5 not only depends on mab-5 expression but also on post-translational interactions with the neighboring HOM-C gene lin-39 and a second, inferred gene activity. Where mab-5 expression overlaps with lin-39 activity, they can interact in two different ways depending on the cell type: They can either effectively neutralize one another where they are both expressed or lin- 39 can predominate over mab-5. As observed for Antennapedia in Drosophila, expression of mab-5 itself is repressed by the next most posterior HOM-C gene, egl-5. Thus, a surprising diversity in HOM-C regulatory mechanisms exists within a small set of cells even in a simple organism.
In C. elegans, the epithelial Pn.p cells adopt either a vulval precursor cell fate or fuse with the surrounding hypodermis (the F fate). Our results suggest that a Wnt signal transduced through a pathway involving the beta-catenin homolog BAR-1 controls whether P3.p through P8.p adopt the vulval precursor cell fate. In bar-1 mutants, P3.p through P8.p can adopt F fates instead of vulval precursor cell fates. The Wnt/bar-1 signaling pathway acts by regulating the expression of the Hox gene lin-39, since bar-1 is required for LIN-39 expression and forced lin-39 expression rescues the bar-1 mutant phenotype. LIN-39 activity is also regulated by the anchor cell signal/let-23 receptor tyrosine kinase/let-60 Ras signaling pathway. Our genetic and molecular experiments show that the vulval precursor cells can integrate the input from the BAR-1 and LET-60 Ras signaling pathways by coordinately regulating activity of the common target LIN-39 Hox.
The Ras signaling pathway specifies a variety of cell fates in many organisms. However, little is known about the genes that function downstream of the conserved signaling cassette, or what imparts the specificity necessary to cause Ras activation to trigger different responses in different tissues. In C. elegans, activation of the Ras pathway induces cells in the central body region to generate the vulva. Vulval induction takes place in the domain of the Hox gene lin-39. We have found that lin-39 is absolutely required for Ras signaling to induce vulval development. During vulval induction, the Ras pathway, together with basal lin-39 activity, up-regulates lin-39 expression in vulval precursor cells. We find that if lin-39 function is absent at this time, no vulval cell divisions occur. Furthermore, if lin-39 is replaced with the posterior Hox gene mab-5, then posterior structures are induced instead of a vulva. Our findings suggest that in addition to permitting vulval cell divisions to occur, lin-39 is also required to specify the outcome of Ras signaling by selectively activating vulva-specific genes.
Hox genes pattern the fates of the ventral ectodermal Pn.p cells that lie along the anteroposterior (A/P) body axis of C. elegans. In these cells, the Hox genes are expressed in sequential overlapping domains where they control the ability of each Pn.p cell to fuse with the surrounding syncytial epidermis. The activities of Hox proteins are sex-specific in this tissue, resulting in sex-specific patterns of cell fusion: in hermaphrodites, the mid-body cells remain unfused, whereas in males, alternating domains of syncytial and unfused cells develop. We have found that the gene egl-27, which encodes a C. elegans homologue of a chromatin regulatory factor, specifies these patterns by regulating both Hox gene expression and Hox protein function. In egl-27 mutants, the expression domains of Hox genes in these cells are shifted posteriorly, suggesting that egl-27 influences A/P positional information. In addition, egl-27 controls Hox protein function in the Pn.p cells in two ways: in hermaphrodites it inhibits MAB-5 activity, whereas in males it permits a combinatorial interaction between LIN-39 and MAB-5. Thus, by selectively modifying the activities of Hox proteins, egl-27 elaborates a simple Hox expression pattern into complex patterns of cell fates. Taken together, these results implicate egl-27 in the diversification of cell fates along the A/P axis and suggest that chromatin reorganization is necessary for controlling Hox gene expression and Hox protein function.
Inactivation of the Caenorhabditis elegans APC-related gene (apr-1) has pointed at two separate functions of apr-1. First, apr-1 is required for the migration of epithelial cells during morphogenesis of the embryo. In this process, APR-1 may act in a Cadherin/alpha-Catenin/beta-Catenin complex as a component of adherens junctions. Second, apr-1 is required for Hox gene expression, most likely by positively regulating the activity of the Wingless signaling pathway. During embryogenesis, apr-1 is required for the expression of ceh-13 labial in anterior seam and muscle cells and during larval development, apr-1 is necessary for the expression of lin-39 deformed in the vulval precursor cells. Thus, APR-1 may positively regulate the activity of the beta-Catenin/Armadillo-related proteins HMP-2 in migrating epithelial cells and BAR-1 in the vulval precursor cells.
