LAD-1, the Caenorhabditis elegans L1CAM homologue, participates in embryonic and gonadal morphogenesis and is a substrate for fibroblast growth factor receptor pathway-dependent phosphotyrosine-based signaling

This study shows that L1-like adhesion (LAD-1), the sole Caenorhabditis elegans homologue of the L1 family of neuronal adhesion molecules, is required for proper development of the germline and the early embryo and embryonic and gonadal morphogenesis. In addition, the ubiquitously expressed LAD-1, which binds to ankyrin-G, colocalizes with the C. elegans ankyrin, UNC-44, in multiple tissues at sites of cell-cell contact. Finally, we show that LAD-1 is phosphorylated in a fibroblast growth factor receptor (FGFR) pathway-dependent manner on a tyrosine residue in the highly conserved ankyrin-binding motif, FIGQY, which was shown previously to abolish the L1 family of cell adhesion molecule (L1CAM) binding to ankyrin in cultured cells. Immunofluorescence studies revealed that FIGQY-tyrosine-phosphorylated LAD-1 does not colocalize with nonphosphorylated LAD-1 or UNC-44 ankyrin but instead is localized to sites that undergo mechanical stress in polarized epithelia and axon-body wall muscle junctions. These findings suggest a novel ankyrin-independent role for LAD-1 related to FGFR signaling. Taken together, these results indicate that L1CAMs constitute a family of ubiquitous adhesion molecules, which participate in tissue morphogenesis and maintaining tissue integrity in metazoans.     

The Caenorhabditis elegans pumilio homolog, puf-9, is required for the 3'UTR-mediated repression of the let-7 microRNA target gene, hbl-1

The Puf family of RNA-binding proteins directs cell fates by regulating gene expression at the level of translation and RNA stability. Here, we report that the Caenorhabditis elegans pumilio homolog, puf-9, controls the differentiation of epidermal stem cells at the larval-to-adult transition. Genetic analysis reveals that loss-of-function mutations in puf-9 enhance the lethality and heterochronic phenotypes caused by mutations in the let-7 microRNA (miRNA), while suppressing the heterochronic phenotypes of lin-41, a let-7 target and homolog of Drosophila Brat. puf-9 interacts with another known temporal regulator hbl-1, the Caenorhabditis elegans ortholog of hunchback. We present evidence demonstrating that puf-9 is required for the 3'UTR-mediated regulation of hbl-1, in both the hypodermis and the ventral nerve cord. Finally, we show that this regulation is dependent on a region of the hbl-1 3'UTR that contains putative Puf family binding sites as well as binding sites for the let-7 miRNA family, suggesting that puf-9 and let-7 may mediate hypodermal seam cell differentiation by regulating common targets.     

C. elegans peb-1 mutants exhibit pleiotropic defects in molting, feeding, and morphology

Caenorhabditis elegans PEB-1 is a novel DNA-binding protein expressed in most pharyngeal cell types and outside the pharynx in the hypodermis, hindgut, and vulva. Previous RNAi analyses indicated that PEB-1 is required for normal morphology of these tissues and growth; however, the peb-1 null phenotype was unknown. Here we describe the deletion mutant peb-1(cu9) that not only exhibits the morphological defects observed in peb-1(RNAi) animals, but also results in penetrant larval lethality characterized by defects in pharyngeal function and molting. Consistent with a function in molting, we found that PEB-1 was detectable in all hypodermal and hindgut cells underlying the cuticle. Comparison to molting-defective lrp-1(ku156) mutants revealed that the peb-1(cu9) mutants were particularly defective in shedding the pharyngeal cuticle, and this defect likely contributed to feeding defects and lethality. Most markers of pharyngeal cell differentiation examined were expressed normally in peb-1(cu9) mutants; however, g1 gland cell expression of a kel-1Colon, two colonsgfp reporter was reduced. As g1 gland cells have prominent functions during molting, we suggest defective gland cell differentiation contributes to peb-1(cu9) molting defects. In comparison, other peb-1 mutant phenotypes, including hindgut abnormalities, appeared independent of the molting defect. Similar phenotypes resulted from late loss of pha-4 function, suggesting that PEB-1 and PHA-4 have common functions in some tissues where they are co-expressed.     

