Abstract
Cohesin, a multiprotein complex, is essential to eukaryotic cell survival because of its roles in cell division, DNA repair and gene regulation. The complex is made up of four core subunits: RAD21, SMC3, SMC1A, and either STAG1 or STAG2. The two STAG-cohesin complexes have semi-redundant roles during cell division but divergent roles in cohesin-mediated gene regulation. The high prevalence of STAG2 mutations in human cancers and developmental disorders has prompted multiple studies on the role of STAG2-cohesin. In contrast, the role of STAG1 is poorly understood.
The zebrafish tailbud is a useful model for cell differentiation owing to the presence of bipotent stem cells that are influenced by signalling pathways to differentiate into mesoderm and ectoderm germ layers in post gastrula embryos. Zebrafish have duplicated stag genes, and homozygous knockouts of stag1a, stag1b and stag2b are viable, likely owing to compensation of other stag genes.
This thesis aimed to identify how loss of Stag1-cohesin affects cell fate determination, using the zebrafish tailbud as a model for differentiation. It also aimed to decipher global gene expression differences between losing Stag1-cohesin and Stag2-cohesin in the zebrafish tailbud, and how these changes influence cell fate determination.
RNA-seq revealed that loss of Stag1b-cohesin causes global transcriptional dysregulation in the zebrafish tailbud. Pathway analysis identified that the cell cycle and dorsoventral patterning are dysregulated in stag1b-/- mutants. Comparison between dysregulated genes in stag1b-/- and stag2b-/- mutant zebrafish identified that there is minimal overlap in gene or pathway dysregulation in the two mutant tailbuds. A viable stag1a-/-;stag1b-/- mutant was generated indicating that, in zebrafish, loss of all Stag1-cohesin is compatible with life.
Phosphohistone H3 labelling of mitotic cells identified reduced proliferation in Stag1-deficient zebrafish tailbuds. HCR-FISH identified that Stag1 deficient zebrafish did not present major tailbud cell differentiation defects. ISH and qPCR experiments confirmed that dorsoventral patterning genes are dysregulated in stag1b-/- and stag1a-/-;stag1b-/- mutant tailbuds, but not in stag2b-/- tailbuds. These findings suggest a new potential role for Stag1-cohesin in maintaining dorsoventral patterning.