Mechanisms of Abnormal Cranial Mesenchyme Morphogenesis in the Hectd1 mutant

项目总结 而推动神经板内形态发生的细胞运动和形状变化是很好的 不同颅脑间充质(CM)谱系的形态发生对神经皱折的贡献 提升以及这些机制的破坏如何导致神经管缺陷仍然是一个悬而未决的问题。 这一应用是基于一种假设，即异常的eHSP90分泌和CM中的变化 Matrisome导致CM行为异常，扰乱了Hectd1突变胚胎中CM的扩张。至 建立CM形态发生异常模型，神经管关闭失败的Hectd1小鼠突变体将用于 确定与CM形态发生相关的谱系关系和基因表达变化 神经管缺陷。 目标1将评估神经脊(NC-CM)和旁中胚层衍生CM(PM-CM)在 Hectd1突变胚胎导致CM形态发生异常和神经皱褶抬高失败。两者都有 CM谱系被认为是神经皱折升高的关键介质。使用SiMView自适应光源进行分析 在正常的CM扩张过程中，薄片显微镜显示PM-CM和NC-CM的明显运动。 厘米。其次，对CM细胞的初步分析表明，NC-CM细胞是主要的迁移细胞。 Hectd1需要在NC-CM中用于神经折叠提升，但PM-CM也无法在Hectd1中扩展 变种人。因此，将使用Hectd1的谱系追踪和条件遗传模型来测试 利用LIVE技术检测NC-CM和PM-CM在Hectd1突变系形态发生异常中的作用 体外CM外植体分析的成像。最后，观察到细胞外HSP90(EHSP90)分泌增加。 在我们的突变小鼠的NC-CM细胞中。这一导致它们异常相互作用的潜在机制 神经管缺陷将通过添加eHSP90的CM外植体的额外实时成像进行测试。 目的2研究神经折叠区抬高过程中CM的基底膜基因表达的变化。中央情报局 细胞被嵌入到细胞外基质(ECM)中，在神经折叠抬高时，ECM经历扩张。 在许多系统中，ECM对形态发生至关重要。关键时间点的胚胎组织将是 利用批量RNA测序分析与CM形态发生有关的细胞外基质基因 神经皱褶抬高。生物信息学分析将识别差异表达的基因，并在原位 杂交实验将绘制不同时间点的关键差异基因表达图谱。成功 这些研究的完成将改变我们对导致神经管缺陷的机制的理解 并解决了我们对神经管形成的理解中的一个关键差距。

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.