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UBC Theses and Dissertations

Tracing the cellular origins of mesenchymal derivatives in craniofacial development Martin, Kathleen Anne


Objectives: Numerous genes have been shown to impact craniofacial development. One such gene, Hypermethylated in cancer 1 (Hic1), is a tumour suppressor gene that encodes for the transcription factor HIC1. Hic1 is broadly expressed within embryonic mesenchyme. Hic1 knockout mice demonstrate multiple craniofacial anomalies, including facial clefting, abnormal closure/truncation of the secondary palate, low-set/underdeveloped ears, abnormal eye development, shortened snouts, acrania, and exencephaly, suggesting a role for Hic1 in craniofacial development. Molecular mechanisms underlying the role of Hic1 in craniofacial development remain largely unknown. Our objective was to elucidate the cellular fate of Hic1⁺ cells during craniofacial development. Methods: The fate of Hic1-expressing cells in craniofacial development was studied using lineage tracing analysis. A Hic1CreERT² mouse, in combination with a Cre-dependent reporter mouse, was used to label and follow Hic1-expressing cells. Reporter gene expression was induced at the onset of Hic1 expression (embryonic age (E) of 10 days). Embryos were collected at E11.5, E13.5, E14.5, E16.5 and E18.5. The distribution and fate of reporter-expressing cells was determined using epi-fluorescence imaging. Micromass cultures of cells derived from the mandibular, frontonasal and maxillary prominences at E11.5 were used to study Hic1-expressing cells in cartilage formation. Samples (n=3) were fixed and stained with Alcian blue or COL2A1 to study the relationship of Hic1⁺ cells to cartilaginous nodules. Single-cell RNA sequencing was performed on cells positive and negative for Hic1-expression in the head. Results: Hic1+ cells contribute to the mesenchyme within facial/masticatory muscles, tendon, tongue, meninges, nasal cartilage/ connective tissue, eyes, salivary gland stroma, teeth (dental papilla/pulp), and to populations of perineural and perivascular cells, including those within the brain. Hic1 is not seen within the palatal shelves, or Meckel’s cartilage. In micromass cultures of facial mesenchyme, most Hic1-expressing cells contribute to the area surrounding cartilaginous nodules, while few were incorporated within the nodules, mimicking the pattern of distribution of Hic1-expressing cells in the nasal structures. Single-cell RNA sequencing suggests Hic1-expressing cells are neural crest-like, and represent mesenchymal progenitors. Conclusions: Hic1-expressing cells contribute to various mesenchymal tissues during craniofacial development, and likely represent a population of cranial neural crest-derived cells or similar.

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