Brn3 POU domain proteins in retinal development

描述（由申请人提供）：本研究的长期目标是阐明控制视网膜神经节细胞（RGC）形成的基因调控网络。虽然许多关键的调控事件，直接视网膜祖细胞（RPC）对一个特定的细胞命运已被确定，RPC仍然是“黑匣子”，其内在属性只是模糊的定义。拟议的实验集中在RPC提交RGC命运的机制。以前，两个关键的转录因子被证明是RGC发展的关键。原神经bHLH因子Math 5对于RPC能力成为RGC是必不可少的，而POU结构域因子POU 4f 2（Brn 3b）在遗传上位于Math 5的下游，并且对于有能力的特定RPC分化成RGC是必需的。math 5-null和pou 4f 2-null视网膜的基因表达谱，获得使用从胚胎视网膜cDNA产生的微阵列揭示了许多外在和内在的调节因子，依赖于Math 5。或POU 4f 2的表达。分析导致的基因调控网络的建设，包括几个层次。在胚胎发生过程中RGCs的基因消融表明，它们不是其他视网膜细胞类型分化所必需的，但对于通过分泌生长因子如Sonic hedgehog来调节上覆RPC的数量是必需的。最重要的假设是，RPC是一个异质性的细胞群体，其性质主要是由bHLH，同源框和其他转录因子的组合定义。具体目标是：（1）使用RGCs的基因消融来鉴定math 5-null、pou 4f 2-null和RGCs消融的视网膜中基因表达的差异，并建立RGC丧失和视神经变性的成年小鼠模型;（2）通过用其他转录调节因子替换Math 5来操纵表达math 5的祖细胞的性质，以确定RPC可以采用新命运的程度;（3）通过鉴定与RGC特异性基因表达相关的顺式调控元件和转录因子，详细阐述RGC基因调控网络的模型;（4）确定RPC中Sonic Hedgehog的异位表达是否恢复了RGC消融视网膜中RPC的正常数量。拟议的实验将导致更好地了解RPC的行为，这将最终提供的手段来操纵RPC的视网膜再生的目的。

DESCRIPTION (provided by applicant): The long-term objective of this research is to elucidate the gene regulatory network that controls the formation of retinal ganglion cells (RGCs). Although many of the critical regulatory events that direct a retinal progenitor cell (RPC) towards a particular cell fate have been identified, RPCs still remain "black boxes" whose intrinsic properties are only vaguely defined. The proposed experiments focus on the mechanisms by which RPCs commit to an RGC fate. Previously, two key transcription factors were shown to be critical for RGC development. The proneural bHLH factor Math5 is essential for RPC competence to become an RGC, while the POU domain factor POU4f2 (Brn3b) is genetically downstream from Math5 and is required for competent, specified RPCs to differentiate into RGCs. Gene expression profiles of math5-null and pou4f2-null retinas obtained using microarrays generated from embryonic retinal cDNAs revealed numerous extrinsic and intrinsic regulatory factors that depend on Math5.or POU4f2 for their expression. The profiling analysis led to the construction of a gene regulatory network consisting of several hierarchial layers. Genetic ablation of RGCs during embryogenesis demonstrated that they are not required for the differentiation of other retinal cell types but are necessary for regulating the number of overlying RPCs by secreting growth factors such as Sonic hedgehog. The overriding hypothesis is that RPCs are a heterogeneous population of cells whose properties are defined largely by combinations of bHLH, homeobox, and other transcription factors. The specific aims are to: (1) use genetic ablation of RGCs to identify differences in gene expression in math5-null, pou4f2-null, and RGC-ablated retinas and to create an adult mouse model for RGC loss and optic nerve degeneration; (2) manipulate the properties of math5-expressing progenitor cells by replacing Math5 with other transcriptional regulators to determine the extent to which RPCs can adopt new fates; (3) elaborate the model for the RGC gene regulatory network by identifying cis-regulatory elements and transcription factors associated with RGC-specific gene expression; and (4) determine whether the ectopic expression of sonic hedgehog in RPCs restores normal numbers of RPCs in RGC-ablated retinas. The proposed experiments will lead to a better understanding of RPC behavior, which will eventually provide the means to manipulate RPCs for the purpose of retinal regeneration.