Regulation of neuronal lineage decisions by the HES-related bHLH protein REF-1

Members of the HES subfamily of bHLH proteins play crucial roles in neural patterning via repression of neurogenesis. In C. elegans, loss-of-function mutations in ref-1, a distant nematode-specific member of this subfamily, were previously shown to cause ectopic neurogenesis from postembryonic lineages. However, while the vast majority of the nervous system in C. elegans is generated embryonically, the role of REF-1 in regulating these neural lineage decisions is unknown. Here, we show that mutations in ref-1 result in the generation of multiple ectopic neuron types derived from an embryonic neuroblast. In wild-type animals, neurons derived from this sublineage are present in a left/right symmetrical manner. However, in ref-1 mutants, while the ectopically generated neurons exhibit gene expression profiles characteristic of neurons on the left, they are present only on the right side. REF-1 functions in a Notch-independent manner to regulate this ectopic lineage decision. We also demonstrate that loss of REF-1 function results in defective differentiation of an embryonically generated serotonergic neuron type. These results indicate that REF-1 functions in both Notch-dependent and independent pathways to regulate multiple developmental decisions in different neuronal sublineages.     

Opposing functions of calcineurin and CaMKII regulate G-protein signaling in egg-laying behavior of C.elegans

Ca(2+)/calmodulin-dependent calcineurin has been shown to have important roles in various Ca(2+) signaling pathways. We have previously reported that cnb-1(jh103) mutants, null mutants of a regulatory B subunit, displayed pleiotropic defects including uncoordinated movement and delayed egg laying in Caenorhabditis elegans. Interestingly, gain-of-function mutants of a catalytic A subunit showed exactly opposite phenotypes to those of cnb-1(null) mutants providing an excellent genetic model to define calcium-mediated signaling pathway at the organism level. Furthermore, calcineurin is also important for normal cuticle formation, which is required for maintenance of normal body size in C.elegans. Genetic interactions between tax-6 and several mutants including egl-30 and egl-10, which are known to be involved in G-protein signaling pathways suggest that calcineurin indeed regulates locomotion and serotonin-mediated egg laying through goa-1(Goalpha) and egl-30(Gqalpha). Our results indicate that, along with CaMKII, calcineurin regulates G-protein-coupled phosphorylation signaling pathways in C.elegans.     

The Caenorhabditis elegans homolog of the putative prostate cancer susceptibility gene ELAC2, hoe-1, plays a role in germline proliferation

The potential prostate cancer susceptibility gene ELAC2 has a Caenorhabditis elegans homolog (which we call hoe-1, for homolog of ELAC2). We have explored the biological role of this gene using RNAi to reduce gene activity. We found that worms subjected to hoe-1 RNAi are slow-growing and sterile. The sterility results from a drastic reduction in germline proliferation and cell-cycle arrest of germline nuclei. We found that hoe-1 is required for hyperproliferation phenotypes seen with mutations in three different genes, suggesting hoe-1 may be generally required for germline proliferation. We also found that reduction of hoe-1 by RNAi suppresses the multivulva (Muv) phenotype resulting from activating mutations in ras and that this suppression is likely to be indirect. This is the first demonstration of a biological role for this class of proteins in a complex eukaryote and adds important information when considering the role of ELAC2 in prostate cancer.     

Characterization of C. elegans RING finger protein 1, a binding partner of ubiquitin-conjugating enzyme 1

In a yeast two-hybrid screen, RING finger protein 1 (RFP-1) and UBR1 were identified as potential binding partners of C. elegans UBC-1, a ubiquitin-conjugating enzyme with a high degree of identity to S. cerevisiae UBC2/RAD6. The interaction of RFP-1 and UBC-1 was confirmed by co-immunoprecipitation experiments. Yeast interaction trap experiments mapped the region of interaction to the basic N-terminal 313 residues of RFP-1. The acidic carboxy-terminal extension of UBC-1 was not required for the interaction with RFP-1. Western blot analysis and indirect immunohistochemical staining show that RFP-1 is present in embryos, larvae, and adults, where it is found in intestinal, nerve ring, pharyngeal, gonadal, and oocyte cell nuclei. Double-stranded RNA interference experiments against rfp-1 indicate that this gene is required for L1 development, vulval development, and for egg laying. By contrast, RNA interference against ubc-1 gave no obvious phenotype, suggesting that ubc-1 is nonessential or is functionally redundant.     

