The connexin43-dependent transcriptome during brain development: importance of genetic background

Use of null mutant mice is a powerful way to evaluate the role of specific proteins in brain function. Studies performed on knockout mice have revealed some unexpected roles of the gap junction proteins (the connexins). Thus, analyses of gene expression in connexin43 (Cx43) null brains indicated that deletion of a single gene (Gja1) induced expression level change of numerous other genes located on all chromosomes and involved in a wide diversity of functional pathways. The significant overlap between alterations in gene expression level, control and coordination in Cx43 knockout and knockdown astrocytes raised the possibility that Gja1 represents a transcriptomic node of gene regulatory networks. However, conditional deletion of Gja1 in astrocytes of two mouse strains resulted in remarkably different phenotypes. In order to evaluate the influence of the genetic background on the transcriptome, we performed microarray studies on brains of GFAP-Cre:Cx43f/f C57Bl/6 and 129/SVEV mice. The surprisingly low number of Cx43 core genes (regulated in all Cx43 nulls regardless of strain) and the high number of differently regulated genes in the two Cx43 CKOs indicate high influence of mouse strain on brain transcriptome. The transcriptomes of WT and Cx43 null brains from both C57Bl/6 and SVEV strains were profiled and compared at perinatal and adult time points to learn more about the strain dependence of the Cx43-null phenotype. For this purpose, differently labeled cDNAs from biological replicas (4 of each genotype) were co-hybridized with Duke MO36K mouse oligonucleotide array spotted with 36k Operon oligonucleotides V4.0.

利用空突变小鼠（null mutant mice）是评估特定蛋白质在脑功能中作用的有力研究手段。针对基因敲除小鼠（knockout mice）开展的相关研究，已揭示出缝隙连接蛋白（gap junction proteins，connexins）的诸多未被预期的功能。对连接蛋白43（connexin43, Cx43）空突变小鼠脑组织的基因表达分析显示，单个基因Gja1的缺失会引发所有染色体上大量其他基因的表达水平改变，这些基因参与了广泛多样的功能通路。Cx43基因敲除与基因敲低（knockdown）的星形胶质细胞（astrocytes）中，基因表达水平、调控与协调的改变存在显著重叠，这提示Gja1可能是基因调控网络（gene regulatory networks）的一个转录组节点（transcriptomic node）。然而，在两种小鼠品系的星形胶质细胞中条件性敲除Gja1，却得到了截然不同的表型（phenotypes）。为评估遗传背景对转录组（transcriptome）的影响，我们对GFAP-Cre:Cx43f/f C57BL/6品系与129/SVEV品系小鼠的脑组织进行了基因芯片（microarray）分析。出人意料的是，仅在所有Cx43空突变小鼠中均被差异调控的Cx43核心基因数量极少，而两种Cx43条件性敲除（conditional knockout, CKO）样本中差异调控基因的数量较多，这表明小鼠品系对脑组织转录组具有极强的影响。为进一步探究Cx43空突变表型的品系依赖性，我们对C57BL/6与SVEV两个品系的野生型（Wild Type, WT）及Cx43空突变小鼠脑组织的转录组，在围产期与成年两个时间点进行了转录组谱分析并加以比较。为此，我们将来自生物学重复（biological replicas，每个基因型设置4个重复样本）的不同标记cDNA，与点样有36k条Operon公司V4.0版寡核苷酸探针的杜克大学MO36K小鼠寡核苷酸芯片进行共杂交。