Six1 role on cell fate decisions within the mouse mandibular arch at single-cell resolution. - ABSTRACT Many craniofacial structural birth defects result from disrupted formation, migration, patterning, or differentiation of neural crest (NC) cells that populate the pharyngeal arches, including the mandibular arch (first arch). Following patterning, cranial NC cells differentiate into various cell fates including bone, cartilage, and connective tissue. SIX1 is a transcription factor that can act as a transcriptional activator or repressor depending on interaction with co-factors. Loss of Six1 in the mouse results in severe craniofacial defects including a smaller and deformed mandible, Meckel’s cartilage, tympanic ring and middle ear ossicles, and mirror image duplication in the upper jaw accompanied by ectopic cartilage on its posterior end. Variants in SIX1 that decrease its dosage (p.Q22X) or alter its function (p.R110W) are linked to branchio-oto-renal (BOR) syndrome, an autosomal dominant disorder characterized by sensorineural and/or conductive hearing loss, pharyngeal arch anomalies, and renal abnormalities. Although published and preliminary data suggest a requirement for SIX1 function in NC differentiation towards osteogenic and chondrogenic cell fates, there is a gap in our knowledge regarding the role of Six1 on NC cell fate decisions within the mandibular arch and the impact of BOR SIX1 variants on NC cell differentiation towards bone and cartilage. In this R03 application I am testing the hypotheses that Six1 transcriptional activity is required for NC cell fate determination within the mandibular arch (Aim 1); and that variants that decrease Six1 dosage and alter its function disrupt craniofacial bone and cartilage development leading to craniofacial dysmorphologies such as BOR (Aim 2). In Aim 1, I am identifying changes in cell populations within the mouse mandibular arch of mouse embryos from a novel knockin line carrying the p.R110W variant and comparing these changes to wild type and Six1 knockout embryos using scRNA-sequencing and spatial transcriptomics. In Aim 2, I am determining the effects of Six1 variants on osteoblast and chondroblast differentiation by using cultured NC cells and guided differentiation towards cartilage or bone followed by quantitative cytochemistry and qPCR. This application will provide novel knowledge regarding SIX1 function during NC cell fate determination within the mandibular arch. Results from this application will characterize a new mouse line (Six1pR110W) that can become an important model to study SIX1-related birth defects. These mice will constitute a critical tool in future studies whose results will provide information for patient diagnosis and care. This application aligns with a Notice of Special Interest (NOSI): Single-Cell Level Spatiotemporal Mapping of Dental and Craniofacial Embryogenesis (NOT-DE-22-003). The proposed experiments in this R03 application will represent the initial phase of my research program, aimed at uncovering the mechanisms by which SIX1 variants contribute to the BOR phenotype and how these variants interfere with interaction with different co-factors (future R01 grant).
