Zebrafish sox9b+ cells function as intestinal stem cells for epithelial homeostasis and regeneration.

Intestinal stem cells (ISCs) are essential for maintaining epithelial homeostasis and driving tissue regeneration. The zebrafish intestine features a distinctive furrow-ruga architecture and lacks the crypt structures characteristic of mammalian intestines, leaving its ISC identity elusive. Characterizing zebrafish stem cell populations and their molecular markers is informative for intestinal physiology and evolution. Here, we identify sox9b+ cells residing within the proliferative zone of intestinal furrows as functional zebrafish ISCs. These cells contribute to epithelial turnover during homeostasis and can differentiate into nearly all characterized epithelial cell lineages. During epithelial regeneration, sox9b+ cells actively respond to injuries and constitute a major source of neogenic epithelium. Functional and transcriptomic analyses further establish sox9b as an essential factor of intestinal stemness and epithelial regeneration. Additionally, we observed that sox9b+ cells gradually acquire regional distribution patterns along the developing intestine. Our work provides a comparative model for understanding ISC function across diverse intestinal architectures.