1. 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.