Poster abstracts
Poster number 5 submitted by Clare Austin
Identification of segmentation clock oscillatory transcripts in the zebrafish presomitic mesoderm
Clare Austin (Molecular Genetics), Thomas Gallagher , Monica Blatnik, Sharon Amacher
Abstract:
The segmentation clock is an oscillatory gene network that regulates somitogenesis, the formation of embryonic somites that develop into vertebrae, axial skeleton, and musculature. To maintain reiterated and rapid oscillations, segmentation clock gene expression is tightly regulated at the transcriptional and post-transcriptional levels. We have focused on the latter and discovered that Proline-rich nuclear receptor cofactor 2 (Pnrc2), a mRNA decay adaptor, is necessary for the degradation of oscillatory gene transcripts in the undifferentiated presomitic mesoderm (PSM) in zebrafish. Given that the number of known PSM oscillatory genes is ten-fold higher in mouse and humans compared to zebrafish, I predict there are many more genes that oscillate in the zebrafish PSM. Because Pnrc2 regulates decay of known zebrafish segmentation clock oscillatory transcripts, I hypothesize some of the yet unidentified oscillatory gene transcripts will also be regulated by Pnrc2. We have conducted RNA-seq on pnrc2 mutant embryos at mid-segmentation (16-somite) stage. To identify additional zebrafish transcripts that oscillate in the PSM, I am focusing on transcripts that are upregulated in pnrc2 mutant embryos in our RNA-seq analysis and also oscillate in the PSM of mice and humans. Using qPCR, I have validated all 15 genes that are both oscillatory in the PSM of humans and mice and upregulated in pnrc2 mutant embryos, and discovered 2 additional transcripts that were not previously thought to be significantly upregulated in the pnrc2 mutant. Using in situ hybridization, I have observed that at least 11 of these transcripts show enhanced and expanded expression in pnrc2 mutant embryos at the mid-segmentation stage. To discover if these genes function as segmentation clock regulators, I am prioritizing the oscillatory genes that show specific and expanded mRNA expression in pnrc2 mutant embryos for CRISPR-mediated knockout to observe how loss of gene function affects somite formation. Additionally, we are mining the RNA-seq data to discover novel PSM oscillatory genes, as well as genes that may oscillate in other tissues. Through these experiments we will determine if conserved oscillators also oscillate in zebrafish and discover novel oscillatory genes, and then determine their functional role in the segmentation clock.
Keywords: RNA oscillation, segmentation clock, somitogenesis