RNA-Sequencing Analysis of Gene-Expression Profiles in the Dorsal Gland of Alligator sinensis at Different Time Points of Embryonic and Neonatal Development

SIMPLE SUMMARY: Skin derivatives, such as integumentary glands, keratin, and bony structures deriving from the epidermis or dermis, are commonly found in various vertebrates species and are suggested to possess a variety of functions that are closely related to the lifestyle of the species. However, most reptile glandular skin derivatives have undergone obvious degeneration due to environmental adaptation to dry land, with the exception of a few skin glands with specific functions. An oval organ/tissue called the dorsal gland spreading from the mid-cervical region to the anterior caudal region in the axial musculature on the dorsal mid-line has been reported in crocodilians, including Alligator sinensis, which is a critically endangered species of the 23 existing crocodilian species. Here, we present the first investigation to focus on particular states of cell proliferation and cell apoptosis, we provide direct molecular evidence which supports the speculation that it might serve as a holocrine secretory, as observed in the other known multicellular exocrine glands. Furthermore, the high-throughput analysis of gene-expression profiles at different timepoints of embryonic and neonatal Alligator sinensis suggested that it might function through the transport and deposition of pigment and lipids via lysosomal exocytosis, these function might relate to an adaptive function in the transition from an amniotic fluid environment to the terrestrial environment around hatching. The corresponding results have considerable importance in enriching our understanding of the intrinsic relationship between the skin derivatives and lifestyles of crocodilians. Furthermore, they have provided a theoretical basis for the rearing and management of newborn Alligator sinensis. ABSTRACT: Significant advances have been made in the morphological observations of the dorsal gland (DG), an oval organ/tissue which lies on both sides of the dorsal midline of the crocodilian. In the current study, RNA sequencing (RNA-seq) was used to identify the changing patterns of Alligator sinesis DGs at different timepoints from the 31st embryonic day (E31) to the newly hatched 1st day (NH1). A comprehensive transcriptional changes of differentially expression gene (DEGs) involved in the melanogenesis, cholesterol metabolism, and cell apoptosis pathways suggested that the DG might serves as a functional secretory gland in formation, transport and deposition of pigment, and lipids secretion via lysosomal exocytosis. Furthermore, the remarkable immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and B-cell lymphoma 2 (Bcl-2)-positive signals in the basilar cells, in parallel with the immuno-reactive TdT-mediated dUTP nick-End labeling(TUNEL) within suprabasal cells, provided direct molecular evidence supporting for the speculation that DG serves as a holocrine secretion mode. Finally, subsequent phylogenetic and immunohistochemical analysis for the PITX2, the identified DEGs in the RNA-seq, was helpful to further elucidate the transcriptional regulatory mechanism of candidate genes. In conclusion, the current results are of considerable importance in enriching our understanding of the intrinsic relationship between the skin derivatives and lifestyles of newborn Alligator sinesis.