Determine the functions of the heterogeneity of embryonic origins in shoulder girdle formation - Project Summary Vertebrate skeletons are ontogenetically heterogeneous with multiple embryonic progenitor cell populations. However, how bone-forming osteoblasts differentiate from distinct embryonic origins and differ in their functional contributions to skeletogenesis remain unknown. We will answer these fundamental but relatively unexplored questions, using the zebrafish pectoral (shoulder) girdle as a powerful model to dissect embryonic heterogeneity in ossification. We have established the zebrafish pectoral girdle as a strikingly simple and transparent experimental model that enables us to dissect the regulation and functions of the embryonic heterogeneity in skeletogenesis, deploying state-of-the-art genomics, genetics, and imaging techniques. To date, we have discovered that 1) at least four distinct cell populations (neural crest cells, lateral plate mesoderm, cardiopharyngeal mesoderm, paraxial mesoderm) differentiate into cleithrum osteoblasts, as determined by single cell RNA-sequencing (scRNA-seq) and genetic cell labeling; 2) the gene expression profiles and chromatin accessibility of osteoblasts derived from diverse embryonic populations differ from each other; and 3) Gli, an indispensable transcription factor for cleithrum formation, is a leading candidate for inducing chromatin accessibility and osteoblast differentiation, as determined by single-nuclear assay for transposase-accessible chromatin followed by high-throughput sequencing (snATAC-seq). Based on these compelling findings, we hypothesize that Gli amplifies progenitor cell chromatin accessibility associated with ossification genes thus generates functionally different osteoblasts in cleithrum morphogenesis. These functionally different osteoblasts likely contribute to various bone morphology in the cleithrum. We will dissect the regulation and functions of heterogeneous cell populations from distinct embryonic origins in pectoral girdle development, growth, and homeostasis through two aims. Aim 1 will determine the distributions and functions of embryonic heterogeneity in pectoral girdle morphogenesis, growth, and homeostasis by genetic labeling and ossification ability tests in vitro and in vivo. We will also optogenetically ablate each cell population specifically in the cleithrum and characterize the phenotype. Aim2 will dissect the molecular mechanisms to generate functionally diverse osteoblasts, reflecting progenitor cell chromatin accessibility. We will identify Gli binding sites and analyze their accessibility changes in an arsenal of Hh-Gli knockout series of fish. Then, we will investigate which cell populations are most susceptible to the changes in the Hh-Gli signaling, investigating the difference of chromatin accessibility and responses associated with ossification genes among osteoblasts derived from diverse embryonic origins. Successful completion of the proposed research will provide fundamental insights into the functions of embryonic heterogeneity in skeletal development at the single-cell level, broadly informing how dysregulation of heterogeneous embryonic origins leads to malformation of human skeletons.
