Morphological and Histological Examinations of the Effects of a Neural Crest-specific Connexin43 Knockout in the Neonatal Mouse Skull

Abstract Body : Introduction and Objective: Connexin43 (Cx43) is a gap junctional protein that is expressed in the constituent cells of bone and in neural crest cells. Loss of Cx43 expression or function has been linked to delayed ossification and aberrant bone remodeling in both germline and conditional mouse models. Furthermore, knockdown of Cx43 in other vertebrates delays neural crest cell migration. However, how loss of Cx43 affects neural crest cell differentiation into osteogenic and chondrogenic fates remains unknown. Thus, we have developed a neural crest-specific Cx43 knockout mouse model to investigate Cx43’s role in neural crest differentiation.
Materials and Methods: Skulls from Wnt1-Cre2;mTmG;Cx43+/+ (Cx43WT) and Wnt1-Cre2;mTmG;Cx43fl/fl (Cx43cKO) mice were collected at post-natal day 0 (P0) for either µCT (N = 8 and 9, respectively) or immunofluorescence (N = 5). Homologous landmark data were collected from µCT images and analyzed with geometric morphometric analyses to quantify and localize size and shape changes to neural crest- and mesoderm-derived regions of the skull.
Chondrocyte and osteoblast differentiation markers (Sox9 and Osx, respectively) were localized and semi-quantitatively analyzed between genotypes. Three regions of interest were assessed: the neural crest-derived nasal cavity, neural crest-derived orbit, and the mesoderm-derived posterior vault. The number of stained cells per mm2 of bone or cartilage was calculated for each area and compared between genotypes.
Results: Skull shape was significantly different between Cx43cKO and Cx43WT neonates (p = 0.0007) and 43% of variation in skull shape appeared to be driven by changes in ossification. When the landmarks are separated into neural crest and mesoderm origins, both areas of the skull see significant shape differences in Cx43cKO neonates (p = 0.0009 and p = 0.0082, respectively). While there was no significant difference in skull size between genotypes (p = 0.3871), there was a trend of Cx43cKO to have smaller skulls than Cx43WT, and when broken down into embryological origins, this effect appears to be driven by neural crest derivatives (p = 0.0778).
Despite skull shape changes, there were no observed differences in the number of Sox9+ chondrocytes or Osx+ osteoblasts per mm2 in all of our regions of interest between genotypes.
Conclusion: Loss of Cx43 in neural crest changes skull shape at P0, but not skull size or expression of osteoblast and chondrocyte differentiation markers.
Significance/Implication: This work shows a phenotypic effect of a neural crest-specific Cx43 knockout on skull morphology at P0 which was primarily linked to differences in ossification. This aligns with phenotypic findings from germline Cx43 mutant and knockout mice; however, germline mutant mice have smaller skulls than wildtype controls at P0, which was not reflected in the Cx43cKO mice. Differences in the shape of mesoderm-derived portions of the skull between Cx43cKO and Cx43WT neonates was unexpected given that these regions express Cx43 in Cx43cKO mice. This may be due to Cx43’s role as a gap junctional protein which passes signals between nearby cells and the proximity of neural crest and mesoderm-derived bones in the cranial vault. We aim to look at chondrocyte and osteoblast differentiation in early embryonic timepoints to identify if and when alterations to neural crest cell differentiation give rise to our observed skull shape differences.
Funding Sources: NSERC CGS-D (ACM), NSERC R5211AO2 (KEW)