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（RGCs）形成的基因调控网络。虽然已经确定了许多指导视网膜祖细胞（RPC）走向特定细胞命运的关键调控事件，但RPC仍然是“黑盒子”，其内在特性只有模糊的定义。拟议的实验侧重于RPCs向RGC承诺命运的机制。在此之前，两个关键的转录因子被证明对RGC的发展至关重要。原生bHLH因子Math5对于RPC能力成为RGC至关重要，而POU结构域因子POU4f2 （Brn3b）在Math5的遗传下游，是有能力的、指定的RPC分化为RGC所必需的。利用胚胎视网膜cdna生成的微阵列获得Math5 -null和pou4f2-null视网膜的基因表达谱，揭示了许多依赖Math5的外在和内在调节因子。或POU4f2表示它们的表达。通过分析，构建了由多个层次组成的基因调控网络。胚胎发生期间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，但通过分泌生长因子（如Sonic hedgehog）来调节覆盖的RPCs的数量是必要的。最重要的假设是，rpc是一种异质细胞群，其特性主要由bHLH、同源盒和其他转录因子的组合决定。具体目的是：(1)利用RGC的基因消融来鉴定math5-null、pou4f2-null和RGC消融视网膜中基因表达的差异，并建立RGC丢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代Math5来操纵表达Math5的祖细胞的特性，以确定rpc可以采用新命运的程度；(3)通过确定与RGC特异性基因表达相关的顺式调控元件和转录因子，构建RGC基因调控网络模型；(4)确定超音hedgehog基因在rgc消融视网膜中异位表达是否能恢复正常的RPCs数量。提出的实验将导致对RPC行为的更好理解，这将最终为视网膜再生的目的提供操纵RPC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.