Regulation of Cranial Mesenchyme Expansion Driving Neural Fold Elevation

项目摘要/摘要 神经管缺陷(NTDS)是人类最常见的结构性出生缺陷之一，并导致长时间的 致残率甚至死亡；然而，潜在的遗传原因在很大程度上仍不清楚。解决这一差距 在理解调节正常和非正常状态的机制的背景下获得知识是最好的 发育不正常。近40年前进行的实验表明，颅骨的扩张 间充质是一种位于神经板下方的细胞群，是抬高颅骨所必需的。 神经皱褶和神经管闭合。然而，关于颅骨间充质的扩张情况还知之甚少。 推动神经皱褶抬高，以及这一过程如何被扰乱以导致NTDS。此外，很少有基因具有 与这一过程有牵连。 在这项建议中，我们提出了一种新的带有Hectd1基因突变的NTDS小鼠模型，并描述了 这些方法将显著提高我们对颅骨间充质扩张是如何 中断了对NTDS的贡献。根据我们之前和初步的数据，我们假设eHSP90 Hectd1突变体分泌的NC-CM刺激CM运动增加干扰扩张 PM-CM和扰乱神经折叠抬高(具体目标1和2)。我们进一步假设 与人类NTDS相关的HECTD1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验证这些假设，这些工具包括：(1)高级 以前所未有的分辨率实时显示颅脑间充质扩张的成像方法 在神经折叠提升过程中，(2)荧光标记的eHSP90探针以阐明空间和时间模式 致病性eHSP90的产生；(3)符合实验条件的体外颅脑间充质细胞外植体实验 操作，(4)一系列新的等位基因小鼠和(5)药物抑制剂，以测试eHSP90 在Hectd1突变胚胎中介导颅骨间充质扩张和神经皱折隆起失败。这些 创新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皱折中的颅脑间充质扩张的方法 在导致NTDS的失败的颅间充质扩张过程中，这一过程是如何出错的 Hectd1突变胚胎。这些信息将被用来确定预测的致病菌的影响 在人类NTD病例中发现HECTD1的序列变异并确定变异是否破坏HECTD1的功能 并导致人类患者的NTDS。

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.