38 - Effects of Early, Acute Prenatal Alcohol Exposure on Cranial Neural Crest Cell Differentiation in the Developing Mouse Skull

Poster Board Number: 38
There are separate poster presentation times for odd and even posters.
Odd poster #s – first hour
Even poster #s – second hour

Co-authors:

Amy Pietrantonio - Graduate Student, Anatomy and Cell Biology, University of Western Ontario; Diana Quinonez - Research Technician, Physiology and Pharmacology, University of Western Ontario; Kevin Barr - Anatomy and Cell Biology - University of Western Ontario; Katherine Willmore - Associate Professor, Anatomy and Cell Biology, University of Western Ontario

Abstract Body : Introduction and Objective: Prenatal alcohol exposure (PAE) contributes to a spectrum of developmental defects classified as fetal alcohol spectrum disorder (FASD). Common diagnostic traits of FASD involve a constellation of craniofacial features, including midface malformations, delayed palatal development, and abnormalities of the eyes and orbits. These craniofacial features are concentrated in the facial skeleton and anterior cranial vault, which are derived from cranial neural crest cells (cNCCs). While there has been much work to assess how PAE affects early cNCC processes such as induction and migration, less is known about how PAE affects how cNCCs differentiate into the bones and cartilages of the skull. Thus, we have employed an oral gavage dosing regime that represents PAE at late gastrulation to understand the impacts of PAE on cNCC differentiation.

Methods: Nulliparous Wnt1-Cre2;mTmG dams (2 – 4 months) were dosed with 2 mL/100 g body weight of either 31.5% v/v ethanol (PAE) or water (vehicle control, VC) via oral gavage at embryonic day (E) 7.5. Skulls from Wnt1-Cre2;mTmG neonates and embryos at E13.5 and E15.5 will be collected for either stain-enhanced µCT (N = 10) or immunofluorescence (IF, N = 3). Skulls collected for stain-enhanced µCT will undergo geometric morphometric analyses to quantify size and shape differences in bone and cartilage. Skulls collected for IF will be stained for the osteoblast differentiation marker Osx and the chondrocyte differentiation marker Sox9 to qualitatively localize differences in cNCC differentiation between groups.

Expected Outcomes and Future Directions: Based on previous research with PAE models, we expect that neural crest-derived bones and cartilages of the facial skeleton and anterior cranial vault of PAE mice will have larger shape differences than the mesoderm-derived bones of the posterior vault and cranial base. In line with expected shape outcomes, we expect that expression of Osx and Sox9 will be different in PAE mice compared to VC mice based on IF data. We are in the process of optimizing cNCC isolation from PAE and VC embryos for single cell RNA sequencing to identify novel targets of ethanol on cNCC differentiation and to compare developmental trajectories of cNCCs between PAE and VC embryos throughout mid-embryogenesis.

Conclusion: This represents the first study of an early, acute PAE regimen on cNCC differentiation and the impacts on cNCC derivatives throughout early ontogeny and will lay the groundwork for in-depth investigations of PAE’s effect on cNCC differentiation.