Refai, Osama M. et al. (2013) International Worm Meeting "Formins Play a Role in the C. elegans Embryonic Elongation."

Pruyne, David, Vanneste, Christopher A., Mains, Paul E., Refai, Osama M.

[International Worm Meeting, 2013]

During the C. elegans embryonic development, the embryo undergoes dramatic changes to elongate from a spheroid into a long, thin worm. The epidermis of the embryo provides the driving force for elongation. The epidermal cytoskeleton is a highly organized structure of actin microfilaments, microtubules and intermediate filaments. The C. elegans Rho-binding kinase (LET-502) and myosin phosphatase (MEL-11) are essential regulators for the actomyosin-mediated embryonic elongation and act in parallel with FEM-2/PP2c phosphatase and PAK-1/p21 activated kinase (GALLY et al. 2009; PIEKNY et al. 2000). Actin nucleation is the rate limiting step of actin filament polymerization. We previously identified fhod-1 (formin homology domain), which is highly similar to the human actin nucleators FHOD1 and FHOD3. Our genetic analysis revealed that fhod-1 acts in the mel-11/let-502 pathway, in parallel to fem-2/pak-1. fhod-1 mutants exhibit less than 25% elongation defects, suggesting redundancy with other formins (or other genes) for actin nucleation. Potential candidates include six other C. elegans formins: daam-1, inft-1, inft-2, frl-1, fozi-1 and cyk-1. Only two of these genes, inft-1and cyk-1, appear to have a role in elongation based on let-502 RNAi in mutant backgrounds. Muscle is required for elongation past the 2 fold stage, and perhaps is required redundantly with let-502/mel-11 at earlier stages. However, we found that the ilk-1 Pat (paralyzed at two fold) mutant did not suppress mel-11.

Refai, Osama M. et al. (2011) International Worm Meeting "A genetic analysis of the axon guidance of the C. elegans pharyngeal neuron M1."

Refai, Osama M., Rohs, Patricia, Gaudet, Jeb, Rollins, Evvi

[International Worm Meeting, 2011]

The guidance of axons to their correct targets within an organ is a critical step in development. We are interested in understanding how individual neurons find their targets in the context of a developing organ. We are using the pharyngeal neuron M1 as a model because it extends a process from the posterior pharyngeal bulb to the anterior end of the pharynx. Furthermore, M1 uses the g1P gland as a substrate to guide its axon. Electron micrographs show that the M1 axon bundles with the g1P gland projection through the anterior pharynx but not in the more posterior part of the pharynx. Ablation of glands results in defects of the M1 trajectory, suggesting that g1P is necessary for M1 guidance. Growth cone defective mutants (e.g. unc-51 and unc-119) showed severe defects of the M1 trajectory at the procorpus, where it contacts g1P, but no defects in the posterior portion of M1, where there is no contact with g1P. These results suggest that the M1 axon develops through two phases: a growth cone-independent phase in the posterior pharynx (as observed for other pharyngeal neurons; Morck et al., 2003) and a growth cone-dependent phase in which the axon is guided by the g1P cell. Tests of mutations of the major guidance pathways (e.g. unc-6, sax-3, vab-1, smp-1 and smp-2), show little or no defect in M1, suggesting the involvement of novel molecules that act via the g1P gland. To identify additional genes involved in M1 guidance, we performed a forward genetic screen to identify mutants that affect M1 axon migration. We isolated 12 mutants with defective M1 trajectories, including mutants that overextend their anterior process, mutants that underextend the anterior process and a single mutant that appears to be missing M1. We are currently in the process of cloning the relevant mutations and will present our findings. Overall, the results are consistent with a model in which growth of the M1 axon involves two phases: a growth cone-independent phase in the posterior pharynx, which likely occurs passively due to pharyngeal elongation; and a growth cone-dependent phase in the anterior pharynx, that establishes the rest of the M1 trajectory and is at least partially dependent on the gland cell extension.

Refai, Osama et al. (2015) International Worm Meeting "Genetic Analysis of the formin fhod-1 and the PP2c phosphatase fem-2 genes during C. elegans embryonic elongation."

Refai, Osama, Mains, Paul

[International Worm Meeting, 2015]

The essential regulators of the C. elegans embryonic elongation (e.g., let-502 and mel-11) are well characterized; however, little is known about their non-myosin downstream targets. fhod-1, a member of the formin family of actin nucleators encodes a protein homologous to human FHOD1 and FHOD3. fhod-1 acts downstream of the let-502/mel-11 pathway. Our genetic analysis implies that fhod-1 is likely the only formin acting during embryonic elongation. FHOD-1 also plays a role in in C. elegans muscle development and maintenance.Immunostaining showed that FHOD-1 is expressed homogenously in epidermal cells before the onset of elongation. FHOD-1 is expressed at higher levels in the lateral epidermal cells that are primarily responsible for driving elongation, but only in late elongation. Ubiquitous epidermal expression has been reported for other elongation related genes, but with gene function being required only in a specific subset of epidermal cells. We recently found that fhod-1 encodes a novel short isoform that omits exon 8 of the current Wormbase gene model. Protein sequence alignments suggest that the alternatively spliced exon encodes a predicted coiled-coil domain with 51% similarity to the human and mouse FHOD3 coiled-coil domains. It is possible that the two isoforms act in a tissue-specific manner. I am testing CRISPR/Cas9 deletion mutations specific to the alternatively spliced exon to see if they effect elongation or muscle function differently. The complementary approach of tissue-specific expression will be used to determine whether short and long fhod-1 isoforms function in different cells.Genetic analysis revealed that C. elegans fem-2, which encodes a PP2c phosphatase, is required for embryonic elongation in addition to its well established role in sexual differentiation. fem-2 acts with pak-1 to mediate elongation in parallel to let-502 and mel-11. I showed that fem-2::gfp, which can rescue fem-2 elongation defects, is expressed ubiquitously but in higher levels in the epidermis at the onset of elongation. Using tissue-specific promoters, I found that fem-2 in dorsal/ventral epidermal cells can mediate embryonic elongation even though it is the lateral cells that are primarily responsible for driving elongation. .