Single-cell transcriptomics identifies a candidate osmoregulatory cell population in the mid-intestine of the saline-alkaline fish Gymnocypris przewalskii.

Saline-alkaline fishes maintain internal homeostasis through effective intestinal ion and acid-base regulation. However, the specific intestinal epithelial cell populations mediating these functions remain poorly characterized. Here, we performed single-cell RNA sequencing on 23,909 mid-intestinal cells from juvenile Gymnocypris przewalskii, a fish native to the saline-alkaline Lake Qinghai, to generate a cell atlas of the mid-intestine. Several major cell classes were identified, including epithelial cells (ECs), enteroendocrine cells (EECs), goblet cells (GCs), and leukocytes (LCs), each exhibiting distinct subpopulation heterogeneity. Within the epithelial cell lineage, we identified a distinct cluster characterized by high expression of best4, cftr, and ca2, suggesting a candidate osmoregulatory cell population (referred to as Best4+ cells). To further evaluate this marker set, we compared fish exposed to freshwater with those exposed to artificial lake water and observed significantly elevated gene expression levels of best4, cftr and ca2 in intestinal cells under lake water conditions. Immunofluorescence detection of BEST4 and CA2 in mid-intestinal sections further provided tissue-level support for the epithelial association of these candidate markers. In addition to this candidate osmoregulatory cell population, the atlas also resolved absorptive epithelial cell states, together with heterogeneity within enteroendocrine cell, goblet cell, and leukocyte lineages. Overall, these findings identify a candidate epithelial cell population relevant to intestinal ion and acid-base regulation in G. przewalskii and clarify the cellular basis of intestinal specialization under saline-alkaline stress.