Cohesin composition and dosage independently affect early development in zebrafish

ABSTRACT Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed ‘cohesinopathies’ are characterized by germline variants of cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear whether mutations in individual cohesin subunits have independent developmental consequences. Here, we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single-cell RNA sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21, mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies.

Normalized transcript counts of rad21a, smc1al, smc3, stag1b, stag2b, stag1a and stag2a taken from RNA-seq of tailbuds and visualized with box plots defining the distribution of expression levels across these genes in the samples.Genotypes are distinguished by color: wild-type samples are displayed in purple, rad21 -/-in blue, rad21 +/-in green, and stag2b -/-samples in yellow.For each panel, the dotted line indicates the level of expression in wild type.Source data available in Tables S1-S3.S4.

Fig
Fig. S4.Overlap of dysregulated genes and pathway enrichment in cohesin mutant tailbuds.(A, B) The Venn diagrams depict the overlap of significantly upregulated (A) and downregulated (B) genes in cohesin-deficient tailbuds.(C, D) Metascape heat maps displaying the top 20 terms enriched among significantly upregulated (C) and downregulated (D) genes in cohesin-deficient tailbuds.Corresponding p-values are indicated by the color scale.*Epithelial cilium movement involved in extracellular fluid movement **Regulation of transmembrane receptor protein serine/threonine kinase signalling pathway

Fig. S5 .
Fig. S5.Reactome analyses of downregulated pathways in rad21 -/-mutant tailbuds.The dot plot shows the top 10 enriched Reactome pathways (out of 26) among the significantly downregulated genes in rad21 -/-tailbuds.The size of each dot indicates the number of genes affected in the pathway, and the dot color represents the adjusted p-value (Padj).

Fig. S6 .
Fig. S6.Expression levels of sox2, tbxta, tbxtb, wnt3a, tbx16 and msgn1 in cohesin mutants.Normalized transcript counts of sox2, tbxta, tbxtb, wnt3a, tbx16 and msgn1 taken from tailbud RNA-seq data with 4 replicates visualised as box plots defining the distribution of expression levels across these genes in the samples.Genotypes are distinguished by color: wild-type samples are displayed in purple, rad21 -/-in blue, rad21 +/-in green, and stag2b -/-samples in yellow.For each panel, the dotted line indicates the level of expression in wild type.

Fig. S7 .
Fig. S7.Expression pattern of marker genes in the tailbud at the 16-somite stage.Schematic depicts the regions in the tailbud and progenitor types where expression of sox2 (yellow), tbxta (magenta) and tbx16 (cyan) expression is expected.

Fig. S8 .
Fig. S8.Cell population analysis of single cell RNA sequencing of stag2b -/-mutant tailbuds compared with wild type.(A) Dot plot depicting the expression of the top 4 marker genes per cell population cluster identified (see Fig. 7A).The dot size scales with the fraction of cells expressing the gene, and the dot color indicates the log fold change between the clusters.(B) Differences in the proportion of cell types between wild-type and stag2b -/-tailbuds among clusters as shown in Fig. 7A.

Fig. S9 .
Fig. S9.Cell cycle phase analysis of single cell data from stag2b -/-and wild-type tailbuds.(A-B) UMAP of wild-type and stag2b -/-single cell data showing cell cycle phases in blue (G0/G1), green (S) and orange (G2/M).Dashed red outline indicates the anterior paraxial mesoderm cluster 1.All cluster annotations for the integrated dataset in (C).(D) Ranked heatmap of z-scores of stag2b -/-cell clusters comparing cell cycle phase proportions.Red indicates an increase and blue a decrease in cell cycle phase compared to wild type.(E) Stacked bar plot of cell cycle phase fractions comparing anterior paraxial mesoderm 1 cluster in wild type with stag2b -/-.(F) Stacked bar plot of cell cycle phase fractions comparing anterior paraxial mesoderm 2 cluster in wild type with stag2b -/-.

Fig. S10 .
Fig. S10.Heat map showing the top 25 differentially up-and downregulated genes between wild-type and stag2b -/-NMP single cell RNA-seq data.For a full list see TableS4.

Fig. S11 .
Fig. S11.Notochord single cell analysis shows upregulation of Hedgehog signaling with an expansion of hypochord cells in stag2b -/-tailbuds.(A) UMAP plot of notochord markers fox2a, tbxta and noto showing their expression in wild-type (top) and stag2b -/-(bottom) notochord cluster.(B).Gene set enrichment analysis of differentially expressed genes derived from pseudo bulk analysis comparing the notochord cluster in wild-type and stag2b -/-tailbud single cell data.(C) UMAP of hypochord markers gdf6a, ccn2a, hapln1a and cdh6 in wild-type (top) and stag2b -/- (bottom) notochord cluster.(D) Bar plot of hypochord-marked cells as proportion of cells in the notochord cluster in wild-type and stag2b -/-tailbud single data.(E) Dot plot of hypochord markers in the notochord cluster comparing wild-type and stag2b -/-tailbuds.