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Regulation of the Ras Signaling Network in C. Elegans Development
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An EGF gradient induces the equipotent C. elegans vulval precursor cells (VPCs) to assume the 3˚-3˚-2˚-1˚-2˚-3˚ pattern of cell fates. EGF triggers the LET-60/Ras-LIN-45/Raf-MEK-2/MEK-MPK-1/ERK canonical MAP kinase cascade to induce 1˚ fate and synthesize of DSL ligands for the lateral Notch signal. In turn, LIN-12/Notch induces neighboring cells to become 2˚. In response to lower dose of EGF signal, LET-60/Ras switches effectors to use the RGL-1/RalGEF-RAL-1/Ral modulatory signaling cascade to promote 2˚ fate in support of LIN-12. The goals of this research are to define principles by which signaling networks function and to identify an effector cascade downstream of RAL-1. RAL-1 signals through EXOC-8/Exo84, an established Ral binding partner, GCK-2, a CNH domain-containing MAP4 Kinase, and PMK-1/p38 MAP kinase to promote 2˚ fate. We also show that RGL-1 plays opposing and genetically separable roles in VPC fate patterning. RGL-1 promotes 2˚ fate via canonical GEF-dependent activation of RAL-1 and 1˚ fate via a non-canonical GEF-independent activity. Our genetic epistasis experiments are consistent with RGL-1 functioning in the modulatory 1˚-promoting AGE-1/PI3-Kinase-PDK-1/PDK-AKT-1/Akt cascade. Animals without RGL-1 experience 15-fold higher rates of VPC patterning errors compared to the wild type. Yet VPC patterning in RGL-1 deletion mutants is not more sensitive to environmental perturbations. We propose that RGL-1 functions as a “Balanced Switch” that orchestrates opposing 1˚- and 2˚-promoting modulatory cascades to decrease developmental stochasticity. To investigate how LET-60/Ras switches effectors to promote different cell fates, we used CRISPR to tag endogenous LIN-45/Raf and RGL-1/RalGEF proteins. We found that they are recruited to different subcellular compartments during VPC induction. LIN-45 is recruited to the basolateral membrane in presumptive 1˚ cells. RGL-1 is recruited to the apical membrane in presumptive 2˚ cells, and this localization depends on functional LET-60. We hypothesize that RGL-1 apical localization in the VPCs is mediated by phosphorylation or scaffold proteins. Our studies delineate a novel Ral-dependent developmental signaling cascade, bifunctional RGL-1 as a “Balanced Switch”, and LET-60 effector segregation mechanism in vivo, thus providing critical insights for understanding Ras biology in cancer and development.
VPC fate patterning
Shin, Hanna (2018). Regulation of the Ras Signaling Network in C. Elegans Development. Doctoral dissertation, Texas A & M University. Available electronically from