Brn3 POU domain proteins in retinal development

描述(申请人提供)：这项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。尽管许多引导视网膜前体细胞(RPC)走向特定细胞命运的关键调控事件已经被识别，但RPC仍然是其固有特性仅被模糊定义的“黑匣子”。拟议的实验集中在RPC承诺RGC命运的机制上。此前，有两个关键的转录因子被证明对RGC的发育至关重要。原性bHLH因子Math5是RPC能力成为RGC所必需的，而POU结构域因子POU4f2(Brn3b)位于Math5的下游，是有能力的、特定的RPC分化为RGC所必需的。利用胚胎视网膜cDNA芯片获得的math5缺失和pou4f2缺失视网膜的基因表达谱揭示了许多依赖于math5或POU4f2表达的外在和内在调节因子。图谱分析导致了由几个层级组成的基因调控网络的构建。胚胎发生过程中RGC的基因消融表明，它们不是其他类型视网膜细胞分化所必需的，但对于通过分泌生长因子(如Sonic Hedgehog)来调节覆盖在上面的RPC的数量是必要的。最重要的假设是，RPC是一个不同种类的细胞群体，其特性主要由bHLH、同源框和其他转录因子的组合决定。其具体目的是：(1)利用视网膜神经节细胞的基因消融来识别math5缺失、pou4f2缺失和RGC缺失的视网膜中基因表达的差异，并建立RGC缺失和视神经变性的成年小鼠模型；(2)通过用其他转录调控因子取代Math5来操纵表达Math5的祖细胞的特性，以确定RPC可以采用新的命运的程度；(3)通过识别与RGC特异性基因表达相关的顺式调控元件和转录因子来阐述RGC基因调控网络的模型；以及(4)确定Sonic Hedgehog在RPC中的异位表达是否能恢复RGC切除的视网膜中RPC的正常数量。拟议中的实验将导致对RPC行为的更好理解，这最终将提供操纵RPC以达到视网膜再生目的的手段。

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.