RUNX transcription factors are essential in maintaining epididymal epithelial differentiation

Apart from the androgen receptor, transcription factors (TFs) that are required for the development and formation of the different segments of the epididymis have remained unknown. We identified TF families expressed in the developing epididymides, of which many showed segment specificity. From these TFs, down-regulation of runt related transcription factors (RUNXs) 1 and 2 expression coincides with epithelial regression in Dicer1 cKO mice. Concomitant deletion of both Runx1 and Runx2 in a mouse epididymal epithelial cell line affected cell morphology, adhesion and mobility in vitro. Furthermore, lack of functional RUNXs severely disturbed the formation of 3D epididymal organoid-like structures. Transcriptomic analysis of the epididymal cell organoid-like structures indicated that RUNX1 and RUNX2 are involved in the regulation of MAPK signaling, NOTCH pathway activity, and EMT-related gene expression. This suggests that RUNXs are master regulators of several essential signaling pathways, and necessary for the maintenance of proper differentiation of the epididymal epithelium. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05211-5.


Figure S1 .
Figure S1.Characterization of Runx1 and Runx2 mutations in mE-Cap18 cells.Schematic picture of the gene structure of Runx1 and Runx2 with untranslated regions (UTR) areas white, coding sequence gray and exon coding DNA binding domain blue.Sequence from exon 5 (Runx1) and e x o n4( Runx2) is marked with box in WT sequence.Different deletions in sequences are marked for cell lines with only Runx1 (dR1) or Runx2 (dR2) deletion and with both deletions (ddR1+R2).For Runx2, point mutations, deletions of nucleotide G in both sites, in one allele of ddR1+R2 cell line are marked with arrows and corresponding nucleotides are underlined in WT sequence.

Figure S2 .
Figure S2.Immunofluorescent staining of tight junction proteins in control (Ctr) and Dicer1 cKO epididymides.Tight junction proteins (TJPs); TJP1 (red), TJP2 (green) and TJP3 (green), and claudins (CLDN, green); CLDN1, CLDN3 and CLD4 were stained in adult 2-monthold Ctr and Dicer1 cKO epididymides.In Ctr TJP1, TJP2 and TJP3 were highly expressed and largely colocalized in the tight junctions in the apical side of the epithelium, whereas in Dicer1 cKO epididymides the expression of TJP1 and TJP2 was markedly reduced, TJP2 being almost undetectable in the tissue sections (left panel, arrowheads).TJP1 staining was discontinuous, suggesting problems in the apical tight junctions of Dicer1 cKO mice.Contrary to TJP1 and -2, TJP3 seemed to be upregulated in Dicer1 cKO, but with similarly discontinuous staining as TJP1 (left panel, arrowheads).CLDN1, -3 and -4 were strongly colocalized with TJP1 in apical tight junctions (middle and right panels, arrowheads) and were detected in the basolateral membrane of epithelial cells in Ctr.CLDN1 and -4 were also detected throughout the plasma membrane of basal cells (arrows).In contrast, no colocalization with TJP1 was observed for any of the claudins in Dicer1 cKO epididymides apical junctions (middle and right panels, arrowheads).Instead the expression of claudins was detected throughout the cytoplasm and basolateral membranes of the Dicer1 cKO epithelium.DNA; blue.Yellow color indicates colocalization.Arrow; basal cell.Scale bars 50 μm.

Figure S3 .
Figure S3.Representative phase-contrast images of the 3D organoid-like structures used in the RNAseq analysis.RNA was harvested on day 12 of culture in Matrigel.Scale bar 50 µm.

FigureFigure S5 .
Figure S4.Morphometric analysis of organotypic cultures (Area measurement).AMIDA analysis from confocal microscope images of organoidlike structures at day 10 of culture.Area describes the size of an individual object.Data visualization and statistical analysis were performed in the R-software environment (www.r-project.org)using Bonferroni-corrected t-tests against Ctr.The number above the box is the number of individual objects analyzed across replicate wells.Data presented as a box and whisker plot (median, black line; mean, red spot).*** p≤0.001.

Figure S6 .
Figure S6.Immunofluorescent staining of vimentin in control (Ctr) and Dicer1 cKO epididymides.Vimentin (red) was stained in 40-day-old and adult 2-monthold Ctr and Dicer1 cKO epididymides.In Ctr VIM was highly expressed in the mesenchymal cells between the tubulus cross sections, whereas in Dicer1 cKO epididymides also some epithelial cells express VIM (inserts, arrows).DNA; blue.Scale bars 50 μm.