Transcriptional control of sonic hedgehog signaling

描述（由申请人提供）：Shh表达的时间和空间控制是确定沿神经管背腹轴离散位置的神经元身份的关键。在Shh功能缺失的情况下，腹侧中线发育受到干扰，导致前脑畸形（HPE），这是一种结构性脑畸形，以及沿前后神经轴长度的神经元模式和寻径缺陷。由于Shh-/-小鼠胚胎中缺陷的严重性，Shh在前脑发育过程中的全部功能尚未实现。在之前的资助期内，我们发现了6个CNS特异性增强子，分布超过500kb，其组合活性覆盖了小鼠神经管中Shh转录的大部分位点，包括腹前脑。我们使用这些Shh调控元件作为工具来进一步剖析Shh在腹侧前脑中的功能。尾侧间脑基底板缺失Shh （ShhDSBE1/-）的小鼠在出生后第一周内发育不良并死亡。初步数据表明，这种表型归因于新定义的吻侧丘脑祖细胞（pTH-R）的错误描述。间脑吻侧缺失Shh （Shh loxp/-; SBE2cre）的小鼠表现出下丘脑、垂体和视神经缺陷，与先天性脑畸形综合征中隔-视神经发育不良（SOD）一致。这一发现表明，来自下丘脑的Shh信号减少可能是SOD发病机制的基础，并且与前脑畸形（HPE）相比，代表了Shh依赖表型的后期表现。本建议中描述的实验将在这些初步观察的基础上继续进行。为了更好地理解Shh在丘脑核发生中的作用，我们提出了一系列实验来研究pTH-R祖细胞在ShhDSBE1/-胚胎中丢失的分子和细胞机制。pTH-R祖细胞的命运将在野生型和ShhDSBE1/-胚胎中进行追踪，并评估其不同的表达、迁移、分化和轴突投射模式。我们还将确定在患有SOD的个体中受影响的下丘脑区域中Shh表达和Shh应答细胞的命运和功能。最后，考虑到Shh信号在下丘脑中的重要性，我们将结合遗传和生化方法确定调节其表达的关键顺式和反式决定因素。这些研究不仅将提高我们对前脑发育的基本理解，而且将为Shh信号减少导致SOD和HPE的致病机制提供新的见解。

DESCRIPTION (provided by applicant): The temporal and spatial control of Shh expression from defined signaling centers is critical for establishing the identity of neurons in discrete positions along the dorsoventral axis of the neural tube. In the absence of Shh function, ventral midline development is perturbed resulting in holoprosencephaly (HPE), a structural brain malformation, as well as neuronal patterning and path finding defects along the length of the anteroposterior neuraxis. Due to the severity of the defects in Shh-/- mouse embryos, the full extent of Shh function during forebrain development has not been realized. In the previous funding period we uncovered six CNS specific enhancers distributed over 500kb whose combined activity covered most sites of Shh transcription in the mouse neural tube including the ventral forebrain. We used these Shh regulatory elements as tools to further dissect Shh function in the ventral forebrain. Mice lacking Shh in the basal plate of the caudal diencephalon (ShhDSBE1/-) failed to thrive and died in the first week after birth. Preliminary data suggest that this phenotype is attributed to the misspecification of a newly defined class of rostral thalamic progenitors (pTH-R). Mice lacking Shh in the rostral diencephalon (Shh loxp/-; SBE2cre) showed hypothalamic, pituitary and optic nerve defects consistent with septo-optic dysplasia (SOD), a congenital brain malformation syndrome. This finding suggests that reduced Shh signaling from the hypothalamus may underlie the pathogenesis of SOD and represent a later manifestation of a Shh dependent phenotype compared to holoprosencephaly (HPE). Experiments described in this proposal will follow up on these initial observations. To better understand the role of Shh in thalamic nucleogenesis we propose a series of experiments to investigate the molecular and cellular mechanisms by which pTH-R progenitors are lost in ShhDSBE1/- embryos. The fate of pTH-R progenitors will be traced in wild type and ShhDSBE1/- embryos and evaluated for their distinct patterns of expression, migration, differentiation and axonal projections. We will also determine the fate and function of Shh expressing and Shh responding cells in regions of the hypothalamus that are affected in individuals with SOD. Finally, given the importance of Shh signaling in the hypothalamus we will identify the critical cis and trans acting determinants regulating its expression using a combination of genetic and biochemical approaches. These studies will not only improve our basic understanding of forebrain development but will provide novel insight into the pathogenic mechanisms by which reduced Shh signaling results in SOD and HPE. PUBLIC HEALTH RELEVANCE: In humans, the developing ventral forebrain is particularly sensitive to the level of Shh expression. A reduction in Shh expression at early stages of forebrain development results in holoprosencephaly (HPE), whereas a slightly later loss of Shh manifests in septo-optic dysplasia (SOD). Experiments outlined in this proposal will not only improve our basic understanding of forebrain development but will provide novel insights into the pathogenic mechanisms by which reduced Shh signaling results in endocrinopathies, such as HPE and SOD.