An optimized in situ hybridization protocol to study early developmental patterns and signaling pathways in paradise fish (Macropodus opercularis)

The Chinese paradise fish (Macropodus opercularis) is an obligate air-breathing species native to the hypoxic freshwater environments of Southeast Asia. While its ethological aspects have been extensively investigated since the 1970 s, the molecular biology of this species remains largely unexplored. Nevertheless, it offers significant potential for research in fields such as behavioral genetics, molecular evolution, and developmental biology. This study aims to optimize the in situ hybridization protocol, a widely used technique in developmental biology proven effective in zebrafish, and adapt it to paradise fish. We applied our optimized protocol to compare the expression of several conserved developmental genes, including chordin (chd), goosecoid (gsc), myogenic differentiation 1 (myod1), T box transcription factor Ta (tbxta), paired box 2a (pax2a), and retinal homebox gene 3 (rx3), in zebrafish and paradise fish embryos. Furthermore, we examined the role of key conserved signaling pathways during early development in both species using small molecular agonists and antagonists. Through these molecular analyses, we can gain deeper insights into the evolutionary conservation of early developmental programs.