Novel Signals Regulate Cell Fate Patterning
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Cell fate patterning of the C. elegans vulva is an ideal system for the study of signal transduction mechanisms.EGF induces six equipotent vulval precursor cells (VPCs) to assume cell fates in a 3Ëš-3Ëš-2Ëš-1Ëš-2Ëš-3Ëš pattern with99.8% accuracy. In the â€œMorphogen Gradientâ€ model for vulval patterning, distance from the EGF sourcedictates the fate of each VPC. In the â€œSequential Inductionâ€ model, the Rasâ†’Rafâ†’MEKâ†’ERK MAP kinasecascade induces 1Ëš fate, elicits DSL ligand production, and thus, via the LIN-12/Notch receptor, induces its twoneighboring VPCs to become 2Ëš. Because of the absence of key molecular details, these two models weredebated for 16 years. We resolved this debate: to interpret the EGF gradient, presumptive 2Ëš cells useRasâ†’RalGEFâ†’Ral rather than the canonical Rasâ†’Raf used in 1Ëšs. Mutating RalGEF or Ral (a cousin of Ras)does not confer strong patterning defects, suggesting that sequential induction is the dominant patterningmechanism. These are clinically relevant molecules: Ras is the most mutated oncoprotein. Rasâ†’RalGEFâ†’Ralis thought to be equally important for oncogenesis as the canonical Rasâ†’Raf. Our central hypothesis is that the Rasâ†’RalGEFâ†’Ral story justifies pursuing unexplored areas of VPCpatterning because clinically important signals are found as positive and negative regulators. The objectives ofthis proposal are to unveil new facets of the molecular basis for the 1Ëš/2Ëš fate choice and its high reproducibilityin vivo, exploiting the strengths of this system for dissection of signaling mechanisms. Our preliminary resultssupport the feasibility of three aims, each with a hypothesis focused on distinct molecular mechanisms. Aim 1: Our CRISPR-generated activating mutation in endogenous Rap1 (Ras proximal) induced ectopic 1Ëšcells. Lack of Rap1 reduces 1Ëš cell induction. We will test the hypothesis that Rap1 is the nexus of twoopposed regulatory inputs that promote and repress ERK activation in presumptive 1Ëš vs 2Ëš cells, respectively. Aim 2: We identified a novel Ral effector, GCK-2/MAP4 kinase, that possibly signals via p38 MAP kinaseto promote 2Ëš fate. We will test the hypothesis that Ralâ†’GCK-2 triggers a p38 cascade. We will also testwhether Ralâ†’GCK-2 signal regulates CCCH RNA binding proteins to stabilize 3â€™UTRs of 2Ëš-promoting genes. Aim 3: MIG-15/MAP4K, the sole paralog of GCK-2/MAP4K, paradoxically inhibits 2Ëš fate. We will test thehypothesis that MIG-15 triggers a JNK MAP kinase cascade, which may be the missing signal that repressesthe Notch receptor in presumptive 1Â° cells. MIG-15 defines a novel class of vulval-specific Notch repressors.We will make use of the results of a screen we have completed for MIG-15-like targets to identify new players. For all three aims we will use CRISPR-engineered endogenous fluorescent reporters to deconvolutespecific signals embedded within the vulval signaling network. Successful completion of these aims will definecomponents and organizational principles of the vulval signaling network that leads to the exceptional fidelity ofpatterning, which are likely to apply both broadly and specifically to other developmental systems.