The Caenorhabditis elegans ems class homeobox gene ceh-2 is required for M3 pharynx motoneuron function

Several homeobox genes, for example those of the ems class, play important roles in animal head development. We report on the expression pattern and function of ceh-2, the Caenorhabditis elegans ems/Emx ortholog. CEH-2 protein is restricted to the nuclei of one type of small muscle cell, one type of epithelial cell, and three types of neurons in the anterior pharynx in the head. We have generated a deletion allele of ceh-2 that removes the homeobox. Animals homozygous for this deletion are viable and fertile, but grow slightly slower and lay fewer eggs than wild type. We assayed the function of two types of pharynx neurons that express ceh-2, the pairs M3 and NSM. M3 activity is substantially reduced in electropharyngeograms of ceh-2 deletion mutants; this defect can account for the observed retardation in larval development, as M3 activity is known to be necessary for effective feeding. NSM function and metabolism are normal based on the assays used. All cells that express ceh-2 in wild type are present in the ceh-2 mutant and have normal morphologies. Therefore, unlike other ems/Emx genes, ceh-2 seems to be important for a late differentiation step and not for neuron specification or regional patterning. Because the CEH-2 homeodomain is well conserved, we tested whether ceh-2 can rescue ems(-) brain defects in Drosophila, despite the apparent differences in biological roles. We found that the C. elegans ems ortholog is able to substitute for fly ems in brain development, indicating that sequence conservation rather than conservation of biological function is important.     

Sensitized genetic backgrounds reveal a role for C. elegans FGF EGL-17 as a repellent for migrating CAN neurons

Although many molecules are necessary for neuronal cell migrations in C. elegans, no guidance cues are known to be essential for any of these cells to migrate along the anteroposterior (AP) axis. We demonstrate that the fibroblast growth factor (FGF) EGL-17, an attractant for the migrating sex myoblasts (SMs), repels the CANs, a pair of neurons that migrate posteriorly from the head to the center of the embryo. Although mutations in genes encoding EGL-17/FGF and a specific isoform of its receptor EGL-15/FGFR had little effect on CAN migration, they enhanced the CAN migration defects caused by mutations in other genes. Two cells at the anterior end of the embryo express EGL-17/FGF, raising the possibility that EGL-17/FGF functions as a repellent for migrating CANs. Consistent with this hypothesis, ectopic expression of EGL-17/FGF shifted the final CAN cell positions away from these novel sites of expression. Cell-specific rescue experiments demonstrated that EGL-15/FGFR acts in the CANs to promote their migration. We also found that the tyrosine phosphatase receptor CLR-1 regulates CAN migration by inhibiting EGL-15/FGFR signaling, and that the FGFR adaptor protein SEM-5/GRB2 may mediate EGL-15/FGFR signaling in CAN migration. Thus, EGL-17/FGF signaling through an EGL-15/FGFR isoform and possibly SEM-5/GRB2 mediates both attraction of the SMs and repulsion of the CANs. This study also raises the possibility that several guidance cues regulate cell migrations along the C. elegans AP axis, and their role in these migrations may only be revealed in sensitized genetic backgrounds.     

Mutations affecting embryonic cell migrations in Caenorhabditis elegans

Four recessive mutations that affect long-range embryonic migration of the two canal-associated neurons (CANs) in C. elegans were isolated and characterized with the goal of identifying genes involved in control of directed cell movement. Mutant animals were identified initially by their "withered" tails, a phenotype associated with abnormal CAN migration; the mutants were then analyzed for abnormal cell migrations by Nomarski microscopy. Based on genetic complementation tests, the mutations were assigned to four different loci, two new (mig-10 III, mig-11 III) and two previously identified (unc-39 V, vab-8 V). Mutations at all four loci affect CAN migration with high to moderate penetrance (the percentage of mutant animals that exhibit the phenotype). In addition, two other bilaterally symmetric pairs of neurons (ALM and HSN), the mesoblast M, and a pair of coelomocyte mother cells are affected by one or more of the mutations, generally with lower penetrance. With the exceptions of HSN and the right coelomocyte mother cell, which occasionally migrate beyond their normal destinations, the cells affected appear to migrate either incompletely or not at all. All the migration phenotypes show incomplete penetrance and variable expressively, although genetic tests suggest that mutations at mig-10 and vab-8 result in complete or nearly complete loss of gene function. The variability in mutant phenotypes allowed tests for interdependence of several of the affected migrations; all those analyzed appeared independent of one another. The possible nature of the mutant defects and possible roles of these four loci in cell migration are discussed.     

