Transcription profiling by array of whisker pads from mice mutant for Trps1

Mutations in TRPS1 cause trichorhinophalangeal syndrome types I and III, which are characterized by sparse scalp hair in addition to craniofacial and skeletal abnormalities. Trps1 is a vertebrate transcription factor containing nine zinc-finger domains, including a GATA-type zinc finger through which it binds DNA. Mice in which the GATA domain of Trps1 has been deleted (Trps1delta gt/delta gt) have a reduced number of pelage follicles and lack vibrissae follicles postnatally. To identify the transcriptional targets of Trps1 in the developing vibrissa follicle, we performed microarray hybridization analysis comparing expression patterns in the whisker pads of wild-type versus Trps1delta gt/delta gt embryos. We identified a number of transcription factors and Wnt inhibitors among transcripts downregulated in the mutant embryos, and several extracellular matrix proteins there were upregulated in the mutant samples. Whole whisker pads were dissected from E12.5 embryos and total RNA was isolated using the RNeasy Mini Kit (Qiagen). Triplicate RNA samples from three independent embryos of each genotype were amplified and labeled for hybridization to Affymetrix GeneChip MOE430A microarrays using Affymetrix reagents and protocols. The data output was analyzed using GeneSpring GX 10.0 commercial software (Agilent Technologies). P-values were calculated using an unpaired t-test. Expression values with a p-value less than or equal to 0.05 and a fold difference of at least 1.5 relative to wild-type baseline expression levels were considered significant.TRPS1基因（TRPS1）的突变可引发I型与III型毛发鼻指综合征（trichorhinophalangeal syndrome），此类综合征除表现为颅面及骨骼异常外，还以头皮毛发稀疏为特征。Trps1是一种脊椎动物转录因子，包含9个锌指结构域（zinc-finger domain），其中含有一个GATA型锌指结构域，可通过该结构域结合DNA。敲除Trps1的GATA结构域的基因工程小鼠（Trps1delta gt/delta gt）出生后被毛毛囊数量减少，且缺失触须毛囊。为鉴定Trps1在发育中触须毛囊内的转录靶标，本研究开展了微阵列杂交（microarray hybridization）分析，对比野生型（wild-type）与Trps1delta gt/delta gt胚胎的触须垫基因表达模式。研究在突变体胚胎的下调转录本中鉴定出多个转录因子与Wnt信号通路抑制剂（Wnt inhibitor），而突变样本中的细胞外基质蛋白（extracellular matrix protein）则呈现上调表达。从胚胎发育第12.5天（E12.5）的胚胎中分离完整触须垫，使用RNeasy Mini Kit（Qiagen）提取总RNA。针对每种基因型，分别采集3个独立胚胎的RNA样本并设置三次技术重复，使用Affymetrix配套试剂与实验流程对RNA进行扩增与标记，随后与Affymetrix GeneChip MOE430A微阵列进行杂交实验。使用商用软件GeneSpring GX 10.0（安捷伦科技，Agilent Technologies）对原始数据输出结果进行分析，采用非配对t检验计算P值。相较于野生型基线表达水平，P值≤0.05且表达倍数变化≥1.5的基因表达值被认定为具有统计学显著性。