BMP–Smad1/9 signaling plays a critical role in regulating zebrafish PGC proliferation
The specification and maintenance of germ cell lineage are vital for reproductive development and heredity. The germ cell fate in some species, such as zebrafish and Xenopus, is determined by maternally supplied germ plasm, whereas in mammals it is induced by bone morphogenetic protein (BMP) signaling after implantation. It remains elusive whether BMP signaling is implicated in zebrafish germ cell development. Here, we demonstrate that, in zebrafish, BMP–Smad1/9 signaling is required for primordial germ cell (PGC) maintenance but not for PGC fate determination or migration. BMP inhibitor treatment or whole-embryo smad1/9 knockdown reduces the number of PGCs. To investigate cell-autonomous roles of BMP–Smad1/9 in PGCs, we generated PGC-specific smad1/9 knockouts using a double-transgenic approach with PGC-specific expression of Cas9 and ubiquitous expression of guide RNAs (gRNAs). Loss of Smad1/9 in PGCs leads to impaired PGC proliferation and increased apoptosis, resulting in reduced PGC numbers and adult sex bias. Moreover, transcriptome analysis of smad1-deficient PGCs revealed no significant changes in the expression of PGC-specific genes, but a marked upregulation of genes involved in cell cycle checkpoints and DNA damage repair. Notably, ectopic ATR–pChk1 activation in smad1-cKO PGCs validates cell cycle defects and DNA replication stress. ATR inhibition restores PGC numbers in mutant embryos. Collectively, these findings underscore a critical role of BMP–Smad1/9 signaling in zebrafish PGC population maintenance, while it is dispensable for PGC fate determination and migration. Overall design: PGCs were sorted from control and germline-specific smad1 knockout (smad1-cKO) zebrafish embryos at the 75%-epiboly stage and 1 day post-fertilization (1 dpf) for transcriptome analysis. Additionally, PGCs were collected from smad1 morphants (smad1-KD) and control morpholino-injected embryos (control MO). PGCs and somatic cells were sorted from control and germline-specific smad1 knockout (smad1-cKO) zebrafish embryos at the 15-somite stage for targeted amplicon sequencing.