The human genome encodes 10 insulin-like genes, whereas the genome remarkably encodes 40 insulin-like genes. Knockout strategies to determine the roles of all the insulin/insulin-like peptide ligands (INS) in has been challenging due to functional redundancy. Here, we individually overexpressed each of the 40 genes pan-neuronally, and monitored multiple phenotypes including: L1 arrest life span, neuroblast divisions under L1 arrest, dauer formation, and fat accumulation, as readouts to characterize the functions of each INS Of the 40 INS peptides, we found functions for 35 INS peptides and functionally categorized each as agonists, antagonists, or of pleiotropic function. In particular, we found that 9 of 16 agonistic INS peptides shortened L1 arrest life span and promoted neuroblast divisions during L1 arrest. Our study revealed that a subset of β-class INS peptides that contain a distinct F peptide sequence are agonists. Our work is the first to categorize the structures of INS peptides and relate these structures to the functions of all 40 INS peptides Our findings will promote the study of insulin function on development, metabolism, and aging-related diseases.

Mutations that affect the single C. elegans Eph receptor tyrosine kinase VAB-1 cause defects in cell movements during embryogenesis. Here, we provide genetic and molecular evidence that the VAB-1 Eph receptor functions with another neuronal receptor, SAX-3/Robo, for proper embryogenesis. Our analysis of sax-3 mutants shows that SAX-3/Robo functions with the VAB-1 Eph receptor for gastrulation cleft closure and ventral epidermal enclosure. In addition, SAX-3 functions autonomously for epidermal morphogenesis independently of VAB-1. A double-mutant combination between vab-1 and slt-1 unmasks a role for the SLT-1 ligand in embryogenesis. We provide evidence for a physical interaction between the VAB-1 tyrosine kinase domain and the juxtamembrane and CC1 region of the SAX-3/Robo receptor. Gene dosage, non-allelic non-complementation experiments and co-localization of the two receptors are consistent with a model in which these two receptors form a complex and function together during embryogenesis.

The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.

The human genome encodes ten insulin-like genes, whereas the C. elegans genome remarkably encodes forty insulin-like genes. The roles of insulin/insulin-like peptide ligands (INS) in C. elegans are not well understood. The functional redundancy of the forty INS genes makes it challenging to address their functions by using knock out strategies. Here, we individually overexpressed each of the forty ins genes pan-neuronally, and monitored multiple phenotypes including: L1 arrest life span, neuroblast divisions under L1 arrest, dauer formation and fat accumulation, as readouts to characterize the functions of each INS in vivo. Of the 40 INS peptides, we found functions for 35 INS peptides and functionally categorized each as agonists, antagonists or of pleiotropic function. In particular, we found that 9 of 16 agonistic INS peptides shortened L1 arrest life span and promoted neuroblast divisions during L1 arrest. Our study revealed that a subset of β-class INS peptides that contain a distinct F peptide sequence are agonists. Our work is the first to categorize the structures of INS peptides and relate these structures to the functions of all forty INS peptides in vivo. Our findings will promote the study of insulin function on development, metabolism, and aging-related diseases.Author SummaryInsulin and insulin-like growth factors are found in all animals and regulate many physiological and developmental processes. The human genome has 10 insulin-like peptides including the well characterized insulin hormone. The nematode C. elegans has 40 insulin-like (INS) peptide genes. All 40 INS peptides have been knocked out but no single INS gene knock out resembles the loss of the C. elegans insulin receptor suggesting that the other INS peptides can compensate when one INS is lost. We have used a genetic approach to overexpress each of the 40 INS peptides in C. elegans and have identified in vivo function for 35 of the 40 INS peptides. Like the human insulin and IGF-1, C. elegans INS peptides are derived from a precursor protein and we have shown that INS peptides with an associated peptide called the F peptide are strong activators of the C. elegans insulin-like receptor. We also identified several INS peptides that inhibit the insulin-like receptor and these inhibitory INS peptides may have therapeutic potential.

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