BACKGROUND About 20-30 distinct Retinal Ganglion Cell (RGC) types transmit visual information from the retina to the brain. The developmental mechanisms by which RGCs are specified are still largely unknown. Brn3a is a member of the Brn3/Pou4f transcription factor family, which contains key regulators of RGC postmitotic specification. In particular, Brn3a ablation results in the loss of RGCs with small, thick and dense dendritic arbors ('midget-like' RGCs), and morphological changes in other RGC subpopulations. To identify downstream molecular mechanisms underlying Brn3a effects on RGC numbers and morphology, our group recently performed a RNA deep sequencing screen for Brn3a transcriptional targets in mouse RGCs and identified 180 candidate transcripts. METHODS We now focus on a subset of 28 candidate genes encoding potential cell type determinant proteins. We validate and further define their retinal expression profile at five postnatal developmental time points between birth and adult stage, using in situ hybridization (ISH), RT-PCR and fluorescent immunodetection (IIF). RESULTS We find that a majority of candidate genes are enriched in the ganglion cell layer during early stages of postnatal development, but dynamically change their expression profile. We also document transcript-specific expression differences for two example candidates, using RT-PCR and ISH. Brn3a dependency could be confirmed by ISH and IIF only for a fraction of our candidates. CONCLUSIONS Amongst our candidate Brn3a target genes, a majority demonstrated ganglion cell layer specificity, however only around two thirds showed Brn3a dependency. Some were previously implicated in RGC type specification, while others have known physiological functions in RGCs. Only three genes were found to be consistently regulated by Brn3a throughout postnatal retina development - Mapk10, Tusc5 and Cdh4.

中文翻译：

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Brn3a / Pou4f1视网膜神经节细胞中细胞类型规范基因的产后发育动力学。

背景技术约20-30种不同的视网膜神经节细胞（RGC）类型将视觉信息从视网膜传递到大脑。指定RGC的发展机制仍然未知。Brn3a是Brn3 / Pou4f转录因子家族的成员，其包含RGC有丝分裂后规格的关键调控因子。特别地，Brn3a切除导致具有小的，浓密的和密集的树突状乔木（“侏儒状” RGC）的RGC丢失，并导致其他RGC亚群的形态发生变化。为了确定Brn3a对RGC数量和形态的影响的下游分子机制，我们小组最近对小鼠RGC中Brn3a转录靶标进行了RNA深度测序筛选，并鉴定了180个候选转录本。方法我们现在集中于28个候选基因的子集，这些候选基因编码潜在的细胞类型决定簇蛋白。我们使用原位杂交（ISH），RT-PCR和荧​​光免疫检测（IIF）在出生和成年之间的五个出生后发育时间点验证并进一步定义它们的视网膜表达谱。结果我们发现，大多数候选基因在产后发育的早期都富集在神经节细胞层中，但会动态改变其表达谱。我们还使用RT-PCR和ISH记录了两个示例候选物的转录物特异性表达差异。ISH和IIF只能对部分候选人确认Brn3a依赖性。结论在我们的候选Brn3a目标基因中，大多数证明了神经节细胞层的特异性，但是只有三分之二的人显示出对Brn3a的依赖性。一些以前涉及RGC类型规范，而另一些具有RGC的已知生理功能。发现整个出生后视网膜发育过程中只有三个基因受到Brn3a的一致调控-Mapk10，Tusc5和Cdh4。