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Developmental genetics of Xenopus

  • Author(s): Lyons, Jessica Burnham
  • Advisor(s): Harland, Richard M
  • et al.
Abstract

Abstract

Developmental genetics of Xenopus

by

Jessica Burnham Lyons

Doctor of Philosophy in Molecular and Cell Biology

University of California, Berkeley

Professor Richard Harland, Chair

The frog Xenopus laevis has been studied for over 200 years. Its advantages as a robust and easily manipulable model organism have been complemented more recently by genetic and genomic studies in Xenopus tropicalis. My thesis work utilized established and cutting-edge techniques to advance our knowledge of developmental genetics in both Xenopus systems.

I used an embryological approach to investigate the roles of the Fgf receptors (Fgfrs) during development. Knockdown and overexpression studies suggested that each Fgfr plays a different role in the specification of mesoderm, and my results are consistent with Fgfr4 playing a role in dampening the Fgf signal. I also showed that X. tropicalis Fgfrs 1-3 are alternatively spliced in D3, the extracellular immunoglobulin domain important for ligand specificity. These isoforms exhibit different temporal and spatial expression patterns, suggesting that control of this alternative splicing plays a role in regulating development.

My thesis work has also harnessed the power of the X. tropicalis system to understand development using a forward genetic approach. Tadpoles homozygous for the recessive lethal mutation curly exhibit ventral edema and curled tails. I used classical genetics to map curly to a 1.9 Mb window on X. tropicalis chromosome 4. The pteg gene, which lies in this region, is misspliced in curly embryos. Isabelle Philipp and I used next-generation (nextgen) sequencing technology to identify differences between the curly mutant DNA and that of the reference genome. Focusing on the region around the pteg gene, we are currently evaluating these differences to find the lesion that causes the curly phenotype.

The efficient application of genetic techniques requires a high-quality genetic map and reference genome, and those available for X. tropicalis were flawed (Wells et al., 2011) (Hellsten et al., 2010). Thus, I developed a genotyping by sequencing (GBS) technique using reduced-representation multiplex nextgen sequencing, and my collaborators in the Rokhsar lab have generated a high-quality SNP map for X. tropicalis based on my data from 192 F2 individuals. This map is being used to construct an improved X. tropicalis genome assembly. By finding regions of correlation for the pigmentation mutation gray, as well as the X. tropicalis sex locus, we demonstrated that this technique can be used for genetic mapping. I applied my GBS method to X. laevis as well, and the SNP map thus generated will facilitate the assembly of a reference genome for this allotetraploid species.

By combining the classical advantages of the Xenopus system with modern techniques, my thesis work has contributed to our understanding of the development, genetics, and genomics of vertebrate biology.

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