From Crest to Bone: Understanding neural crest skeletogenic capacity along the neural axis - ABSTRACT The evolution of vertebrates has been significantly shaped by the emergence of the neural crest, a critical innovation believed to have bolstered predatory prowess and fostered the expansion of craniofacial structures. During development of vertebrate embryos, distinct neural crest subpopulations arise along the body axis. While these subpopulations share a basic neural crest gene regulatory network (GRN), superimposed upon this are distinct regional subcircuits that imbue subpopulations with distinct developmental potentials. Indeed, only the cranial neural crest in amniotes possesses the ability to generate ectomesenchymal derivatives like osteoblasts. Recent studies have demonstrated that axial level specific GRN subcircuits play a critical role in enabling neural crest cells to develop specific cell types, with amniotes possessing a cranial-specific circuit capable of reprogramming trunk neural crest cells to form craniofacial cartilage. In our recent research, we discovered that trunk neural crest cells in sturgeons, a primitive group of bony fish, possess the remarkable ability to create bony structures in the postcranial region, suggesting that the ancient neural crest has the ability to contribute to the formation of skeletogenic tissues along the entire neural axis. By utilizing this unique model organism that has dermal bones from head-to-tail, my goal is to undercover the underlying fundamental GRN driving neural crest- derived bone formation. I hypothesize that this basic GRN may have served as a scaffold that became elaborated and increased in complexity during vertebrate evolution, culminating in craniofacial bone formation in mammals. This proposal will determine the presence of genes akin to amniote cranial-specific gene regulatory circuit in sturgeon. To investigate this, hybridization chain reaction and CRISPR/Cas9-mediated mutagenesis will be used to examine coexpression and linkages within the GRN subcircuits in migrating neural crest cells and their derivatives. Next, single cell RNA-sequencing and unbiased comparative transcriptomic analysis will be performed on sturgeon neural crest cells at cranial and trunk levels and compared with chick and lamprey datasets to uncover genetic circuits characteristic of sturgeon neural crest subpopulations, particularly those associated with skeletogenesis. Finally, ATAC-seq assays at various axial levels and developmental stages during sturgeon neural crest development will be used to pinpoint potential enhancers and assess alterations in open chromatin patterns based on axial location and developmental timing. Together, the results of these aims will illuminate the mechanisms underlying the origin of new cell types, such as osteoblasts, within the neural crest. In addition to identifying useful targets for therapeutic intervention preventing craniofacial defects, this award will provide me with necessary training as I prepare to begin my independent career. Dr. Marianne Bronner's laboratory at the Caltech and the assembled advisory council offer essential resources, knowledge, and training atmosphere required to effectively pursue the proposed objectives and help me establish an independent research career.
