Abstract
Fish air breathing is crucial for the transition of vertebrates from water to land. So far, the genes involved in fish air breathing have not been well identified. Here, we performed gene enrichment analysis of positively selected genes (PSGs) in loach (Misgurnus anguillicaudatus, an air-breathing fish) in comparison to Triplophysa tibetana (a non-air-breathing fish), haplotype-resolved genome assembly of the loach, and gene evolutionary analysis of air-breathing and non-air-breathing fishes and found that the PSG mex3a originated from ancient air-breathing fish species. Deletion of Mex3a impaired loach air-breathing capacity by inhibiting angiogenesis through its interaction with T-box transcription factor 20. Mex3a overexpression significantly promoted angiogenesis. Structural analysis and point mutation revealed the critical role of the 201st amino acid in loach Mex3a for angiogenesis. Our findings innovatively indicate that the ancient mex3a is a fish air-breathing gene, which holds significance for understanding fish air breathing and provides a valuable resource for cultivating hypoxia-tolerant fish varieties.