Transcription profiling of jejunums from wild type and Klf9-/- mice to investigate the involvement of Klf9 in small intestine crypt-villus cell renewal and lineage determination

Kruppel-like factor 9 (Klf9), a zinc-finger transcription factor, is implicated in the control of cell proliferation, cell differentiation and cell fate in brain and uterus. Using Klf9 null mutant mice, we have investigated the involvement of Klf9 in small intestine crypt-villus cell renewal and lineage determination. We report the predominant expression of Klf9 gene in small intestine smooth muscle (muscularis externa). Jejunums null for Klf9 have shorter villi, reduced crypt stem/transit cell proliferation, and altered lineage determination as indicated by decreased and increased numbers of Goblet and Paneth cells, respectively. A stimulatory role for Klf9 in villus cell migration was demonstrated by BrdU labeling. Results suggest that Klf9 controls the elaboration, from small intestine smooth muscle, of molecular mediator(s) of crypt cell proliferation and lineage determination, and of villus cell migration. Experiment Overall Design: Total RNA was extracted in parallel from the jejunums of five WT and five Klf9-/- male mice (PND 30) using TRIzol reagent (Invitrogen, Carlsbad, CA). Conversion of each RNA preparation to corresponding fragmented cRNA. Fifteen ug of each cRNA was hybridized for 16 hours to an Affymetrix mouse 430A GeneChip. Ten GeneChips (each corresponding to a single animal) were hybridized, washed and scanned in parallel. Following the wash, signal amplification, and signal detection steps, GeneChips were scanned (Agilent GeneArray laser scanner) and the resultant images quantified using Affymetrix MAS 5.0 software.Krüppel样因子9（Kruppel-like factor 9, Klf9）是一种锌指转录因子，参与调控脑与子宫内的细胞增殖、细胞分化及细胞命运决定。本研究利用Klf9基因敲除突变小鼠，探究了Klf9在小肠隐窝-绒毛细胞更新与谱系决定中的作用。研究发现，Klf9基因在小肠平滑肌（外膜肌层）中呈优势表达。Klf9纯合敲除小鼠的空肠组织绒毛长度缩短，隐窝干细胞/转运细胞增殖能力降低，且谱系决定发生改变——杯状细胞与潘氏细胞（Paneth cells）的数量分别呈现下降与上升趋势。通过5-溴脱氧尿嘧啶核苷（BrdU）标记实验，证实Klf9对绒毛细胞迁移具有促进作用。研究结果表明，Klf9可通过小肠平滑肌，调控隐窝细胞增殖、谱系决定及绒毛细胞迁移的分子介导因子的表达。 实验整体设计：从5只野生型（Wild Type, WT）与5只Klf9纯合敲除（Klf9-/-）雄性小鼠（出生后第30天，PND 30）的空肠组织中同步提取总RNA，所用试剂为TRIzol试剂（Invitrogen公司，美国加利福尼亚州卡尔斯巴德市）。将每份RNA样品制备为对应的片段化互补RNA（cRNA）。取15 μg每份cRNA与Affymetrix小鼠430A基因芯片进行16小时杂交。共设置10张基因芯片（每张对应单只小鼠），同步完成杂交、洗涤与扫描操作。在完成洗涤、信号扩增及信号检测步骤后，使用安捷伦基因阵列激光扫描仪（Agilent GeneArray laser scanner）对基因芯片进行扫描，并通过Affymetrix MAS 5.0软件对所得图像进行定量分析。