next-generation sequencing approaches to identify genotype-phenotype relationships during miRNA tuning of neural crest osteogenesis

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PROJECT SUMMARY Research: Every 4 ½ minutes, a baby is born with a birth defect, including such that affect the craniofacial skeleton. These facial malformations cause disfigurement leading to long-term psychological impact and are often associated with physical dysfunction imposing tremendous burden on the affected individuals and their families. Although the causes for most of these severe developmental pathologies are still unknown, it is clear that the inappropriate formation and differentiation of neural crest (NC) cells, the cells that normally give rise to the osteoblasts in the affected craniofacial skeleton, plays a large part. Given the causality between miscontrolled osteoblast differentiation from the NC and the tissue malformations arising from it, an understanding of the molecular networks underlying NC development is crucial for the potential treatment or prevention of craniofacial defects, yet far from complete. For instance, the contribution of epigenetic regulators, such as microRNAs to the formation of craniofacial bones remains understudied, despite their potential as diagnostic markers. For this K18 application it is proposed to use data sets generated already under 5R01DE025330 to examine the epigenetic regulation of NC development by a specific microRNA, miR361, which is pro-osteogenic in vitro and expressed in the developing NC in vivo. Assessment of skeletal phenotypes when miR361 is knocked out together with single cell RNA-sequencing using an innovative and effective in vitro differentiation model based on human pluripotent stem cells will validate the impact of miR361 on NC-osteogenesis. Bulk RNA sequencing and ChIP sequencing will identify NC-specific loci that are transcriptionally regulated downstream of miR361. Together, these data will define the relationship between this miRNA, transcriptional activation in NC cells and their subsequent propensity for osteoblast differentiation. Goals/Training plan/environment: The goals for this K18 application are two-fold: 1) to train in the design, execution and specifically the analysis of next-generation sequencing data sets, and 2) to acquire hands-on experience with imaging-modality-assisted analysis of skeletal phenotypes. The attainment of such skills, in addition to attending several workshops focused on bioinformatics throughout the year of support will prepare the applicant for future funding and mentoring opportunities. The applicant has assembled a strong team of local mentors and regional collaborators who have extensive experience in the area of the proposed work and a strong record of training scientists to become successful independent investigators. In summary, the proposed project will allow the applicant to enrich her long-standing track record in stem cell biology with bioinformatics and phenotypic skeletal analysis, which will foster the perspectives and augment the skills necessary to be a successful craniofacial biologist.