Forward Genetic Screen for Genes Required for Embryonic Morphogenesis in C. elegans Alexander Miller1, Molly Jud1, Thalia Padilla1, Josh Lowry2, Bruce Bowerman1 1University of Oregon Department of Molecular Biology; 2 University of Utah Department of Human Genetics Abstract III. Identify the Mutant Genes Methods and Results Obtaining homozygous mutant worms for whole genome sequencing and SNP mapping Our research in the Bowerman laboratory focuses on embryonic morphogenesis in Caenorhabditis elegans. Morphogenesis is the coordinated movement of cells during embryonic development in all animals, and defects in this process can lead to human disorders including neural tube closure, vascular, and limb development defects. C. elegans is a powerful genetic tool to investigate basic cellular events necessary for proper morphogenesis during human development. My project is to perform a forward genetic screen for genes required for morphogenesis, utilizing a collection of roughly 1,000 temperature-sensitive embryonic lethal (TS-EL) mutants created by the Bowerman lab. First, TS-EL mutants are terminally phenotyped to identify mutants with penetrant defects in morphogenesis. Next, I genetically characterize the TS-EL mutants to isolate homozygous recessive, single mutant alleles. We then identify the mutant gene by a combination of SNP mapping and whole genome sequencing to find candidate genes, followed by complementation testing. Last, mutants are sent to our collaborators, the Zhirong Bao laboratory at the Sloan Kettering Cancer Center, for cell-fate lineaging, to characterize the cell movement defects at a single cell level. I have genetically characterized five mutant alleles: or542ts, or566ts, or1216ts, or614ts, and or1121ts. All these alleles are homozygous recessive, single mutants with penetrant morphogenesis defects. Moving forward, I will continue the forward genetic screen to identify genes essential for morphogenesis. My studies will help to develop a deeper understanding of the basic genetic pathways and cell biological changes required for embryonic morphogenesis in C. elegans and improve our comprehension of human development. A. Hawaiian Crosses Scheme B. Example data: hlh-1 (or1312ts) I. Identify Morphogenesis-Defective Mutants Image and score for failure during embryonic morphogenesis Terminal phenotyping protocol for identifying morphogenesis mutants Genes required For morphogenesis Introduction A. SNP mapping of chromosome II for allele or566 B. Structure and function of let-19, or566’s gene identity let-19 Cell movement and shape changes comprise morphogenesis The majority of mutants fail morphogenesis early mn19 os33 or566 A. Morphogenesis during development A. Nomarski images of morphogenesis-defective mutant embryos B. Quantification of morphogenesis failure * C. Alleles and their Identified mutated genes Allele Gene ID Sequence Change Protein Change Human Ortholog Gene Function or566 let-19 cCa→ cTa P2376L TRAP240 Transcriptional Coactivation Subunit or542 chaf-1 Gcc → Acc A437T CHAF1A Chromatin Assembly Factor or614 zwl-1 gGa → gAa G285E ZWILCH Kinetochore Protein * Conclusion Terminally phenotyped 13 mutants, three of which are either too leaky or not morphogenesis-defective. Genetically characterized 6 of these 13 alleles, all of which are recessive single mutants Identified the genetic identity of mutant or566, let-19, a transcriptional co-activator subunit. Mutant alleles terminally phenotyped by myself are marked with an asterisk. Penetrant > 70% same phenotype 50% < Variable < 70% same phenotype B. Cell shape changes during epidermal enclosure Penetrant Variable II. Genetically Characterize Mutant Alleles Performing dominance and segregation tests on mutant alleles Future Directions A. Wild-type cross scheme B. Results of genetic characterization Allele Dom. Test (n) Seg. Freq. (n) % Embryonic Lethal 15˚ C (n) 26˚ C (n) or1107 - or1121* 1.70% (530) 26.3% (80) 0.26% (391) 99.0% (405) or542* 0.70% (416) 29.1% (79) 0.80% (593) 100% (635) or1216* 2.50% (471) 17.3% (75) 1.40% (419) 99.7% (324) or1688 2.70% (259) 19.3% (88) 56.6.% (806) 87.7% (929) or566* 2.60% (380) 23.0% (78) 5.70% (436) 100% (236) or614* 1.35% (369) 23.1% (78) 13.5% (208) 99.0% (244) or906 or909 or1033 Continue with the genetic screen, characterizing and identifying more genes required for embryonic morphogenesis Complete genetic characterization of recently terminally phenotyped mutants Send identified mutant genes to our collaborating lab (Bao Laboratories) for cell-fate lineaging in order to better describe the mutant phenotype C. Temperature-sensitive mutant proteins Acknowledgements Summer Program for Undergraduate Research at University of Oregon (SPUR), National Institutes of Health (NIH), The Zhirong Bao Laboratories, and collective members of the Bowerman Laboratory Goal: To perform a forward genetic screen – using the Bowerman Lab collection of ~1000 temperature-sensitive embryonic lethal mutants – to identify genes that regulate cytoskeleton and cell adhesion required for cell shape changes and cell migration, the bases of morphogenesis. Alleles genetically characterized by myself are marked with an asterisk Research reported in this poster was supported by Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under award number R25HD0708. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.