SWI/SNF complexes are required for retinal pigmented epithelium differentiation and for the inhibition of cell proliferation and neural differentiation programs

ABSTRACT During embryonic development, tissue-specific transcription factors and chromatin remodelers function together to ensure gradual, coordinated differentiation of multiple lineages. Here, we define this regulatory interplay in the developing retinal pigmented epithelium (RPE), a neuroectodermal lineage essential for the development, function and maintenance of the adjacent retina. We present a high-resolution spatial transcriptomic atlas of the developing mouse RPE and the adjacent ocular mesenchyme obtained by geographical position sequencing (Geo-seq) of a single developmental stage of the eye that encompasses young and more mature ocular progenitors. These transcriptomic data, available online, reveal the key transcription factors and their gene regulatory networks during RPE and ocular mesenchyme differentiation. Moreover, conditional inactivation followed by Geo-seq revealed that this differentiation program is dependent on the activity of SWI/SNF complexes, shown here to control the expression and activity of RPE transcription factors and, at the same time, inhibit neural progenitor and cell proliferation genes. The findings reveal the roles of the SWI/SNF complexes in controlling the intersection between RPE and neural cell fates and the coupling of cell-cycle exit and differentiation.


Development • Supplementary information
Table S1.Summary of the samples analyzed by Geo-seq.Each tab includes embryo number and genotype and the information on the length of the samples collected by lasercapture microdissection for the Geo-seq analysis.
Table S2.List of the differentially expressed genes (DEGs) and statistical results based on DESeq2 analysis of the control samples.The DEGs for each indicated comparison are presented in a separate tab.
Table S3.List of genes in each cluster based on K-means clustering of the control samples.
Table S4.Functional enrichment results for the genes in each of the presented clusters (ClusterProfiler).
Table S5.List of regulons identified in the control samples based on the SCENIC analysis.
Table S6.Predicted targets for the regulons identified in control samples by SCENIC analysis.
Click here to download Table S1 Click here to download Table S2 Click here to download Table S3 Click here to download Table S4 Click here to download Table S5 Click here to download Click here to download Table S7 Click here to download Table S8 Click here to download Table S9 Click here to download Table S10 Click here to download :10.1242/dev.201488:Supplementary information Development • Supplementary information

Fig. S2 .
Fig. S2.Geo-seq data quality control and analyses of E14.5 PE and Me control and FcKO samples.(A) Violin plots showing the detected gene number (red), mapping ratio (blue), and total sequenced reads (green) for all samples.(B) Annotation tree based on pairwise correlation analysis between all samples.(C) Football plot presenting the average relative positional expression of PE (Mitf, Sox9, Pax6) and Me (Dkk2, Pitx2, Col3a1) genes.Expression levels are represented on a scale of green (low) to red (high).(D) Volcano plot representing the differentially expressed genes in control PE vs. Me samples.GSEA plots of overrepresented terms in control PE (E) or Me (F) genes.
Fig. S4.The expression of several retinal genes is not elevated in the SWI/SNF-deficient PE.Football plots representing the mean relative expression of Rax (A) and Six6 (B) in the control and FcKO embryos.(C-H) Antibody labeling of E14.5 control and FcKO samples, staining for (C, D) VSX2, (E, F) BRN3b, and (G, H) ISL1.Higher magnifications of right and left boxed regions (C-H) are presented to the right and left of the main panels, respectively.

Fig. S5 .
Fig. S5.The change in the expression of cell-adhesion, extracellular matrix and WNTpathway genes in the control compared to FcKO PE and Me.(A) Heat map showing the average spatial expression pattern of genes related to the blood-retinal barrier and Bruch's membrane.(B) Heat map showing the average spatial expression pattern of genes associated with canonical and noncanonical WNT signaling.(C) Football plots for expression of selected genes from each group in the PE of control and FcKO.ONH, optic nerve head.

Table S11 . Predicted targets for each of the regulons identified by SCENIC analysis in FcKO compared to control samples. Table S7. List of the differentially expressed genes (DEGs) and statistical results based on DESeq2 analysis of the FcKO compared to control samples.
The DEGs for each indicated comparison are presented in a separate tab.