Candidate Regulatory Genes for Hindlimb Development in the Embryos of the Chinese Alligator (Alligator sinensis)

Crocodilians, which are a kind of animal secondary adaptation to an aquatic environment, their hindlimb can provide the power needed to engage in various life activities, even in low-oxygen water environments. The development of limbs is an important aspect of animal growth and development, as it is closely linked to body movement, support, heat production, and other critical functions. For the Chinese alligator, the hindlimb is one of the main sources of power, and its development and differentiation will directly influence the survival ability in the wild. Furthermore, a better understanding of the hindlimb developmental process will provide data support for the comparative evolutionary and functional genomics of crocodilians. In this study, the expression levels of genes related to hindlimb development in the Chinese alligator embryos during fetal development (on days 29, 35, 41, and 46) were investigated through transcriptome analysis. A total of 1675 differentially expressed genes (DEGs) at different stages were identified by using limma software. These DEGs were then analyzed using weighted correlation network analysis (WGCNA), and 4 gene expression modules and 20 hub genes were identified that were associated with the development of hindlimbs in the Chinese alligator at different periods. The results of GO enrichment and hub gene expression showed that the hindlimb development of the Chinese alligator embryos involves the development of the embryonic structure, nervous system, and hindlimb muscle in the early stage (H29) and the development of metabolic capacity occurs in the later stage (H46). Additionally, the enrichment results showed that the AMPK signaling pathway, calcium signaling pathway, HIF-1 signaling pathway, and neuroactive ligand–receptor interaction are involved in the development of the hindlimb of the Chinese alligator. Among these, the HIF-1 signaling pathway and neuroactive ligand–receptor interaction may be related to the adaptation of Chinese alligators to low-oxygen environments. Additionally, five DEGs (CAV1, IRS2, LDHA, LDB3, and MYL3) were randomly selected for qRT-PCR to verify the transcriptome results. It is expected that further research on these genes will help us to better understand the process of embryonic hindlimb development in the Chinese alligator.