Transcriptional control of sonic hedgehog signaling

描述(由申请人提供)：对特定信号中心Shh表达的时间和空间控制对于确定神经管背腹轴上离散位置的神经元的身份至关重要。在缺乏Shh功能的情况下，腹侧中线发育受到干扰，导致无前脑畸形(HPE)，这是一种结构性脑畸形，以及沿前后神经轴的神经元模式和寻路缺陷。由于Shh-/-小鼠胚胎缺陷的严重性，Shh在前脑发育过程中的作用尚未被充分认识。在之前的资助期间，我们发现了六个分布在500kb以上的中枢神经系统特异性增强子，它们的结合活性覆盖了小鼠神经管中包括腹侧前脑在内的大部分Shh转录位点。我们使用这些Shh调节元件作为工具来进一步剖析腹侧前脑中的Shh功能。在尾侧间脑基板缺乏Shh的小鼠(ShhDSBE1/-)无法茁壮成长，并在出生后第一周死亡。初步数据表明，这种表型归因于对一类新定义的嘴丘脑前体细胞(PTH-R)的错误指定。在吻侧间脑中缺乏Shh的小鼠(Shh loxP/-；SBE2cre)显示出下丘脑、垂体和视神经缺陷，符合隔视发育不良(SOD)，这是一种先天性脑畸形综合征。这一发现表明，来自下丘脑的Shh信号减少可能是SOD发病的基础，与全前脑(HPE)相比，Shh依赖的表型表现为更晚的表现。这项提案中描述的实验将在这些初步观察结果的基础上进行。为了更好地了解Shh在丘脑核发生中的作用，我们提出了一系列实验来研究ShhDSBE1/-胚胎中PTH-R前体丢失的分子和细胞机制。将在野生型和ShhDSBE1/-胚胎中追踪PTH-R前体的命运，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Shh表达和Shh反应细胞在下丘脑受影响的区域的命运和功能。最后，鉴于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作用决定因素。这些研究不仅将提高我们对前脑发育的基本了解，而且将为减少Shh信号导致SOD和HPE的发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.