Investigating the Effects of Histone H3.3 Point Mutations in Neural Crest Cells and Craniofacial Development

Abstract

Craniofacial development is an intricate process that requires the meticulous and coordinated expression of many genes. Histone proteins which are fundamental to both the structural integrity and organization of DNA into nucleosomes and consequently, chromatin play an important role in coordinated gene expression. Furthermore, the modification of histone tails is now known to be an essential mechanism for coordinated expression of a large number of genes. Recently, a point mutation in the gene h3f3a which encodes the histone variant Histone H3.3, was identified in a forward genetic screen in zebrafish. H3.3A was found to be important for neural crest cell induction and generation of cranial neural crest cell derived head bones. The authors proposed that incorporation of histone H3.3 was required for expression of genes important for cranial neural crest cell specification, whereas remodelling of this histone tail may be important for later stages in neural crest cell differentiation. However, it remains to be confirmed if disrupted modification of histone tails is also important in neural crest cells. We postulate that modification of the H3.3 tail is required for coordinated expression of genes important for neural crest migration and/or differentiation. To test this hypothesis, we used CRISPR/Cas9 to generate a mouse line with a conditional missense mutation in H3f3a. The mutation generated was previously shown to result in an abnormal increase in methylation of K27 and a decrease in methylation of K36 of the histone H3.3 tail. In this poster, we will present preliminary data using the Wnt‐1 Cre2 mouse line to drive expression of this dominant negative mutation in neural crest cells. Alcian Blue Staining will be used to analyze cartilage in E14.5 embryos and alizarin red will be used to stain bones formed in heads of E17.5 embryos. Bones will be analyzed in control and mutant embryos. Our result will enable us to uncover if K27 or K36 methylation is important in neural crest cell formation, migration and/or differentiation. This work will begin to unravel the role of epigenetics in neural crest cell formation.Support or Funding Information