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

项目摘要/摘要 神经脊是起源于正在形成的中枢神经的干细胞群。 系统。NC细胞通过经历时空调节的上皮细胞从神经上皮剥离- 间充质转化(EMT)以退出神经管。颅骨NC细胞，长于颅骨头区 胚胎和是体内唯一具有分化为头面部骨骼和 软骨，是面部发育不可缺少的；突变影响NC发育导致 影响头面部结构的多种疾病和畸形。我博士后工作的重点 一直在研究控制和促进颅脑NC EMT的机制。在我的第一阶段 博士后培训，我已经证明了这个发展中的EMT项目是由时间限制控制的 WNT拮抗剂德拉新的表达。德拉欣在EMT中作用的一个特点是它的瞬时表达 和快速降调；德拉欣的耐受通过对颅脑NC EMT的有害影响 规范的Wnt信号下游靶标的失调。通过K99的支持，我发现 DRAXIN在颅脑NC中的瞬时表达是通过其3‘-未翻译的转录后介导的 区域(Utr)。重要的是，Draxin在迁徙前被RNA结合蛋白Eavl1/Hur稳定 阶段，然后靶向胞质处理体(P体)进行衰变，以驱动适当的颅骨NC EMT。 总而言之，这些发现开始解开一种新的机制，通过这种机制，颅脑NC EMT受到调控 通过转录后调节机制平衡分子变阻器的稳定性和衰变， 德拉新。新冠肺炎研究的限制和大学的关闭严重推迟了我的职业计划 发展。在K99的支持下，我完成了我的目标1和3中提出的许多目标 最初的建议，试图说明Drasin和Wnt信号之间的相互作用，以及 分别调控Draxin的表达。然而，新冠肺炎的研究限制严重推迟了 目标1的完成和目标2的进展，目标2寻求应用延时和先进显微镜 技术(例如，FRET)，以更全面地探索Drasin函数。此外，完成目标3并出版 这些研究需要在单分子成像和RIP-SEQ方面进行额外的实验。资金延期 将使我在高级显微镜和RIP-SEQ方面发展关键的新技能，以获得机械 了解颅脑NC EMT中的Draxin活动，并允许我补充修改实验 有必要发表在家长K99奖下完成的工作，以帮助我获得终身教职 定位，在发育信号领域建立充满活力的独立研究计划，并更好地 给我必要的知识，让我过渡到研究颅脑NC的发展和移植。

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.