Characterization of the roles and regulation of Draxin in cranial neural crest

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PROJECT SUMMARY/ABSTRACT The neural crest (NC) is a stem cell population that originates within the forming central nervous system. NC cells delaminate from the neuroepithelium by undergoing a spatiotemporally regulated epithelial— mesenchymal transition (EMT) to exit from the neural tube. Cranial NC cells, which arise in the head region of the embryo and are the only NC population in vivo with the ability to differentiate into craniofacial skeleton and cartilage, are indispensable for the development of the face; mutations affecting NC development result in numerous diseases and malformations affecting the craniofacial structures. The focus of my postdoctoral work has been to study the mechanisms that control and facilitate cranial NC EMT. During the first phase of my postdoctoral training, I have shown that this developmental EMT program is controlled by temporally restricted expression of the Wnt antagonist, Draxin. A hallmark of Draxin's function during EMT is its transient expression and rapid downregulation; perdurance of Draxin has deleterious effects on cranial NC EMT through dysregulation of downstream targets of canonical Wnt signaling. Through the support of the K99, I discovered that the transience of Draxin expression in cranial NC is mediated post-transcriptionally via its 3'-untranslated region (UTR). Importantly, Draxin is stabilized by the RNA-binding protein Elavl1/HuR at the premigratory stage, then targeted to cytoplasmic processing bodies (P-bodies) for decay to drive proper cranial NC EMT. Collectively, these discoveries begin to unravel a new mechanism whereby cranial NC EMT is regulated through post-transcriptional regulatory mechanisms balancing stability and decay of a molecular rheostat, Draxin. COVID-19 research restrictions and university closures severely delayed my career plans and development. Through the support of the K99, I completed many of the goals proposed in Aims 1 and 3 of my original proposal, which sought to illuminate the interaction between Draxin and Wnt signaling, and the regulation Draxin expression, respectively. However, COVID-19 research restrictions severely delayed the completion of Aim 1 and progress of Aim 2, which sought to apply time-lapse and advanced microscopy techniques (e.g. FRET) to more fully explore Draxin function. Further, completion of Aim 3 and publication of these studies requires additional experiments in single-molecule imaging and RIP-seq. A funding extension would allow me to develop critical new skills in advanced microscopy and RIP-seq to gain a mechanistic understanding of Draxin activity during cranial NC EMT, and allow me to comple the revision experiments necessary to publish the work performed under the parent K99 award to help me secure a tenure-track faculty position, establish a vibrant independent research program in the developmental signaling field, and better equip me with the knowledge necessary to transition into the study of cranial NC development and migration.