Transcriptomic Analysis Reveals Multilayer Regulation Underlying Development and Pregnancy in the Malayan Pangolin

The critically endangered Malayan pangolin (Manis javanica) exhibits distinctive physiological adaptations; nevertheless, its molecular mechanisms underlying development and reproduction remain inadequately characterized, thereby impeding effective conservation initiatives. In this study, we conducted a comprehensive transcriptomic analysis of six tissues (intestine, kidney, liver, lung, muscle, and spleen) derived from adult, fetal, and pregnant individuals. The fetal stage demonstrated the greatest abundance of stage-specific genes, which were predominantly enriched in developmental pathways such as epithelial organization and transmembrane signaling. Differential gene expression (DGE) analysis revealed that genes upregulated in adults were primarily associated with metabolic and immune functions, whereas those upregulated in fetuses were linked to developmental processes and cell cycle regulation. Co-expression network analysis identified critical hub genes, including GLDC in adults and NCAM1 in fetuses, that are implicated in stage-specific regulatory mechanisms. Additionally, extensive differential alternative splicing (DAS) events were observed, with exon skipping (SE) representing the most frequent splicing pattern. Integrative analyses suggested that DGE and DAS largely constitute independent regulatory layers; however, a subset of hub genes, such as OBSL1 and TNIP1, exhibited concurrent differential expression and splicing, indicating their potential central roles in coordinating developmental processes. This study presents a detailed transcriptomic atlas of Malayan pangolin development and pregnancy, providing candidate biomarker genes for evaluating reproductive health and developmental status, thereby informing future conservation strategies.