Hox genes control the choice of cell fates along the anteroposterior (AP) body axis of many organisms. In C. elegans, two Hox genes, lin-39 and mab-5, control the cell fusion decision of the 12 ventrally located Pn.p cells. Specific Pn.p cells fuse with an epidermal syncytium, hyp7, in a sexually dimorphic pattern. In hermaphrodites, Pn.p cells in the mid-body region remain unfused whereas in males, Pn.p cells adopt an alternating pattern of syncytial and unfused fates. The complexity of these fusion patterns arises because the activities of these two Hox proteins are regulated in a sex-specific manner. MAB-5 activity is inhibited in hermaphrodite Pn.p cells and thus MAB-5 normally only affects the male Pn.p fusion pattern. Here we identify a gene, ref-1, that regulates the hermaphrodite Pn.p cell fusion pattern largely by regulating MAB-5 activity in these cells. Mutation of ref-1 also affects the fate of other epidermal cells in distinct AP body regions. ref-1 encodes a protein with two basic helix-loop-helix domains distantly related to those of the hairy/Enhancer of split family. ref-1, and another hairy homolog, lin-22, regulate similar cell fate decisions in different body regions along the C. elegans AP body axis.
During larval development in C. elegans, some of the cells of the ventral epidermis, the Pn.p cells, fuse with the growing epidermal syncytium hyp7. The pattern of these cell fusions is regulated in a complex, sexually dimorphic manner. It is essential that some Pn.p cells remain unfused in order for some sex-specific mating structures to be generated. The pattern of Pn.p cell fusion is regulated combinatorially by two genes of the C. elegans Hox gene cluster: lin-39 and mab-5. Some of the complexity in the Pn.p cell fusion pattern arises because these two Hox proteins can regulate each other's activities. We describe a zinc-finger transcription factor, REF-2, that is required for the Pn.p cells to be generated and to remain unfused. REF-2 functions with the Hox proteins to prevent Pn.p cell fusion. ref-2 may also be a transcriptional target of the Hox proteins.
Ê General mechanisms by which Hox genes establish cell fates are known. However, a few Hox effectors mediating cell behaviors have been identified. Here we found the first effector of LIN-39/HoxD4/Dfd in Caenorhabditis elegans. In specific vulval precursor cells (VPCs), LIN-39 represses early and late expression of EFF-1, a membrane protein essential for cell fusion. Repression of eff-1 is also achieved by the activity of CEH-20/Exd/Pbx, a known cofactor of Hox proteins. Unfused VPCs in lin-39(-);eff-1(-) double mutants fail to divide but migrate, executing vulval fates. Thus, lin-39 is essential for inhibition of EFF-1-dependent cell fusion and stimulation of cell proliferation during vulva formation.
Development of the vulva in C. elegans is mediated by the combinatorial action of several convergent regulatory inputs, three of which, the Ras, Wnt and Rb-related pathways, act by regulating expression of the lin-39 Hox gene. LIN-39 specifies cell fates and regulates cell fusion in the mid-body region, leading to formation of the vulva. In the lateral seam epidermis, differentiation and cell fusion have been shown to be regulated by two GATA-type transcription factors, ELT-5 and -6. We report that ELT-5 is encoded by the egl-18 gene, which was previously shown to promote formation of a functional vulva. Furthermore, we find that EGL-18 (ELT-5), and its paralogue ELT-6, are redundantly required to regulate cell fates and fusion in the vulval primordium and are essential to form a vulva. Elimination of egl-18 and elt-6 activity results in arrest by the first larval stage; however, in animals rescued for this larval lethality by expression of ELT-6 in non-vulval cells, the post-embryonic cells (P3.p-P8.p) that normally become vulval precursor cells often fuse with the surrounding epidermal syncytium or undergo fewer than normal cell divisions, reminiscent of lin-39 mutants. Moreover, egl-18/elt-6 reporter gene expression in the developing vulva is attenuated in lin-39(rf) mutants, and overexpression of egl-18 can partially rescue the vulval defects caused by reduced lin-39 activity. LIN-39/CEH-20 heterodimers bind two consensus HOX/PBC sites in a vulval enhancer region of egl-18/elt-6, one of which is essential for vulval expression of egl-18/elt-6 reporter constructs. These findings demonstrate that the EGL-18 and ELT-6 GATA factors are essential, genetically redundant regulators of cell fates and fusion in the developing vulva and are apparent direct transcriptional targets of the LIN-39 Hox protein.