Gene expression in E10.5 Maxillary Arch Mesenchyme from control and Lhx6-/-;Lhx8-/- mutant embryos

We used laser capture microdissection to isolate maxillary arch mesenchyme from E10.5 embryos. This tissue was collected from both control (3x) and Lhx6-/-;Lhx8-/- mutant (3x) samples. Transcriptional profiling was performed using Affymetrix GeneChip Mouse Genome 430 2.0 arrays. The mutant mice are of mixed genetic background of C57BL/6J, 129 and CD-1 strains. The head of E10.5 embryos was collected and embedded in Optimal Cutting Temperature resin (Tissue-Tek) by flash freezing on dry ice. The frozen sections were collected on polyethylene naphthalate membrane slides (Leica). Leica LMD6000 Laser Micro-Dissection System was used to cut out the normal expression domain of Lhx6 and Lhx8 in the maxillary arch mesenchyme. The tissue was collected from the entire antero-posterior extent of the maxillary arches. Total RNA was extracted using RNeasy Micro Kit (Qiagen). Subsequent steps of transcriptional profiling were performed by the New York University Genome Technology Center, beginning with the amplification of RNA by Ovation Nano Amplification system (NuGen). RNA samples from three wild-type and three Lhx6-/-;Lhx8-/- mutantmutant embryos, all somite count- and sex-matched (females), were analyzed with Affymetrix GeneChip Mouse Genome 430 2.0 arrays. A list of Lhx-regulated genes were generated from the microarray result based on the following criteria: fold change in the average expression between wild types and Lhx6-/-;Lhx8-/- mutant mutants is >1.5, the difference is statistically significant (P < 0.05) and the average intensity of the probe signal is >100 for wild-type and/or mutant samples. The resulting list of 212 genes was used for a gene ontology analysis with DAVID (27,28).本研究采用激光捕获显微切割（laser capture microdissection, LCM）技术，从胚胎发育第10.5天（E10.5）的胚胎中分离上颌弓间充质组织。实验样本分为对照组（3个生物学重复）与Lhx6-/-;Lhx8-/-双突变体组（3个生物学重复）。转录组分析采用Affymetrix GeneChip Mouse Genome 430 2.0小鼠基因组芯片完成。所用突变小鼠的遗传背景为C57BL/6J、129及CD-1品系的混合背景。收集E10.5胚胎的头部，经干冰快速冷冻后，包埋于最优切割温度包埋树脂（Optimal Cutting Temperature resin，商品名Tissue-Tek）中。将冰冻切片铺于聚萘二甲酸乙二醇酯（polyethylene naphthalate, PEN）膜载玻片（徕卡Leica产品）上，使用徕卡LMD6000激光显微切割系统，切割获取上颌弓间充质中Lhx6与Lhx8的正常表达区域，收集的组织覆盖上颌弓的前后全长。总RNA提取采用Qiagen的RNeasy Micro试剂盒。后续转录组分析步骤由纽约大学基因组技术中心完成，首先通过NuGen的Ovation Nano扩增系统对RNA进行扩增。本次分析使用了3只野生型与3只Lhx6-/-;Lhx8-/-双突变体胚胎的RNA样本，所有样本均按体节数及性别匹配（均为雌性），并采用Affymetrix GeneChip Mouse Genome 430 2.0芯片进行检测。基于以下标准从芯片结果中筛选得到Lhx调控基因列表：野生型与Lhx6-/-;Lhx8-/-双突变体的平均表达倍数差异>1.5、差异具有统计学意义（P<0.05），且野生型和/或突变体样本的探针信号平均强度>100。最终得到的212个基因列表被用于DAVID（27,28）的基因本体（Gene Ontology, GO）富集分析。