Mechanisms of Abnormal Cranial Mesenchyme Morphogenesis in the Hectd1 mutant

Hectd1 突变体异常颅间充质形态发生的机制

• 批准号: 10686499
• 负责人: Claire Marie Moran
• 金额: $ 4.44万
• 依托单位: CHILDREN'S RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-15 至 2025-03-14
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10686499
• 关键词: Address; Anencephaly; Anterior; Behavior; Bioinformatics; Biological Assay; Cell Lineage; Cell Surface Receptors; Cells; Cellular biology; Cephalic; Cessation of life; Child; Client; Complex; Core Facility; Data; Defect; Developmental Biology; Doctor of Philosophy; Dorsal; Dyskinetic syndrome; Embryo; Embryonic Development; Exencephalies; Experimental Genetics; Extracellular Matrix; Failure; Fibronectins; Foundations; Gene Expression; Genes; Genetic Models; Genetic Transcription; Genomics; Heat-Shock Proteins 90; Image; Image Analysis; In Situ; In Situ Hybridization; In Vitro; Laboratories; Learning; Light; Maps; Measures; Mediator; Mentors; Mentorship; Mesenchyme; Metalloproteases; Methods; Microscope; Modeling; Molecular; Molecular Chaperones; Morphogenesis; Movement; Mutant Strains Mice; Mutation; Neural Crest; Neural Fold; Neural Tube Closure; Neural Tube Defects; Neural tube; Paraxial Mesoderm; Play; Process; Proteins; Publishing; Research Institute; Research Training; Role; Scientist; Shapes; Spinal Dysraphism; Structural Congenital Anomalies; System; Testing; Time; Tissue Expansion; Tissue-Specific Gene Expression; Training; Ubiquitination; Validation; Visualization; Work; career; career development; cell behavior; cell motility; cellular imaging; differential expression; disability; embryo tissue; experimental study; extracellular; innovation; malformation; migration; mouse genetics; mutant; mutant mouse model; neural plate; novel; transcriptome sequencing; ubiquitin ligase

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.

项目摘要 虽然细胞运动和形状变化驱动神经板中形态发生的变化很好 表征，不同颅间质（CM）谱系的形态发生如何有助于神经褶皱 高程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。 该应用基于以下假设：异常EHSP90分泌和CM的变化 女生成体导致CM行为异常，破坏了Hectd1突变体胚胎中CM的扩张。到 模型异常CM形态发生，一种失败神经管闭合的Hectd1小鼠突变体将用于 确定谱系关系和基因表达变化，而CM形态发生前导 到神经管缺陷。 AIM 1将评估神经波峰（NC-CM）和近期中胚层衍生的CM（PM-CM）如何相互作用 HECTD1突变胚导致CM形态发生异常和神经折叠升高的失败。两个都 CM谱系被认为是神经折叠抬高的关键介体。使用SimView自适应光分析 正常CM膨胀期间的薄板显微镜显示了PM-CM和NC-的不同运动 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。 NC-CM中需要HATD1才能进行神经折叠的高度，但是PM-CM也未能在Hectd1中扩展 突变体。因此，HECTD1的谱系追踪和条件遗传模型将用于测试 NC-CM和PM-CM对使用Live的Hectd1突变线在HECTD1突变线中的异常形态发生的贡献 体外CM外植体分析的成像。最后，观察到增加的细胞外HSP90（EHSP90）分泌 在我们突变小鼠的NC-CM细胞中。这种可能异常相互作用的潜在机制导致 通过添加EHSP90，将通过CM外植体的其他实时成像对神经管缺陷进行测试。 AIM 2将评估神经褶皱升高期间CM的源组基因表达的变化。 CM 细胞嵌入细胞外基质（ECM）中，该基质在神经褶皱升高期间经历膨胀。 ECM对于许多系统中的形态发生至关重要。关键时间点的胚胎组织将是 使用大量RNA测序分析以鉴定与CM形态发生有关的细胞外基质基因 神经褶皱高程。生物信息学分析将确定差异表达的基因，原位 杂交实验将在不同时间点绘制临界差异基因表达。成功的 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时的关键差距。