Thursday, December 26, 2024
12/26/2024
PROJECT SUMMARY While the cellular movements and shape changes that drive morphogenesis within the neural plate are well characterized, how morphogenesis of different cranial mesenchyme (CM) lineages contributes to neural fold elevation and how disruption of these mechanisms results in neural tube defects remains an open question. This application is based on the hypothesis that aberrant eHSP90 secretion and changes in the CM matrisome result in abnormal CM behavior, disrupting CM expansion in the Hectd1 mutant embryo. To model abnormal CM morphogenesis, a Hectd1 mouse mutant that fails neural tube closure will be used to determine both lineage relationship and gene expression changes that occur with CM morphogenesis leading to the neural tube defect. Aim 1 will assess how the neural crest (NC-CM) and paraxial mesoderm derived CM (PM-CM) interact in the Hectd1 mutant embryo leading to abnormal CM morphogenesis and failure of neural fold elevation. Both CM lineages are implicated as critical mediators of neural fold elevation. Analysis using SiMView adaptive light sheet microscope during normal CM expansion demonstrated distinct movements of the PM-CM and the NC- CM. Secondly, preliminary analysis of CM cells indicates that NC-CM cells are the primary migratory cells. Hectd1 is required in the NC-CM for neural fold elevation, but the PM-CM also fails to expand in the Hectd1 mutant. Therefore, lineage tracing and conditional genetic models of Hectd1 will be used to test the contribution of the NC-CM and PM-CM to abnormal morphogenesis in the Hectd1 mutant line using live imaging of in vitro CM explant assays. Lastly, increased extracellular HSP90 (eHSP90) secretion is observed in NC-CM cells of our mutant mouse. This potential mechanism for their abnormal interaction leading to the neural tube defect will be tested with additional live imaging of CM explants with the addition of eHSP90. Aim 2 will assess the changes in matrisome gene expression of the CM during neural fold elevation. The CM cells are embedded in an extracellular matrix (ECM), which undergoes expansion during neural fold elevation. The ECM is critical to morphogenesis in many systems. Embryonic tissues from key timepoints will be analyzed using bulk RNA sequencing to identify extracellular matrix genes implicated in CM morphogenesis in neural fold elevation. Bioinformatics analyses will identify differentially expressed genes, and in situ hybridization experiments will map critical differential gene expression across different time points. Successful completion of these studies will transform our understanding of the mechanisms leading to neural tube defects and address a crucial gap in our understanding of neural tube formation.
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