School

School of Humanities and Sciences

Department

Biology

Department

Biochemistry

ICC Theme

Other

Date

2-4-2019 12:10 PM

Abstract

In humans, fibroblast growth factor receptors (FGFRs) play an important role in many developmental pathways that control cell proliferation, differentiation, migration, and survival. Mutations in FGFRs can cause developmental disorders including Achondroplasia (dwarfism), Craniosynostosis (abnormal growth of the skull), and different types of cancer.

We use Caenorhabditis elegans, a transparent nematode, as a model to investigate FGFR signaling. In addition to being cheap and easy to maintain, C. elegans also have a short life cycle, produce many progeny and share 35% of genes with humans. The sole FGFR in C. elegans is EGL-15 which is required for many functions, one of which is fluid homeostasis. EGL-15 is negatively regulated by the protein CLR-1. When CLR-1 function is disrupted, EGL-15 is hyperactivated which results in an increased accumulation of fluid in worms and the Clr (Clear) phenotype. Clr worms appear clear, transparent, and plump. This Clr phenotype and the hyperactivation of EGL-15 can be suppressed with either mutations in egl-15 or other downstream components of this pathway, such as sem-5.

Previous research has shown that the interaction between SEM-5 and EGL-15 requires the phosphorylation of two tyrosines (Y1009 and Y1087). Complete loss of EGL-15 or the complete loss of SEM-5 can suppress the Clr phenotype suggesting that the hyperactivation of EGL-15 is suppressed. However, when Y1009 and Y1087 in EGL-15 cannot be phosphorylated, this EGL15 mutant cannot suppress the Clr phenotype suggesting that the EGL-15 SEM-5 interaction is being mediated by an additional site on EGL-15 or an additional yet to be identified component.

To identify these missing components, we conducted a soc (suppressor of Clr) screen to identify components that can suppress the Clr phenotype in the absence of the two previously identified EGL-15 SEM-5 binding sites. Components that can suppress the Clr phenotype identified in this screen could represent components that can mediate fluid homeostasis independent of the previously identified SEM-5 binding sites. One potential component that has been identified by our screen and whole genome sequencing is the gene cca-1.

We are currently taking a two-pronged approach to verify the role of cca-1 in the EGL-15 signaling pathway. Our first approach is to use a technique called RNA interference (RNAi) to alter gene function. We are using RNAi to knock down specific genes in C. elegans. In addition, to knocking down cca-1 function, we are also using L4440 as a negative control, and egl-15 as a positive control. It has been previously shown that loss of egl-15 can suppress the Clr phenotype and L4440 has no effect. We have currently completed two rounds of this RNAi experiment. Unfortunately, our positive control has not been as robust as expected. As a result, we are in the process of improving our methods increase the strength of the RNAi.

Our second approach is to create a genetic triple mutant that contains the clr-1, egl-15 (no SEM-5 binding sites), and cca-1 mutations. This will allow us to test if loss of cca-1 gene function can suppress the Clr phenotype, in the absence of the known SEM-5 binding sites. To create this triple mutant, we will cross a male worm containing the clr-1 and egl-15 mutations with a hermaphrodite worm containing a cca-1 mutation. Once the triple mutant is obtained, we will determine if these triple mutants are Clr or non-Clr. If they are Clr, we would conclude that cca1 cannot suppress the Clr phenotype and is unlikely to be the missing link between EGL-15 and SEM-5 we have been searching for. If the triple mutant is non-Clr, we would conclude that cca1 can suppress the Clr phenotype and is a viable candidate to be the missing link between EGL15 and SEM-5 and we will continue to characterize the role of CCA-1 in the EGL-15 signaling pathway.

The identification of novel components, such as CCA-1, will further our understanding of FGFR signaling in C. elegans. Given the similarities between C. elegans FGFR signaling and human FGFR signaling, information gained by using C. elegans as a model could also help us better understand FGFR signaling in humans. Therefore, our data would help shed light on the mechanisms involved with developmental disorders caused by mutations in the FGFR pathway in humans.

