Event Title

A Genetic Screen To Identify New FGFR Signaling Components In C. Elegans

Location

SU 214

Start Date

19-4-2019 12:00 PM

Department

Biology

Session

Session 5

Description

Fibroblast growth factor receptors (FGFRs) belong to a family of receptor tyrosine-kinase (RTK) cell-surface receptors which phosphorylate specific tyrosine residues to trigger downstream responses such as cell proliferation, migration, and differentiation. The study of the EGL-15 FGFR in the nematode Caenorhabditis elegans has long been used as a paradigm to understand principles of RTK signaling, since defects in the processes mediated by EGL-15 result in striking phenotypes that provide powerful genetic tools. One such process is the regulation of fluid homeostasis. EGL-15 hyperactivation causes excessive accumulation of clear fluid inside the worm’s body (the Clear (Clr) phenotype). The isolation of Clr suppressors, termed Suppressor of Clr (soc) mutants, has led to the identification of many of the core components of EGL-15 signaling. For example, the original set of soc mutations identified the Grb2/SEM-5 adaptor protein that links RTK signaling to the activation of the RAS/MAPK pathway. EGL-15 can directly bind SEM-5 via a pair of binding sites on its carboxy-terminal domain (CTD). Although SEM-5 is required for EGL-15 signaling, the egl-15(n1457) mutation that truncates the CTD (ΔCTD) and eliminates the known SEM-5 binding sites on EGL-15 does not confer a Soc phenotype. These data suggest the existence of an alternate pathway that links EGL-15 to SEM-5/Grb2 in its role in mediating fluid homeostasis. To identify components of this alternative pathway, we repeated the genetic screen for Soc mutants, this time with the ΔCTD mutation in the background. This “enhancer” screen resulted in the isolation of 33 mutations. 17 of these mutations have been initially characterized and assigned to complementation groups. Seven of these enhancer mutations are new alleles of the known soc genes egl-15 (5 alleles) and soc-1 (2 alleles). Five enhancer mutations define a new soc gene on the X chromosome, soc-3; another enhancer mutation, soc(ay167), maps to another novel position on the X chromosome, potentially identifying an additional new soc gene. Four other mutations define an autosomal soc complementation group that is not one of the major soc genes identified in the original Soc screen. Sixteen enhancer mutations are still being characterized. Our preliminary characterization of these enhancer alleles indicates that the screen was successful in two ways. First, the behavior of the enhancer alleles supports two cooperative pathways that transduce EGL-15 signaling: egl-15(ay150), soc-1(ay172), and all alleles of soc-3 show only partial effects on fluid homeostasis on their own and are enhanced by the ΔCTD mutation. Second, the characterization of an initial subset of these soc enhancer mutations suggests that they define up to four new soc genes that potentially define the alternate pathway. These results are consistent with the original hypothesis that two downstream pathways collaborate to affect fluid homeostasis regulation mediated by the EGL-15 FGFR. Further genetic analysis and whole-genome sequencing will be used to identify the molecular identities of these new FGFR signaling genes.

Comments

Cindy Voisine, Michael Stern, and Te-Wen Lo are the faculty sponsors of this project.

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Apr 19th, 12:00 PM

A Genetic Screen To Identify New FGFR Signaling Components In C. Elegans

SU 214

Fibroblast growth factor receptors (FGFRs) belong to a family of receptor tyrosine-kinase (RTK) cell-surface receptors which phosphorylate specific tyrosine residues to trigger downstream responses such as cell proliferation, migration, and differentiation. The study of the EGL-15 FGFR in the nematode Caenorhabditis elegans has long been used as a paradigm to understand principles of RTK signaling, since defects in the processes mediated by EGL-15 result in striking phenotypes that provide powerful genetic tools. One such process is the regulation of fluid homeostasis. EGL-15 hyperactivation causes excessive accumulation of clear fluid inside the worm’s body (the Clear (Clr) phenotype). The isolation of Clr suppressors, termed Suppressor of Clr (soc) mutants, has led to the identification of many of the core components of EGL-15 signaling. For example, the original set of soc mutations identified the Grb2/SEM-5 adaptor protein that links RTK signaling to the activation of the RAS/MAPK pathway. EGL-15 can directly bind SEM-5 via a pair of binding sites on its carboxy-terminal domain (CTD). Although SEM-5 is required for EGL-15 signaling, the egl-15(n1457) mutation that truncates the CTD (ΔCTD) and eliminates the known SEM-5 binding sites on EGL-15 does not confer a Soc phenotype. These data suggest the existence of an alternate pathway that links EGL-15 to SEM-5/Grb2 in its role in mediating fluid homeostasis. To identify components of this alternative pathway, we repeated the genetic screen for Soc mutants, this time with the ΔCTD mutation in the background. This “enhancer” screen resulted in the isolation of 33 mutations. 17 of these mutations have been initially characterized and assigned to complementation groups. Seven of these enhancer mutations are new alleles of the known soc genes egl-15 (5 alleles) and soc-1 (2 alleles). Five enhancer mutations define a new soc gene on the X chromosome, soc-3; another enhancer mutation, soc(ay167), maps to another novel position on the X chromosome, potentially identifying an additional new soc gene. Four other mutations define an autosomal soc complementation group that is not one of the major soc genes identified in the original Soc screen. Sixteen enhancer mutations are still being characterized. Our preliminary characterization of these enhancer alleles indicates that the screen was successful in two ways. First, the behavior of the enhancer alleles supports two cooperative pathways that transduce EGL-15 signaling: egl-15(ay150), soc-1(ay172), and all alleles of soc-3 show only partial effects on fluid homeostasis on their own and are enhanced by the ΔCTD mutation. Second, the characterization of an initial subset of these soc enhancer mutations suggests that they define up to four new soc genes that potentially define the alternate pathway. These results are consistent with the original hypothesis that two downstream pathways collaborate to affect fluid homeostasis regulation mediated by the EGL-15 FGFR. Further genetic analysis and whole-genome sequencing will be used to identify the molecular identities of these new FGFR signaling genes.