Regulation of Cranial Mesenchyme Expansion Driving Neural Fold Elevation

项目概要/摘要 神经管缺陷（NTD）是人类最常见的结构性出生缺陷之一，并导致长期的 长期残疾甚至死亡;然而，潜在的遗传原因在很大程度上仍然未知。弥补这一差距， 在理解调节正常和 异常发育近40年前进行的实验表明， 间充质是位于神经板下的细胞群，是颅面隆起所必需的。 神经褶皱和神经管闭合。然而，人们对颅间充质如何扩张知之甚少 驱动神经折叠升高，以及这一过程如何被破坏导致NTD。此外，很少有基因 参与了这个过程。 在这个建议中，我们提出了一种新的NTDs小鼠模型，Hectd 1基因突变，并描述了 这些方法将大大促进我们对颅间充质扩张如何能够 导致NTD的原因基于我们先前和初步的数据，我们假设eHSP 90 从Hectd 1突变体NC-CM分泌的刺激增加的CM运动干扰扩增 的PM-CM和破坏神经折叠的提升（具体目标1和2）。我们进一步假设， 与人类NTD相关的HECTD 1序列变体采用相同的致病机制 （具体目标3）。我们将使用一系列创新工具来测试这些假设，包括：（1）高级 成像方法以前所未有的分辨率在真实的时间内可视化颅间充质的扩张 在神经褶升高过程中，（2）荧光标记的eHSP 90探针，以阐明空间和时间模式 致病性eHSP 90的产生，（3）离体颅间充质外植体测定适合实验 操作，（4）一种新的等位基因系列的小鼠系和（5）药理学抑制剂，以测试是否eHSP 90 介导Hectd 1突变胚胎中颅间充质扩张和神经折叠升高的失败。这些 创新的工具将与成熟的条件遗传学、组织学和胚胎学相结合， 以前所未有的细节描绘正常神经褶中颅间充质扩张的方法 升高以及在导致NTD的颅间充质扩张失败期间， Hectd 1突变胚胎。这些信息将用于确定预测的致病性 在人类NTD病例中鉴定的HECTD 1序列变体，并确定变体是否破坏HECTD 1功能 并导致人类患者的NTD。

Project Summary/Abstract Neural tube defects (NTDs) are among the most common structural birth defects in humans and result in long- term disability or even death; yet, the underlying genetic causes remain largely unknown. Addressing this gap in knowledge is best achieved in the context of understanding the mechanisms mediating both normal and abnormal development. Experiments conducted nearly 40 years ago indicate that expansion of the cranial mesenchyme, a cell population that resides beneath the neural plate, is required for elevation of the cranial neural folds and neural tube closure. Yet, little is known regarding how expansion of the cranial mesenchyme drives neural fold elevation and how this process can be disrupted to cause NTDs. Moreover, few genes have been implicated in this process. In this proposal we present a novel mouse model of NTDs with a mutation in the Hectd1 gene and describe approaches that will significantly advance our understanding of how cranial mesenchyme expansion can be disrupted contributing to NTDs. Based on our previous and preliminary data we hypothesize that eHSP90 secreted from Hectd1 mutant NC-CM stimulates increase movement of the CM interfering with expansion of the PM-CM and disrupting neural fold elevation (Specific Aims 1 & 2). We further hypothesize that HECTD1 sequence variants associated with human NTDs employ the same pathogenic mechanism (Specific Aim 3). We will test these hypotheses using a combination of innovative tools including: (1) advanced imaging approaches to visualize, at unprecedented resolution expansion of the cranial mesenchyme in real time during neural fold elevation, (2) fluorescently labeled eHSP90 probes to elucidate spatial and temporal patterns of pathogenic eHSP90 production, (3) an ex vivo cranial mesenchyme explant assay amenable to experimental manipulation, (4) a novel allelic series of mouse lines and (5) pharmacological inhibitors to test whether eHSP90 mediates failure of cranial mesenchyme expansion and neural fold elevation in Hectd1 mutant embryos. These innovative tools will be combined with well-established conditional genetic, histological and embryological approaches to delineate, in unprecedented detail, expansion of cranial mesenchyme in normal neural fold elevation and how this process goes awry during failed cranial mesenchyme expansion responsible for NTDs in the Hectd1 mutant embryo. This information will be used to determine the impact of predicted pathogenic sequence variants of HECTD1 identified in human NTD cases and ascertain if variants disrupt HECTD1 function and contribute to NTDs in human patients.