The LIM homeodomain transcription factor Lhx2 is required for Muller glia development in the vertebrate retina

Purpose: Muller glia are the only glial cell type produced by the neuroepithelial progenitor cells which generate the vertebrate retina. Muller glia are required to maintain retinal homeostasis and support the survival of retinal neurons. Furthermore, they function as an adult stem cell, mediating retinal regeneration among select vertebrate classes. However, the mechanisms which regulate Muller development are poorly understood as considerable overlap exists in gene expression between retinal progenitor cells and differentiated Muller glia. We investigate the functional role of the LIM homeodomain transcription factor Lhx2 in the specification and development of Muller glia in the mouse. Methods: RNA-Seq was performed in collaboration with the Johns Hopkins School of Medicine Deep Sequencing and Microarray Core Facility. Libraries were prepared using Illumina TruSeq RNA Sample kit (Illumina, San Diego, CA) following manufacturer's recommended procedure. The PCR amplified library was purified using RNAClean XP magnetic beads (Agencourt, Beverley, MA) and run out on a High Sensitivity DNA Chip (Agilent, Santa Clara, CA) for quality check. We used STAR to align RNA-Seq reads onto Ensembl mouse genome GRCm38, release 72. To generate the stand attribute for alignments containing splice junctions, we used the outSAMstrandField intronMotif program. The spliced alignments without strand definition were removed. Number of reads mapped to exons was counted by htseq-count. Genes expressed at very low levels were omitted from further analysis. Gene expression differences between wildtype and mutant samples, significance (p-value) and false discovery rate (FDR) were computed using the generalized linear models based EdgeR. Results: We observed a substantial reduction in expression of Notch pathway genes including Notch1, the Notch ligands Dll1 and Dll3, as well as gliogenic Notch effector genes such as Hes1, Hes5, Id1 and Sox8 and the Muller-gliogenic factor Rax. We likewise observe a substantial reduction in expression of progenitor-specific genes such as Vsx2 and Fgf15. Furthermore, we observed a decrease in the expression of early-onset glial markers such as Crym , Spon1, and Car2. Retinal mRNA profiles of post-natal day 0.5 (P0.5) Lhx2 wild type (N=3) and Lhx2lox/lox; Pdgfr-Cre deltacKO (N=3) mice were generated using Illumina TruSeq and analyzed with Agilent high sensitivity DNA analsis kit.研究目的：米勒胶质细胞（Muller glia）是生成脊椎动物视网膜的神经上皮祖细胞（neuroepithelial progenitor cells）所产生的唯一一类胶质细胞。米勒胶质细胞对于维持视网膜稳态、支持视网膜神经元存活不可或缺。此外，在部分脊椎动物类群中，它们可作为成体干细胞介导视网膜再生。然而，由于视网膜祖细胞与分化的米勒胶质细胞之间存在显著的基因表达重叠，调控米勒胶质细胞发育的分子机制目前仍未被充分阐明。本研究聚焦于LIM同源结构域转录因子（LIM homeodomain transcription factor）Lhx2，探讨其在小鼠米勒胶质细胞特化与发育过程中的功能作用。 实验方法：本研究联合约翰·霍普金斯医学院深度测序与微阵列核心设施开展RNA测序（RNA-Seq）实验。参照制造商推荐流程，使用Illumina TruSeq RNA样本试剂盒（Illumina公司，美国加利福尼亚州圣地亚哥）构建测序文库。采用RNAClean XP磁珠（Agencourt公司，美国马萨诸塞州比弗利）纯化PCR扩增后的文库，并通过安捷伦高灵敏度DNA芯片（Agilent公司，美国加利福尼亚州圣克拉拉）进行质量质控。本研究使用STAR比对工具将RNA测序读段比对至Ensembl数据库发布的小鼠基因组GRCm38版本72。为生成包含剪接位点的比对结果链属性，我们调用outSAMstrandField内含子基序程序，并移除无链定义的剪接比对结果。通过htseq-count工具统计比对至外显子的读段数量，剔除表达水平极低的基因以开展后续分析。基于广义线性模型的EdgeR软件包，计算野生型与突变型样本间的基因表达差异、显著性p值（p-value）及错误发现率（FDR）。 实验结果：我们观测到Notch通路相关基因的表达量显著下调，包括Notch1、Notch配体Dll1与Dll3，以及胶质发生相关Notch效应基因Hes1、Hes5、Id1、Sox8和米勒胶质细胞发生因子Rax。同时，祖细胞特异性基因Vsx2与Fgf15的表达水平同样显著降低。此外，早期胶质细胞标志物Crym、Spon1及Car2的表达量也出现下降。本研究通过Illumina TruSeq测序技术，获取了出生后0.5天（P0.5）的Lhx2野生型（N=3）与Lhx2lox/lox; Pdgfr-Cre deltacKO（N=3）小鼠的视网膜mRNA表达谱，并采用安捷伦高灵敏度DNA分析试剂盒完成后续分析。