The Role of Connexin-43 in Cranial Neural Crest Cell Differentiation

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.L7700.

ABSTRACT

BACKGROUND: Most features of the craniofacial complex that are necessary for feeding derive from cranial neural crest cells (cNCCs). These pluripotent stem cells originate from the dorsal neural folds and undergo a series of coordinated processes such as induction, epithelial-to-mesenchymal transition (EMT), migration, and differentiation. While most of these processes have been well-studied for their roles in development, much remains to be known about the final step cNCCs must undergo, differentiation. Connexin-43 (Cx43) is a gap junctional protein that is widely expressed, evolutionarily conserved, and has been well-studied for its impacts on neural crest and bone. Previous research has shown that loss of Cx43 function impacts neural crest EMT and migration and can delay early osteoblast and chondrocyte differentiation; however, it is unknown how Cx43 deficiency in cNCCs impacts cNCC differentiation into osteochondrogenic lineages and the resultant impacts on craniofacial morphology. Therefore, the purpose of this project is to determine the effects of a neural crest-specific loss of Cx43 on osteogenic and chondrogenic differentiation, and how these effects may alter skull phenotype.

METHODS: We have developed a mouse model wherein the gene encoding for Cx43 has been conditionally knocked out in neural crest cells and their derivatives using the Wnt1-Cre2 driver (Wnt1-Cre2^+/- ;mTmG^+/+ ;Cx43^fl/fl , herein Cx43cKO). To compare the developing skull phenotype between Cx43cKO mice and littermates in which the Cx43 gene is not floxed (Wnt1-Cre2^+/- ; mTmG^+/+ , herein Cx43WT), we will collect homologous landmark data from microCT images of newborn skulls and use geometric morphometric analyses to quantify and visualize shape differences in neural crest- and mesoderm-derived portions of the skull between genotypes. To determine the localized effects of a neural-crest specific loss of Cx43 function on osteoblast and chondrocyte differentiation, we will use immunofluorescence staining. Expression of chondrocyte, osteoblast, and osteocyte differentiation markers will be localized and semi-quantitatively compared between genotypes and between neural crest- and mesoderm-derived cells. With these assays, we aim to understand the relationship between Cx43, cNCC differentiation, and the subsequent impacts on skull morphology.

PRELIMINARY RESULTS: Cx43cKO neonates show decreased ossification compared to Cx43WT littermates, which is most prominent in facial bones such as the nasal bones, and cranial vault bones such as the frontal and parietal bones. Potential driving forces for this decreased ossification in neural crest- and mesoderm-derived bones are delayed differentiation of the osteoblast-osteocyte lineage and disrupted signaling between cells in neighbouring bones.

SIGNIFICANCE & FUTURE DIRECTIONS: This study will help elucidate the role of cNCC differentiation in skull development, and how loss of Cx43 function impacts skull development. We will add embryonic and post-natal timepoints to further understand the impacts of neural crest-specific deletion of Cx43 throughout development; pilot data with Cx43cKO adult mice show that alterations to skull shape persist and overtly affect the face and mandible.

PMID:35555906 | DOI:10.1096/fasebj.2022.36.S1.L7700