HSF4 microarray gene expression analysis in the newborn mouse lens

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 Three biological replicate experiments were performed with HSF null and wildtype lenses.本研究旨在鉴定热休克因子4（HSF4）的潜在下游靶基因，故检测了HSF4缺陷型新生小鼠与野生型晶状体中HSF4的差异表达水平。为探究Brg1在小鼠晶状体发育中的功能，研究人员借助晶状体特异性αA-晶状体蛋白（αA-crystallin）启动子，在有丝分裂后晶状体纤维细胞中表达显性负性Brg1（dnBrg1）转基因构建体。形态学研究结果显示，转基因晶状体出现晶状体纤维细胞分化异常，最终诱发白内障形成。电子显微镜观察发现，晶状体缝线结构异常，且晶状体纤维细胞存在不完全核溶解（脱核）过程缺陷。为鉴定受Brg1调控的基因，本研究对胚胎第15.5天（E15.5）的野生型与dnBrg1转基因晶状体开展了RNA表达谱分析。此外，通过比对dnBrg1转基因、Pax6杂合子以及Hsf4纯合子晶状体中的差异表达基因，本研究鉴定出多个受Brg1、HSF4与Pax6共同调控的基因。其中，脱氧核糖核酸酶IIb（DNase IIb）——晶状体纤维细胞脱核过程所需的关键酶——在Pax6、Brg1与Hsf4模型体系中均呈现下调表达。采用条件性基因靶向技术构建的Brg1晶状体特异性敲除实验证实，Brg1对晶状体纤维细胞分化为必需条件，且间接参与视网膜发育，但并非晶状体谱系形成所必需。关键词：差异mRNA表达。本研究针对HSF4缺陷型与野生型晶状体完成了3次生物学重复实验。