BioProject

Two types of osteoblasts are required to assemble the zebrafish embryonic skeleton: classical osteoblasts homologous to the mammalian situation, and notochord sheath cells, which serve as non-classical osteoblasts. More...

Two types of osteoblasts are required to assemble the zebrafish embryonic skeleton: classical osteoblasts homologous to the mammalian situation, and notochord sheath cells, which serve as non-classical osteoblasts. The gene entpd5a is critically required for ossification via both types of osteoblasts. Despite the usefulness of zebrafish models in vertebrate research, the genetic regulation of bone formation, as well as mechanisms of transcriptional control of entpd5a, remain largely unknown. Here, using a newly generated transgenic line, we isolate classical and non-classical osteoblasts from zebrafish embryos and performed both ATAC-Seq and RNA-Seq. We analysed results independently and integratively to understand those chromatin dynamics and accompanying transcriptomic changes that occur in different skeletal cells. We show that although Dlx family factors are playing important roles in classical osteoblast regulation, Hox family factors are involved in governing spinal ossification via non-classical osteoblasts. We further present a resource-driven analysis of the entpd5a promoter, experimentally validating the ATAC-Seq dataset and proposing mechanisms of regulating the complex entpd5a expression pattern during zebrafish osteogenesis. Our results thus provide a necessary comprehensive resource for the field of bone development and indicate spatio-temporally regulated promoter/enhancer interactions taking place in the entpd5a locus. Overall design: We used the newly generated entpd5a:Gal4FF;UAS:GFP;R2col2a1a:mCherry transgenic line to isolate via FACS head (classical) and trunk (non-classical) osteoblasts from zebrafish embryos at 15dpf. We also extracted adjacent head cartilage and intersegmental spinal chondrocytes as controls. With these four isolated cell populations we performed both ATAC-Seq and RNA-Seq and analysed data at first individually, to establish open chromatin regions and gene expression profiles, respectively. We then integrated the data to enable us to produce a list of candidate transcription factors likely to function in vivo in each of the cell types during zebrafish skeletogenesis. Less...