Transcriptional control of sonic hedgehog signaling

描述（由申请人提供）：来自限定的信号传导中心的Shh表达的时间和空间控制对于确立沿神经管的背腹轴沿着离散位置的神经元的身份是关键的。在Shh功能的情况下，腹侧中线发育受到干扰，导致前脑无裂畸形（HPE），一种结构性脑畸形，以及神经元图案化和沿沿着前后神经轴长度的路径发现缺陷。由于Shh-/-小鼠胚胎中缺陷的严重性，在前脑发育过程中Shh功能的完整程度尚未实现。在上一个资助期，我们发现了六个CNS特异性增强子，分布在500 kb以上，其组合活性覆盖了小鼠神经管中包括腹侧前脑在内的Shh转录的大多数位点。我们使用这些Shh调控元件作为工具，进一步剖析腹侧前脑中的Shh功能。在间脑尾侧基板中缺乏Shh的小鼠（ShhDSBE 1/-）无法茁壮成长，并在出生后第一周死亡。初步数据表明，这种表型是由于一个新定义的类喙丘脑祖细胞（PTH-R）的错误指定。在头端间脑中缺乏Shh的小鼠（Shh loxp/-; SBE 2cre）显示出与视隔发育不良（SOD）（一种先天性脑畸形综合征）一致的下丘脑、垂体和视神经缺陷。这一发现表明，减少Shh信号从下丘脑可能是SOD的发病机制，并代表一个Shh依赖性表型相比，前脑无裂（HPE）的后期表现。本提案中所述的实验将在这些初步观察的基础上进行。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ShhDSBE 1/-胚胎中pTH-R祖细胞丢失的分子和细胞机制。将在野生型和ShhDSBE 1/-胚胎中追踪pTH-R祖细胞的命运，并评价其不同的表达、迁移、分化和轴突投射模式。我们还将确定在SOD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信号在下丘脑的重要性，我们将确定关键的顺式和反式作用的决定因素，调节其表达的遗传和生化方法相结合。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。 公共卫生相关性：在人类中，发育中的腹侧前脑对Shh表达水平特别敏感。在前脑发育的早期阶段，Shh表达的减少导致前脑无裂畸形（HPE），而稍晚的Shh缺失则表现为视隔发育不良（SOD）。本提案中概述的实验不仅将提高我们对前脑发育的基本理解，而且将为减少Shh信号导致内分泌疾病（如HPE和SOD）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.