Document Type

Poster

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Apr 2nd, 12:10 PM

Classification of Novel Components of the Fibroblast Growth Factor Receptor

In humans, fibroblast growth factor receptors (FGFRs) play an important role in many developmental pathways that control cell proliferation, differentiation, migration, and survival. Mutations in FGFRs can cause developmental disorders including Achondroplasia (dwarfism), Craniosynostosis (abnormal growth of the skull), and different types of cancer.

We use Caenorhabditis elegans, a transparent nematode, as a model to investigate FGFR signaling. In addition to being cheap and easy to maintain, C. elegans also have a short life cycle, produce many progeny and share 35% of genes with humans. The sole FGFR in C. elegans is EGL-15 which is required for many functions, one of which is fluid homeostasis. EGL-15 is negatively regulated by the protein CLR-1. When CLR-1 function is disrupted, EGL-15 is hyperactivated which results in an increased accumulation of fluid in worms and the Clr (Clear) phenotype. Clr worms appear clear, transparent, and plump. This Clr phenotype and the hyperactivation of EGL-15 can be suppressed with either mutations in egl-15 or other downstream components of this pathway, such as sem-5.

Previous research has shown that the interaction between SEM-5 and EGL-15 requires the phosphorylation of two tyrosines (Y1009 and Y1087). Complete loss of EGL-15 or the complete loss of SEM-5 can suppress the Clr phenotype suggesting that the hyperactivation of EGL-15 is suppressed. However, when Y1009 and Y1087 in EGL-15 cannot be phosphorylated, this EGL15 mutant cannot suppress the Clr phenotype suggesting that the EGL-15 SEM-5 interaction is being mediated by an additional site on EGL-15 or an additional yet to be identified component.

To identify these missing components, we conducted a soc (suppressor of Clr) screen to identify components that can suppress the Clr phenotype in the absence of the two previously identified EGL-15 SEM-5 binding sites. Components that can suppress the Clr phenotype identified in this screen could represent components that can mediate fluid homeostasis independent of the previously identified SEM-5 binding sites. One potential component that has been identified by our screen and whole genome sequencing is the gene cca-1.

We are currently taking a two-pronged approach to verify the role of cca-1 in the EGL-15 signaling pathway. Our first approach is to use a technique called RNA interference (RNAi) to alter gene function. We are using RNAi to knock down specific genes in C. elegans. In addition, to knocking down cca-1 function, we are also using L4440 as a negative control, and egl-15 as a positive control. It has been previously shown that loss of egl-15 can suppress the Clr phenotype and L4440 has no effect. We have currently completed two rounds of this RNAi experiment. Unfortunately, our positive control has not been as robust as expected. As a result, we are in the process of improving our methods increase the strength of the RNAi.

Our second approach is to create a genetic triple mutant that contains the clr-1, egl-15 (no SEM-5 binding sites), and cca-1 mutations. This will allow us to test if loss of cca-1 gene function can suppress the Clr phenotype, in the absence of the known SEM-5 binding sites. To create this triple mutant, we will cross a male worm containing the clr-1 and egl-15 mutations with a hermaphrodite worm containing a cca-1 mutation. Once the triple mutant is obtained, we will determine if these triple mutants are Clr or non-Clr. If they are Clr, we would conclude that cca1 cannot suppress the Clr phenotype and is unlikely to be the missing link between EGL-15 and SEM-5 we have been searching for. If the triple mutant is non-Clr, we would conclude that cca1 can suppress the Clr phenotype and is a viable candidate to be the missing link between EGL15 and SEM-5 and we will continue to characterize the role of CCA-1 in the EGL-15 signaling pathway.

The identification of novel components, such as CCA-1, will further our understanding of FGFR signaling in C. elegans. Given the similarities between C. elegans FGFR signaling and human FGFR signaling, information gained by using C. elegans as a model could also help us better understand FGFR signaling in humans. Therefore, our data would help shed light on the mechanisms involved with developmental disorders caused by mutations in the FGFR pathway in humans.

 

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