Abstract
Cohesinopathies are multisystem disorders caused by germline mutations in cohesin subunit genes. Cohesin has non-overlapping roles in cell division and gene expression. Cohesinopathies are caused by interplay of both cohesin roles; however, their individual contribution is not understood. We aimed to investigate cell division and gene expression in cohesin deficiency to better understand the molecular pathology of cohesionopathies.
Cohesin subunit Rad21 is essential for cell division, whereas Stag2 controls gene expression without affecting cell division. We used zebrafish embryos with knockout mutations in rad21 or stag2 to investigate consequences for stem cell fate choice. Multipotent cells in the embryonic tailbud, called neuromesodermal progenitors (NMPs), form mesoderm or neurons under the control of Wnt signalling. We performed RNA-sequencing of wild type, stag2 and rad21 mutant tailbuds (4 replicates of tailbud pools per condition).
Thousands of genes were dysregulated in stag2 and rad21 mutant tailbuds (Padj <0.05). Genes downregulated in rad21 mutants were strongly cell cycle associated (ngenes=95, P=2*10-7). Mesodermal induction was disrupted, likely due to sox2 upregulation (LFC = 0.53, Padj = 6*10-4) and tbxta downregulation (LFC = -0.42, Padj = 0.01). Expression of the vimr2 gene was downregulated (LFC = -1.23, Padj = 5.54*10-5) in stag2 mutants indicating the disruption of the mesodermal induction, however, markers of differentiated mesoderm (ngenes=24, P=0.0013) were upregulated. Expression of endothelial marker genes were downregulated in stag2 mutants (ngenes=11, P=2*10-7). Muscle forms when Wnt signalling is high, conversely, low Wnt signalling produces endothelial cells. We suggest that stag2 mutants compensate for the lack of mesodermal induction by upregulation of the Wnt signalling pathway (ngenes=3, P=6*10-7). Increased Wnt reporter expression was observed by confocal microscopy. To stimulate mesoderm induction in cohesin mutants, we treated embryos with a Wnt agonist and performed RNA-sequencing of the tailbuds. External stimulation of Wnt signalling rescued transcriptional signatures in stag2, but not in rad21 mutants (interaction analysis).
Our results show that developmental outcomes depend on which cohesin subunit is mutated leading to a better understanding of the molecular pathology of Cohesinopathies.