Fig. 6: ETV1 deletion inhibits pancreatic progenitor in vitro specification into endocrine cells.

A Single-cell sequencing reveals distinct cell populations within WT and KO samples. UMAP plots visualize the distribution of 5218 and 8810 WT and KO cells, respectively. Clusters are identified as follows: PP1 (red) – pancreatic progenitors 1, PP2 (green) – pancreatic progenitors 2, prolifPP (yellow) – proliferating pancreatic progenitors, EP_EC (blue) – endocrine progenitors and endocrine cells, LP (orange) – liver progenitors, mes (purple) – mesenchyme cells. B Feature plots confirm cluster identities based on marker expression: PDX1, NKX6-1, and SOX9 for PP1, PP2, and prolifPP; NEUROG3 for endocrine progenitors and endocrine cells; AFP for liver progenitors, COL3A1 for mesenchyme cells. C Dot plot represents the average expression and percentage of cells expressing markers associated with a specific cell type or cluster. PDX1, NKX6-1, and SOX9 are highly expressed in PP1, PP2, and prolifPP clusters. PP1 and prolifPP clusters show increased expression of genes related to cell adhesion, while the prolifPP cluster shows an elevated expression of proliferation markers (HIST1H1B, TOP2A, and MKI67). The PP2 cluster demonstrates the expression of genes connected to the Notch signaling pathway, axon guidance, and cell migration. The EP_EC cluster expresses NEUROD1, NEUROG3, CHGA, PAX4 and ARX. Liver progenitors (LP) show increased expression of genes related to liver function, including AFP, FGB, and CFTR. Enhanced expression of COL3A1, COL5A2, and VIM is observed in mesenchyme subpopulation (mes). D Violin plots show the differential expression of SLIT3, NOTCH2, SEMA3A, and SEMA6D between WT pancreatic progenitors 1 (PP1) and pancreatic progenitors 2 (PP2) clusters. The expression of SLIT3 and NOTCH2 is higher in the PP2 cluster compared to the PP1 cluster, while the expression of SEMA3A and SEMA6D is increased in the PP1 compared to the PP2 cluster. E Table and bar plot show the cluster frequencies in WT and KO cells. In KO a decreased frequency is observed for the pancreatic progenitors 1 (PP1, red), and endocrine progenitors and endocrine cells (EP_EC, blue) clusters. Conversely, PP2 (PP2 green), liver progenitors (LP, orange) and mesenchyme (mes, purple) clusters show increased frequencies in KO cells compared to WT cells. The frequency of proliferating pancreatic progenitors (prolifPP, yellow) is similar for both WT and KO cells. F Bar plot shows significantly enriched functional terms (adjusted p-value ≤ 0.05, KEGG pathway database) related to the PI3K/AKT signaling pathway, cell adhesion, and cell migration among genes upregulated in pancreatic progenitor 2 (PP2) cluster. G Violin plot demonstrates increased expression of VCL in KO (green) compared to WT (yellow) cells. PP1 – pancreatic progenitors 1, PP2 – pancreatic progenitors 2, prolifPP – proliferating pancreatic progenitors, EP_EC – endocrine progenitors and endocrine cells, LP (orange) – liver progenitors, mes – mesenchyme cells. H Violin plot demonstrates increased expression of COL4A in KO (green) compared to WT (yellow) cells. PP1 – pancreatic progenitors 1, PP2 – pancreatic progenitors 2, prolifPP – proliferating pancreatic progenitors, EP_EC – endocrine progenitors and endocrine cells, LP (orange) – liver progenitors, mes – mesenchyme cells. I ATAC-seq tracks demonstrate VCL (left) and COL4A (right) loci in WT (yellow) and KO (green) PPs. The peaks represent normalized and combined biological replicates (N = 2). J Representative images of VCL (green) immunofluorescence staining in KO and WT PP spheres. DAPI marks the nuclei (blue). Scale bar = 200 μm. K Representative images of COL4A (green) immunofluorescence staining in WT and KO PP spheres. DAPI marks the nuclei (blue). Scale bar = 200 μm. K Quantification of fluorescence intensity from immunofluorescence staining in WT (yellow) and KO (green) PPs (day 12). N = 3 biological replicates. Two-sided student’s t-test was used to determine the p-values shown on the graph. Data are presented as the mean ± SDs.