Mechanisms of Complex Tissue Regeneration and the Functional Outcomes of Craniofacial Dysmorphology
Louie, Ke'ale
2020
Abstract
There is a clinical need to restore craniofacial tissues affected by disorders of traumatic and/or congenital etiology. Effective therapies within this specialized region must achieve anatomic regeneration while also restoring function. The work in this dissertation attempts to identify mechanisms underlying the former while simultaneously elucidating the latter using mastication as a readout of craniofacial tissue interactions. The global hypothesis is that perturbation of gene expression during adult craniofacial tissue regeneration has functional outcomes that extend beyond the immediately dysmorphic structures. It was additionally hypothesized that: (1) genes differentially expressed at early time points play unique roles during cellular reprogramming and adult muscle regeneration and (2) fundamental changes in dental and craniofacial morphology decrease chewing efficiency. Zebrafish possess robust regenerative capabilities as demonstrated by the rapid (i.e., 7-10 day) anatomic and functional regeneration of individual craniofacial muscles following >50% surgical resection. Analysis of differentially expressed genes (DEG) at two time points (i.e., 9 and 18 hours post injury(hpi)) within actively regenerating craniofacial muscle identified over 6000 candidates that could play a role during craniofacial muscle regeneration. Subsequent clustering of these DEG by temporal expression patterns then revealed subsets of genes which were transiently upregulated at only the 9hpi (early) or 18hpi (late) time points. Among the early expressed genes were known epigenetic regulators including ezh2, prmt5, and prmt7. In vivo knockdown experiments revealed that these genes were necessary for both cellular reprogramming and anatomic regeneration. This differed from late expressed genes such as fn1a which was necessary for anatomic regeneration but not cellular reprogramming. Also among the early expressed genes were twist transcription factors, elements known to affect craniofacial muscle development and epithelial to mesenchymal transitions during tumorigenesis. Of the zebrafish twist paralogs, only twist3 was necessary for cellular reprogramming and anatomic regeneration in adult zebrafish while twist1a, twist1b, and twist2 only affected embryonic muscle development. Ellis-van Creveld syndrome (EVC) is a rare genetic condition that affects the craniofacial region. However, the etiology of cephalometric differences and the functional outcomes of dental and craniofacial dysmorphology are poorly described in the literature. Use of an animal model for EVC in which a causative gene ortholog (i.e., Evc2) was conditionally knocked out (cKO) in neural crest cells revealed that changes in facial profile advanced with age and were secondary effects of a shortened anterior skull base. Further application of our Evc2-cKO model revealed that mastication parameters such as bite force and chewing rate were reduced as were molar surface area and complexity. No changes in chewing effectiveness suggested that behaviors such as longer feeding bouts may effectively compensate for anatomic deficits in the craniofacial region. Investigation into the temporal regulation of adult tissue regeneration and gene subfunctionalization has led to the identification of multiple pathways that represent potentially druggable targets for future regenerative therapies. Assessment and characterization of the functional outcomes in states of congenital craniofacial dysmorphology provide insight into form-function relationships and represent potential metrics by which successful rehabilitation may be measured. However, future studies must further elucidate the additional mechanisms (e.g., histone modification) underlying tissue regeneration as well as assess adaptation by evaluating chewing function at additional developmental and/or post-acute injury time points.Deep Blue DOI
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craniofacial development Ellis-van Creveld syndrome mastication regeneration
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