Molecular Control of Shh-Gli Signaling in the Vertebrate CNS

成年脊椎动物中枢神经系统（CNS）的正常功能主要取决于胚胎发生过程中在正确的时间和位置产生特定的神经元和胶质细胞类型。这个过程在很大程度上是由提供空间和时间线索的分泌信号蛋白和转导这些信号以影响细胞类型特异性遗传程序的转录因子之间的相互作用控制的。在发育中的中枢神经系统中，分级的Sonic hedgehog （Shh）信号通过3个Gli转录因子发挥作用，通过调节神经元和胶质祖细胞分裂过程中特定基因的表达来控制神经管中的细胞命运规范。目前的数据支持一种模型，即3种Gli蛋白通过产生“Gli活性梯度”来转导分级Shh信号。然而，越来越清楚的是，这种途径不能完全解释这种结构中细胞的复杂动态和空间发育。最近，我们发现不同腹侧神经元祖结构域的正确建立涉及“典型”sh - gli和Wnt-Tcf信号元件的转录整合。这些结果与我们的初步数据相结合，表明其他sh - gli靶基因也可以被Tcf蛋白调节，并且在其他地方发表的工作表明，sh - gli和Wnt-Tcf途径之间的转录交叉对话可能是控制组织模式和细胞命运规范的共同机制。本研究将以发育中的脊椎动物脊髓为模型系统，研究在特定中枢神经系统谱系中调节这种相互作用的分子机制。

The proper functioning of the adult vertebrate central nervous system (CNS) depends critically on the generation of specific neuronal and glial cell types at the correct times and positions during embryogenesis. This process is largely controlled by interactions between secreted signaling proteins that provide spatial and temporal cues and the transcription factors that transduce these signals to effect cell type-specific genetic programs. In the developing CNS, graded Sonic hedgehog (Shh) signaling, acting through the 3 Gli transcription factors, controls cell fate specification in the neural tube by regulating the expression of specific sets of genes in dividing neuronal and glial progenitor cells. Current data supports a model where the 3 Gli proteins transduce graded Shh signaling by producing a “Gli activity gradient”. However, it is becoming increasingly clear that this pathway cannot fully account for the complex dynamic and spatial development of cells in this structure. Recently we have found that the proper establishment of distinct ventral neuronal progenitor domains involves the transcriptional integration of “canonical” Shh-Gli and Wnt-Tcf signaling elements. These results, taken with our preliminary data showing that additional Shh-Gli target genes can also be regulated by Tcf proteins, and work published elsewhere, suggests that transcriptional cross-talk between the Shh-Gli and Wnt-Tcf pathways is likely to be a common mechanism for controlling tissue patterning and cell fate specification. This proposal will examine the molecular mechanisms that regulate this interaction in specific CNS lineages, using the developing vertebrate spinal cord as a model system. The experiments in Aim 1 will address the requirement for Tcf4, a key transcriptional regulator of canonical Wnt signaling, in neural development by examining targeted Tcf4 mouse mutants and by using genetic lineage tracing and target gene enhancer analysis approaches to address its role in regulating the expression of cell fate determinant genes that are also controlled by Shh-Gli signaling. In Aim 2, we will employ sensitive in vivo luciferase assays using identified Shh-Gli target gene enhancer elements to study the dynamic aspects of the transcriptional integration of Shh-Gli and Wnt-Tcf signaling in spinal cord cells. This approach allows both graded concentration and temporal effects to be evaluated under conditions of altered activator and/or repressor activity. In Aim 3, we will test the idea that positive Wnt/b-catenin/Tcf signaling controls the development of a subset of oligodendrocytes by regulating the expression of an important factor, Nkx2.2, which is also a target of Shh-Gli signaling. These experiments are united by the hypothesis that both negative and positive Wnt signaling inputs regulate Shh-Gli target gene expression at different times and in distinct CNS lineages. Hedgehog-Gli signaling is widely recognized as playing a critical role in both development and in an ever increasing number of human diseases. A common theme in most cases is that the pathway becomes deregulated, i.e., pathway activation becomes uncoupled from the many levels of control that normally restrict the responses to the appropriate times and tissues. This is thought to lead to the inappropriate up-regulation of target genes whose constitutive activities initiate or contribute to abnormal growth (e.g., cancerous tumors) or patterning (e.g., holoprosencephaly). Similarly, abnormalities in Wnt signaling are also known to be involved in many human disorders, and the proper control over these signals is equally critical for normal development and adult function. The possibility that transcriptional regulators of the Shh-Gli and Wnt-Tcf pathways may converge to control a specific set of shared target genes has important clinical and diagnostic implications. By elucidating the transcriptional mechanisms regulating the expression of shared target genes in the CNS, our studies will open new diagnostic and treatment avenues that are likely to improve the outcome in human patients suffering from Shh or Wnt signaling related diseases.