HSF4 microarray gene expression analysis in the newborn mouse lens.. Mus musculus

Differential expression of HSF4 in null newborn mouse and wildtype lenses was examined to identify putative downstream targets of HSF4. To examine roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific aA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in E15.5 embryonic wild type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous, and Hsf4 homozygous lenses identified multiple genes co-regulated by Brg1, Hsf4 and Pax6. Among them DNase IIb, a key enzyme required for lens fiber cell denucleation, was found downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was required for lens fiber cell differentiation and indirectly for retinal development but was not essential for lens lineage formation. Keywords: Differential mRNA Expression Overall design: Three biological replicate experiments were performed with HSF null and wildtype lenses.

为鉴定热休克因子4（HSF4）的潜在下游靶基因，本研究检测了HSF4缺失型新生小鼠与野生型小鼠晶状体中HSF4的差异表达水平。为探究Brahma相关蛋白1（Brg1）在小鼠晶状体发育中的调控作用，研究人员利用晶状体特异性αA-晶状体蛋白（aA-crystallin）启动子，在有丝分裂后晶状体纤维细胞中表达了显性负性Brg1（dnBrg1）转基因构建体。形态学研究显示，该转基因小鼠的晶状体出现异常的晶状体纤维细胞分化，进而诱发白内障。电子显微镜观察发现，转基因晶状体存在晶状体缝线结构异常及晶状体纤维细胞不完全核溶解（脱核，denucleation）现象。为鉴定受Brg1调控的靶基因，研究人员对胚胎发育第15.5天（E15.5）的野生型小鼠与dnBrg1转基因小鼠的晶状体进行了RNA表达谱分析。此外，通过对比dnBrg1转基因、配对盒基因6（Pax6）杂合子及Hsf4纯合子小鼠晶状体中的差异表达基因，研究人员筛选出多个受Brg1、Hsf4与Pax6共同调控的基因。其中，脱氧核糖核酸酶IIb（DNase IIb）作为晶状体纤维细胞脱核过程的关键酶，在Pax6、Brg1及Hsf4三种模型系统中均呈现表达下调。利用条件性基因靶向技术构建晶状体特异性Brg1缺失模型，结果表明Brg1对晶状体纤维细胞分化是必需的，且间接参与视网膜发育，但并非晶状体谱系形成所必需的调控因子。关键词：差异mRNA表达 整体实验设计：本研究针对HSF4缺失型与野生型小鼠晶状体开展了3次生物学重复实验。