SAX-7/L1CAM and HMR-1/cadherin function redundantly in blastomere compaction and non-muscle myosin accumulation during Caenorhabditis elegans gastrulation

Gastrulation is the first major morphogenetic movement in development and requires dynamic regulation of cell adhesion and the cytoskeleton. Caenorhabditis elegans gastrulation begins with the migration of the two endodermal precursors, Ea and Ep, from the surface of the embryo into the interior. Ea/Ep migration provides a relatively simple system to examine the intersection of cell adhesion, cell signaling, and cell movement. Ea/Ep ingression depends on correct cell fate specification and polarization, apical myosin accumulation, and Wnt activated actomyosin contraction that drives apical constriction and ingression (Lee et al., 2006; Nance et al., 2005). Here, we show that Ea/Ep ingression also requires the function of either HMR-1/cadherin or SAX-7/L1CAM. Both cadherin complex components and L1CAM are localized at all sites of cell-cell contact during gastrulation. Either system is sufficient for Ea/Ep ingression, but loss of both together leads to a failure of apical constriction and ingression. Similar results are seen with isolated blastomeres. Ea/Ep are properly specified and appear to display correct apical-basal polarity in sax-7(eq1);hmr-1(RNAi) embryos. Significantly, in sax-7(eq1);hmr-1(RNAi) embryos, Ea and Ep fail to accumulate myosin (NMY-2∷GFP) at their apical surfaces, but in either sax-7(eq1) or hmr-1(RNAi) embryos, apical myosin accumulation is comparable to wild type. Thus, the cadherin and L1CAM adhesion systems are redundantly required for localized myosin accumulation and hence for actomyosin contractility during gastrulation. We also show that sax-7 and hmr-1 function are redundantly required for Wnt-dependent spindle polarization during division of the ABar blastomere, indicating that these cell surface proteins redundantly regulate multiple developmental events in early embryos.     

ARI-1, an RBR family ubiquitin-ligase, functions with UBC-18 to regulate pharyngeal development in C. elegans

The LIN-35 retinoblastoma protein homolog and the ubiquitin-conjugating enzyme UBC-18 function redundantly to control an early step of pharyngeal morphogenesis in C. elegans. In order to identify ubiquitin-ligases acting downstream of UBC-18, we carried out a two-hybrid screen using UBC-18 as the bait molecule. Our screen identified three putative ubiquitin-ligases, one of which, ARI-1, showed genetic interactions leading to defective pharyngeal development that were identical to that previously observed for UBC-18. ARI-1 is a member of the RBR family of ubiquitin-ligases and contains a C-terminal motif that places it within the highly conserved Ariadne subfamily of RBR ligases. Our analyses indicate that ARI-1 is the principal Ariadne family member in C. elegans that is involved in the control of pharyngeal development with UBC-18. Using GFP reporters, we find that ARI-1 is expressed dynamically in a wide range of tissues including muscles and neurons during embryonic and postembryonic development. We also provide evidence that dsRNA species containing 14 or fewer base pairs of contiguous identity with closely related mRNAs are sufficient to mediate off-target silencing in C. elegans.     

Caenorhabditis elegans DAZ-1 is expressed in proliferating germ cells and directs proper nuclear organization and cytoplasmic core formation during oogenesis

The deleted in azoospermia (DAZ) family genes encode potential RNA-binding proteins that are expressed exclusively in germ cells in a wide range of metazoans. We have previously shown that mutations in daz-1, the only DAZ family gene in Caenorhabditis elegans, cause pachytene stage arrest of female germ cells but do not affect spermatogenesis. In this study, we report that DAZ-1 protein is most abundantly expressed in proliferating female germ cells, in a manner independent of the GLP-1 signaling pathway. DAZ-1 is dispensable in males but it is expressed also in male mitotic germ cells. Detailed phenotypic analyses with fluorescence microscopy and transmission electron microscopy have revealed that loss of daz-1 function causes multiple abnormalities as early as the onset of meiotic prophase, which include aberrant chromatin structure, small nucleoli, absence of the cytoplasmic core, and precocious cellularization. Although the reduced size of nucleoli is indicative of a low translational activity in these cells, artificial repression of general translation in the germline does not phenocopy the daz-1 mutant. Thus, we propose that DAZ-1 in C. elegans plays essential roles in female premeiotic and early meiotic germ cells, probably via regulating the translational activity of specific target genes required for the progression of oogenesis.