Fig. 7

a Schematic summarizing changes in splicing patterns identified by rMATS between sibling and smad1-cKO PGCs at 75% epiboly stage. Changed events were filtered for FDR < 0.05 and absolute percent spliced in (ΔPSI) > 0.1. ES, exon skipping; IR, intron retention; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice site; MXE, mutually exclusive exon. The proportions of each alternatively spliced (AS) event were shown in parentheses. b Left, Venn diagram showing overlaps among AS genes in cKO and KD RNA-seq datasets with DEGs in PGCs. P-value was determined via a hypergeometric test. Right, enriched GO terms for all AS genes in cKO and KD PGCs. c RT-PCR validation of altered splicing of selected AS genes in smad1-cKO PGCs at 75% epiboly stage. The compositions of exons for the major products were indicated on the right, with the blue exon being alternatively spliced. d Co-immunoprecipitation (co-IP) of Smad1 with spliceosome components Snrpa and Snrpb in HEK293T cells. e Proximity ligation assay (PLA) of pSmad1/5/9 and Snrpb in 75% epiboly embryos. Top: schematic of the PLA principle. Bottom left: representative images showing PLA probe signals between pSmad1/5/9 and Snrpb in PGCs (kop:GFP, green). Bottom right, quantification of nuclear PLA signals normalized to cytoplasmic levels in PGCs. Each dot represented one PGC. Nc, total number of observed PGCs. Error bars indicated SD, with the mean indicated by the center line. Statistical significance was determined by a two-sided Student’s t-test. f Model of BMP–Smad signaling regulating zebrafish PGC population. WT PGCs proliferate and survive properly, contributing to balanced sex differentiation. smad1/9-cKO PGCs exhibit altered splicing and DNA damage response, resulting in ATR activation, reduced proliferation, and cell death, consequently inducing decreased PGC numbers and biased sex differentiation toward males. Source data are provided as a Source Data file.