Molecular mechanisms of differentiation of habenular and thalamic neurons

描述(申请人提供)：缰核和丘脑在胚胎发育过程中来自两个密切相关的祖细胞区域，它们在调节成熟大脑的前脑回路方面发挥着关键作用。丘脑和缰核的不同连接以及丘脑轴突向特定皮质区域的地形图投射的发育机制在转录和轴突指导水平上仍然知之甚少。我们的长期目标是确定调节丘脑和缰核的特性和连通性的分子机制，并最终了解由丘脑和缰核的异常形成和/或功能引起的大脑疾病。这项应用的总体目标是确定在丘脑轴突最初生长及其随后导航到皮质期间对指导决策的分子控制。我们的中心假设是，Gbx2通过调节硫酸乙酰肝素的修饰和引导受体Robo1和Robo2的表达，控制丘脑轴突对其通往皮质的引导线索的内在反应性。因此，我们提出的研究将为了解硫酸乙酰肝素在调节轴突引导中的重要但知之甚少的作用提供新的见解。此外，阐明Gbx2如何控制丘脑轴突的内在反应性将加深我们对丘脑连通性建立的理解，包括丘脑皮质轴突的地形图。这一假说是基于我们的初步研究结果提出的，包括确定了几个可能介导Gbx2功能的下游靶点。在强大的初步数据的指导下，这一假说将通过追求两个具体目标来检验：1)确定硫酸乙酰肝素修饰在发育中的丘脑中的作用；2)确定调节丘脑皮质投射地形的分子机制。我们将结合体外研究(脑切片和外植体培养、分子生物学和生物化学)和小鼠遗传学(嵌合体、基因镶嵌、可诱导遗传命运定位和条件敲除)。这种方法是创新的，因为它使用了最先进的小鼠遗传学，并使用不同的荧光蛋白来识别所有丘脑轴突和Gbx2缺失的轴突，以研究丘脑皮质投射的地形图。这项拟议的研究有望显著提高我们对丘脑向皮质投射的转录调控和轴突引导信号的理解。最终，这些知识将有助于我们理解癫痫、精神分裂症、双相情感障碍和自闭症等神经疾病的发病机制或易感性。

DESCRIPTION (provided by applicant): The habenula and thalamus arise from two closely related progenitor domains during embryogenesis and they play crucial roles in modulating the forebrain circuitry in a mature brain. The developmental mechanisms underlying the distinct thalamic and habenular connectivity, and the topographic projection of thalamic axons to specific cortical areas are still poorly understood at both transcriptional and axon guidance levels. Our long-term goal is to determine the molecular mechanisms that regulate thalamic and habenular identity and connectivity, and ultimately understand the brain disorders resulting from abnormal formation and/or function of the thalamus and habenula. The overall objective of this application is to determine the molecular control of the guidance decisions during the initial outgrowth of thalamic axons and their subsequent navigation to the cortex. Our central hypothesis is that Gbx2 controls the intrinsic responsiveness of thalamic axons to guidance cues encountered in their path to the cortex by regulating the modification of heparan sulfate and the expression of guidance receptors Robo1 and Robo2. Therefore, our proposed study will provide new insight into the important but less understood role of heparan sulfate in regulating axonal guidance. Furthermore, the elucidation of how Gbx2 controls the intrinsic responsiveness of thalamic axons will enhance our understanding on the establishment of the thalamic connectivity, including the topography of thalamocortical axons. The hypothesis has been formulated on the basis of results from our preliminary studies, including identifications of several downstream targets that may mediate Gbx2 function. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Determine the role of modifications of heparan sulfate in the developing thalamus; and 2) Determine the molecular mechanism that regulates the topography of thalamocortical projections. We will combine in vitro studies (brain slice and explant culture, molecular biology, and biochemistry) and mouse genetics (chimeric, genetic mosaic, inducible genetic fate mapping and conditional knock-out). The approach is innovative, because it employs state-of-the-art mouse genetics, and utilizes different fluorescence proteins to identify all thalamic axons and those with Gbx2 deletion to study the topography of thalamocortical projections. The proposed studies are expected to significantly enhance our understanding on the transcriptional regulation and axonal guidance signaling in the establishment of thalamic projections to the cortex. Ultimately, such knowledge will contribute to our understanding on the pathogenesis or susceptibility of neurological illnesses, such as epilepsy, schizophrenia, bipolar disorder, and autism.