Graded cold stress impairs zebrafish larval development by disrupting growth signaling and mitochondrial function.

Temperature critically regulates early development in fish, yet the cellular and molecular mechanisms underlying cold-induced developmental disruption in fish remain poorly defined. Here, we investigated the effects of graded low-temperature exposure on zebrafish embryonic and larval development using integrated organismal, cellular, and molecular analyses. We generated EGFP-transgenic zebrafish to enable direct in vivo fluorescence visualization and evaluation of multiple organs. Wild-type and EGFP-labeled embryos were reared at 28 °C (control) or reduced temperatures (22 °C, 16 °C). Fluorescence imaging showed 22 °C caused developmental delay, reduced growth, impaired eye and cardiac development, and abnormal caudal vein morphology, while 16 °C induced severe developmental arrest and early lethality. At the molecular level, low temperature suppressed growth and cardiac-related genes (igf1, nkx2.5, gata4, tbx5) and upregulated inflammation-, proapoptosis- and antioxidant-related genes (tnfa, il1b, bax, sod1), with concurrent reduced mitochondrial membrane potential indicating impaired mitochondrial function. Collectively, low temperature disrupts zebrafish embryogenesis via coordinated effects on growth signaling, stress responses, and mitochondrial function. These findings link environmental temperature stress to altered gene expression and cellular phenotypes during early zebrafish development, providing insights into mechanisms of cold-induced developmental disruption in this model.