Understanding the role of Hdacs in zebrafish craniofacial development

One third of all birth defects are associated with craniofacial malformation including lack of suture closure, cleft palate, or failure of skeletal formation. Progress in understanding the origin of these diseases depends on understanding the cellular and genetic mechanisms that produce normal craniofacial patterning. The zebrafish

is an excellent model system for studying the process of craniofacial development, as it is possible to observe all stages of jaw formation, from development of cranial neural crest (CNC) cells, which migrate into the pharyngeal arches and differentiate into skeletal cells, all the way through generation of bone and cartilage in a

few days. The ability to generate genetic mutations and transgenic lines in zebrafish allows for precise dissection of molecular mechanisms involved in craniofacial patterning. Histone deacetylases (Hdacs) are enzymes that function to regulate transcription during development. Human disorders and animal models

demonstrate that Hdacs are involved in the development of the CNC-derived skeleton. For example, Cornelia de Lange syndrome, cleft palate disease, and Fetal Alcohol Syndromes all involve defects to CNC cells or derivatives, and are associated with abnormal Hdac function. The long-term objective of this study is to reveal

how individual Hdacs interact with known and novel gene pathways to regulate specification, migration, differentiation, and activity of CNC cells and their derivatives to pattern the craniofacial skeleton. The proposed studies will focus on investigating the function of Hdac1 and protein complexes involving Hdac1 in zebrafish,

using data generated using RNA-Seq and ATAC-Seq and mutant lines with unknown craniofacial phenotypes (i.e. phf21aa;ab, zmym2) to test the hypotheses that Hdac1 patterns post-migratory CNC cells through regulation of patterning genes and growth of cartilage and bone. Using a chemical inhibitor screen, this study

will further elucidate the potential roles of other Hdacs (i.e. Hdac3, 6, 8) in development of the jaw skeleton in the zebrafish model. Overall, the approach will include 1) investigation of genes and chromatin modifications associated with patterning and skeletal development in hdac1 mutants 2) Analysis of the roles of factors in the

chromatin modifying Lsd1-CoREST-Hdac1 and Braf/Hdac complexes in skeletal development, 3) A reductive strategy to test specific Hdac inhibitors applied to embryos (e.g. Romidepsin, TMP-195) in which the role of particular classes or individual Hdacs on craniofacial development will be established, leading future reverse

genetics projects to generate novel Hdac mutants using CRISPR-Cas9. Research will involve undergraduates, with a goal of exposing students to research design and to train them in critical thinking, molecular techniques, data analysis, and communication skills.