Transcriptome analyses reveal molecular mechanisms that regulate endochondral ossification in amphibian Bufo gargarizans during metamorphosis.

Background: A developmental transition from aquatic to terrestrial existence is one of the most important events in the evolution of terrestrial vertebrates. Amphibian metamorphosis is a classic model to study this transition. The development of the vertebrate skeleton can reflect its evolutionary history. Endochondral ossification serves a vital role in skeletal development. Thus, we sought to unravel molecular mechanisms that regulate endochondral ossification during Bufo gargarizans metamorphosis.

Methods: The alizarin red-alcian blue double staining method was used to visualize the skeletal development of B. gargarizans during metamorphosis. RNA sequencing (RNA-seq) was used to explore the transcriptome of B. gargarizans in four key developmental stages during metamorphosis. Real-time quantitative PCR (RT-qPCR) was used to validate the expression patterns of endochondral ossification related genes.

Results: Endochondral ossification increased gradually in skeletal system of B. gargarizans during metamorphosis. A total of 137,264 unigenes were assembled and 44,035 unigenes were annotated. 10,352 differentially expressed genes (DEGs) were further extracted among four key developmental stages. In addition, 28 endochondral ossification related genes were found by searching for DEG libraries in B. gargarizans. Of the 28 genes, 10 genes were validated using RT-qPCR.

Conclusions: The exquisite coordination of the 28 genes is essential for regulation of endochondral ossification during B. gargarizans metamorphosis.

General Significance: The present study will not only provide an invaluable genomic resource and background for further research of endochondral ossification in amphibians but will also aid in enhancing our understanding of the evolution of terrestrial vertebrates.