Fig 2: Improper maturation of the hiPSC-derived NCCs after intracameral injection. a qRT-PCR analysis of the corneal endothelial-related genes (Na+/K+-ATPase, ZO-1, SLC4A11, AQP1, N-cadherin, and COL8A2) and the NCC-related genes (P75, TFAP2A, TFAP2B, and SOX10) in the hiPSC-derived NCCs before transplantation (Pre), 7 days and 14 days after transplantation (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001. b Immunofluorescence staining of the hiPSC-derived NCCs before transplantation, 7 days and 14 days after transplantation. DNA was visualized by staining with DAPI (blue) and Human Nuclei (HuNu)

Fig 3: Inactivation of Tfap2a and Tfap2b by CRISPR/Cas9 leads to defects in nephric duct morphogenesis and progenitor cell identity.a Schematic representation of Tfap2a and Tfap2b exons targeted by CRISPR/Cas9 technology and the location of the sgRNAs used. b Wholemount GFP fluorescence of control (Pax2-GFP) and allelic series of Tfap2a/2b;Pax2-GFP mutant embryos at E9.5 and E10.5. White arrows denote nephric duct integrity defects whereas yellow arrow and inset magnification highlight ectopic Pax2-GFP positive cells. Scale bar of the inset = 50 µm. c Quantification of nephric duct (ND) elongation in Tfap2a/2b;Pax2-GFP double KO embryos at E9.5. n = 15 (Control) and n = 4 (Tfap2a/2b double KO) biologically independent samples. The graphs represent mean ± SD, assessed by a two-tailed Mann–Whitney test. Source data are provided as a Source Data file. d Immunostaining for the nephric duct marker Gata3 in transverse sections of E9.5 Tfap2a/2b double mutant shows an elongation defect and the presence of ectopic Gata3 positive (nephric duct) cells (yellow arrows). Nephric duct cells are denoted by white dotted lines. Scale bar 25 µm for all pictures. e Quantification of Gata3+ cells in the rostral region of E9.5 control and Tfap2a/2b double KO embryos (n = 7). The graph represents mean ± SD, determined by a two-tailed unpaired t-test. Source data are provided as a Source Data file. f Immunostaining for the markers Hoxb9 (NdPr1), and intermediate mesoderm (Wt1) in transversal sections of the nephric duct in E9.5 control and Tfap2a/2b double mutant embryos. Yellow arrows denote Gata3 positive cells expressing Hoxb9 or Wt1 markers. White dotted lines denote the nephric duct. Scale bar 25 µm for all the pictures. g Quantification of Hoxb9+,Gata3+ cells number in sections of E9.5 control and Tfap2a/2b double KO embryos. n = 11 (Control) and n = 6 (Tfap2a/2b double KO) independent sections from four different embryos per genotype. h Quantification of the number of Wt1+,Gata3+ cells in sections of E9.5 control and Tfap2a/2b double KO embryos. n = 15 (Control) and n = 11 (Tfap2a/2b double KO) independent sections from four different embryos per genotype. The graphs represent mean ± SD, compared by a two-tailed Mann–Whitney test. Source data are provided as a Source Data file.

Fig 4: EWSR1-ATF1 displays a distinct binding pattern compared to wt ATF1.a Genomic distribution of 2385 consensus EWSR1-ATF1 binding sites shared between DTC1 and SU-CCS-1 cell lines, illustrating preferential EWSR1-ATF1 binding to distal genomic regions. b DNAse I hypersensitivity profile comparison between 2011 distal EWSR1-ATF1 sites and 516 wt ATF1 bound distal sites across 113 different cell types, showing a more restricted DNA accessibility pattern for the fusion protein binding sites in other cell types. c Top panel: heatmaps depicting EWSR1 N, ATF1 C, H3K4me1, H3K27ac, and ATAC signal intensities at 2011 EWSR1-ATF1 bound distal sites in DTC1 and SU-CCS-1 cell lines, as well as three primary CCS tumors. Bottom panel: EWSR1 N, ATF1 C, H3K4me3, H3K27ac, and ATAC signal intensities at 374 TSS-associated EWSR1-ATF1 binding sites. For each heatmap 20 kb regions centered on the EWSR1-ATF1 peaks are shown. Signals are ranked by ATF1 C intensity. d De novo motif enrichment analysis for 2011 distal EWSR1-ATF1 binding regions. The top three motifs identified are shown. Binomial p-values are given by the motif enrichment software HOMER. e, ChIP-seq track at MERTK and ID4 genomic loci, illustrating similar EWSR1-ATF1 binding and chromatin activity profiles between DTC1 and primary CCS tumor cells, and the presence of TFAP2A, SOX10, and MITF at EWR1-ATF1 sites.

Fig 5: TFAP2A expression and activity profile in the NMuMG EMT model. a-b NMuMG cells were treated with 2 ng/mL of TGFß1 for 72 h and were stained for TFAP2A and F-Actin (a) and TFAP2A and E-cadherin (b). The merged panels represent colocalization of the imaged markers with the nucleus which was stained with DAPI and compared to controls. Scale bar represents 50 µm. c NMuMG cells were treated for 14 days with 2 ng/mL of TGFß1. Quantitative RT-PCR of Tfap2a during the time course of this treatment indicates that Tfap2a mRNA levels are reduced upon EMT. The EMT markers E-cadherin (Cdh1), Fibronectin (Fn1), Occludin (Ocln), and Vimentin (Vim) follow the expected trend. d Two mRNA-seq samples from independent wells were prepared from a time course of NMuMG cells treated for 14 days with 2 ng/mL of TGFß1, and the data was consequently analyzed with ISMARA [30]. The figure depicts the dynamics of TFAP2A/C transcriptional activity during the time course. The sequence logo of the TFAP2A/C binding motif is also indicated. e-f Lysates from NMuMG/E9 cells treated with 2 ng/mL of TGFß1 for 72 h were probed for TFAP2A, GAPDH and Lamin B expression by WB and their levels compared with the expression levels of Actin and also to the Ponceau-stained membrane (e). The bar plot represents the densitometric quantification of the TFAP2A protein levels upon treatment compared to the control (f) ** indicates a p-value < 0.01 in the paired t-test (P = 0.0014)