Two Hox cofactors, the Meis/Hth homolog UNC-62 and the Pbx/Exd homolog CEH-20, function together during C. elegans postembryonic mesodermal development

The TALE homeodomain-containing PBC and MEIS proteins play multiple roles during metazoan development. Mutations in these proteins can cause various disorders, including cancer. In this study, we examined the roles of MEIS proteins in mesoderm development in C. elegans using the postembryonic mesodermal M lineage as a model system. We found that the MEIS protein UNC-62 plays essential roles in regulating cell fate specification and differentiation in the M lineage. Furthermore, UNC-62 appears to function together with the PBC protein CEH-20 in regulating these processes. Both unc-62 and ceh-20 have overlapping expression patterns within and outside of the M lineage, and they share physical and regulatory interactions. In particular, we found that ceh-20 is genetically required for the promoter activity of unc-62, providing evidence for another layer of regulatory interactions between MEIS and PBC proteins.     

The conserved kinase UNC-51 acts with VAB-8 and UNC-14 to regulate axon outgrowth in C. elegans

Directional cues guide growth cones. While molecules like UNC-6/netrin direct migrations along the dorsoventral axis of many organisms, it is unclear how anteroposterior guidance is achieved. We describe a physical interaction between VAB-8, a protein both necessary and sufficient for posteriorly directed migrations in C. elegans, and UNC-51, a conserved serine/threonine kinase that functions generally in axon outgrowth. We show that both proteins function in the CAN neurons to direct their axons posteriorly. Expression in the CANs of peptides predicted to interfere with interactions between UNC-51 and both VAB-8 and UNC-14, a second protein that interacts physically with UNC-51, disrupts CAN axon outgrowth. We provide genetic evidence that VAB-8 functions in an UNC-51 pathway for posteriorly directed CAN axon guidance and show that VAB-8 and UNC-14 can be targets of UNC-51 kinase activity. Taken together, our results suggest that VAB-8 and UNC-14 are substrates that mediate the function of UNC-51 in axon outgrowth.     

Behavioral and synaptic defects in C. elegans lacking the NK-2 homeobox gene ceh-28

C. elegans pharyngeal behavior consists of two distinct types of muscle contractions, termed pumping and peristalsis. Pumping ingests and concentrates bacteria in the anterior pharyngeal lumen, and it is occasionally followed by a transient peristaltic contraction that carries ingested bacteria through the posterior pharyngeal isthmus. These behaviors are controlled by a small pharyngeal nervous system consisting of 20 neurons that is almost completely independent of the extra-pharyngeal nervous system. The cholinergic motor neuron M4 controls peristalsis via synapses with the posterior isthmus muscles. Here we show that the NK-2 family homeobox gene ceh-28 is expressed in M4, where it regulates synapse assembly and peristalsis. ceh-28 mutants exhibit frequent and prolonged peristalses, and treatment with agonists or antagonists of muscarinic acetylcholine receptors can phenocopy or suppress ceh-28 mutant defects, respectively. Synapses in ceh-28 mutant M4 cells are irregularly spaced and sized, and they are abnormally located along the full length of the isthmus. We suggest that CEH-28 inhibits synaptogenesis, and that ceh-28 mutant behavioral defects result from excessive or ectopic stimulation of muscarinic acetylcholine receptors in the isthmus muscles.     

A role for Drosophila IAP1-mediated caspase inhibition in Rac-dependent cell migration

Border cell migration in the Drosophila ovary is a relatively simple and genetically tractable model for studying the conversion of epithelial cells to migratory cells. Like many cell migrations, border cell migration is inhibited by a dominant-negative form of the GTPase Rac. To identify new genes that function in Rac-dependent cell motility, we screened for genes that when overexpressed suppressed the migration defect caused by dominant-negative Rac. Overexpression of the Drosophila inhibitor of apoptosis 1 (DIAP1), which is encoded by the thread (th) gene, suppressed the migration defect. Moreover, loss-of-function mutations in th caused migration defects but, surprisingly, did not cause apoptosis. Mutations affecting the Dark protein, an activator of the upstream caspase Dronc, also rescued RacN17 migration defects. These results indicate an apoptosis-independent role for DIAP1-mediated Dronc inhibition in Rac-mediated cell motility.     

Overlapping roles of two Hox genes and the exd ortholog ceh-20 in diversification of the C. elegans postembryonic mesoderm

Members of the Hox family of homeoproteins and their cofactors play a central role in pattern formation of all germ layers. During postembryonic development of C. elegans, non-gonadal mesoderm arises from a single mesoblast cell M. Starting in the first larval stage, M divides to produce 14 striated muscles, 16 non-striated muscles, and two non-muscle cells (coelomocytes). We investigated the role of the C. elegans Hox cluster and of the exd ortholog ceh-20 in patterning of the postembryonic mesoderm. By examining the M lineage and its differentiation products in different Hox mutant combinations, we found an essential but overlapping role for two of the Hox cluster genes, lin-39 and mab-5, in diversification of the postembryonic mesoderm. This role of the two Hox gene products required the CEH-20 cofactor. One target of these two Hox genes is the C. elegans twist ortholog hlh-8. Using both in vitro and in vivo assays, we demonstrated that twist is a direct target of Hox activation. We present evidence from mutant phenotypes that twist is not the only target for Hox genes in the M lineage: in particular we show that lin-39 mab-5 double mutants exhibit a more severe M lineage defect than the hlh-8 null mutant.