Expression data from mouse embryo during neural tube closure

This data series was used for two separate studies. The initial study was aimed to identify expression changes brought about by the Cecr2Gt45Bic mutation during neural closure. The study included two different strains, BALB/cCrl in which Cecr2GT45Bic shows a neural tube defect phenotype and FVB/N in which Cecr2Gt45Bic does not manifest neural closure defects. The second was to idenify strain specific expression differences present during neural closure of the mouse embryo between BALB/cCrl and FVB/N in order to identify candidate modifiers of the Cecr2Gt45Bic neural tube defect. Relevant abstracts are included below. The initial study ; BACKGROUND: Over 200 mouse genes are associated with neural tube defects (NTDs), including Cecr2, the bromodomain-containing subunit of the CERF chromatin remodeling complex. METHODS: Gene-trap mutation Cecr2Gt45Bic results in 74% exencephaly (equivalent of human anencephaly) on the BALB/c strain. Gene expression altered during cranial neural tube closure by the Cecr2 mutation was identified through microarray analysis of 11-14 somites stage Cecr2Gt45Bicembryos. RESULTS: Analysis of Affymetrix Mouse 430 2.0 chips detected 60 transcripts up-regulated and 54 transcripts down-regulated in the Cecr2Gt45Bic embryos (fold > 1.5, p < 0.05). The Cecr2 transcript was reduced only ~ 7- to 14-fold from normal levels, suggesting the Cecr2Gt45Bic is a hypomorphic mutation. We therefore generated a novel Cecr2 null allele (Cecr2tm1.1Hemc). Resulting mutants displayed a stronger penetrance of exencephaly than Cecr2Gt45Bic in both BALB/c and FVB/N strains, in addition to midline facial clefts and forebrain encephalocele in the FVB/N strain. The Cecr2 transcript is reduced 260-fold in the Cecr2tm1.1Hemc line. Subsequent qRT-PCR using Cecr2tm1.1Hemc mutant heads confirmed downregulation of transcription factors Alx1/Cart1,Dlx5, Eya1, and Six1. CONCLUSIONS: As both Alx1/Cart1 and Dlx5 mouse mutations result in exencephaly, we hypothesize that changes in expression of these mesenchymal/ectodermal transcription factors may contribute to NTDs associated with Cecr2. Birth Defects Research (Part A), 2010. 010 Wiley-Liss, Inc. The second study: ABSTRACT:Although neural tube defects (NTDs) are common in humans, little is known about their multifactorial genetic causes. While most mouse models involve NTDs caused by a single mutated gene, we have previously described a multigenic system involving susceptibility to NTDs. In mice with a mutation in Cecr2, the cranial NTD exencephaly shows strain specific differences in penetrance, with 74% penetrance in BALB/cCrl and 0% penetrance in FVB/N. Whole genome linkage analysis showed that a region of chromosome 19 was partially responsible for this difference in penetrance. We now reveal by genetic analysis of three subinterval congenic lines that the chromosome 19 region contains more than one modifier gene. Analysis of embryos showed that although a Cecr2 mutation causes wider neural tubes in both strains, FVB/N embryos overcome this abnormality and close. A microarray analysis comparing neurulating female embryos from both strains identified differentially expressed genes within the chromosome 19 region, including Arhgap19, which is expressed at a lower level in BALB/cCrl due to a stop codon specific to that substrain. Modifier genes in this region are of particular interest because it is syntenic to human chromosome 10q25, the site of a human susceptibility locus. (MANUSCRIPT PENDING SUBMISSION) Theiler STAGE 13-14 (11-14 somite) female embryos where dissected away from extraembryonic membranes for RNA extraction and hybridization on Affymetrix microarrays. A single embryo was processed per array, with four biological replicates per genotype BALB/cCrl, Cecr2GT45Bic BALB/cCrl, FVB/N, and Cecr2Gt45Bic FVB/N.本数据集系列应用于两项独立研究。 第一项研究旨在探究Cecr2Gt45Bic突变在神经闭合过程中引发的基因表达变化。该研究涉及两种不同的小鼠品系：携带Cecr2GT45Bic突变后会表现出神经管缺陷（Neural Tube Defects, NTDs）表型的BALB/cCrl品系，以及携带该突变却不会出现神经闭合缺陷的FVB/N品系。第二项研究则旨在鉴定BALB/cCrl与FVB/N小鼠胚胎神经闭合过程中的品系特异性表达差异，以筛选出能够调控Cecr2Gt45Bic相关神经管缺陷的候选修饰基因。下文附上相关研究摘要。 ### 第一项研究 **背景**：目前已有超过200个小鼠基因与神经管缺陷（NTDs）相关，其中包括Cecr2——CERF染色质重塑复合物的溴结构域亚基。 **方法**：Cecr2Gt45Bic基因陷阱突变在BALB/c品系中可导致74%的胎鼠出现露脑畸形（对应人类的无脑畸形）。本研究通过对11~14体节期的Cecr2Gt45Bic胚胎进行微阵列分析，鉴定了Cecr2突变在颅神经管闭合过程中改变的基因表达谱。 **结果**：对Affymetrix Mouse 430 2.0芯片的分析显示，Cecr2Gt45Bic胚胎中共有60个转录本上调、54个转录本下调（倍数变化>1.5，p<0.05）。Cecr2转录本的表达量仅较正常水平降低7~14倍，提示Cecr2Gt45Bic属于亚效突变（hypomorphic mutation）。为此，我们构建了一种新型的Cecr2无效等位基因（Cecr2tm1.1Hemc）。相较于Cecr2Gt45Bic突变体，该等位基因的突变体在BALB/c和FVB/N两个品系中均表现出更高的露脑畸形外显率，且在FVB/N品系中还出现了中线面部裂及前脑脑膨出。Cecr2tm1.1Hemc品系的Cecr2转录本表达量降低了260倍。后续通过对Cecr2tm1.1Hemc突变胎鼠头部组织进行实时定量反转录聚合酶链反应（qRT-PCR）验证，发现转录因子Alx1/Cart1、Dlx5、Eya1及Six1的表达均出现下调。 **结论**：鉴于Alx1/Cart1和Dlx5的小鼠突变均可导致露脑畸形，我们推测这些间充质/外胚层转录因子的表达变化可能参与了Cecr2相关神经管缺陷的发生。《出生缺陷研究（A辑）》，2010年，© 2010 Wiley-Liss公司版权所有。 ### 第二项研究 **摘要**：尽管神经管缺陷（NTDs）在人类中较为常见，但其多因素遗传致病机制仍鲜为人知。多数小鼠模型的神经管缺陷由单基因突变引发，而我们此前曾报道过一套涉及神经管缺陷易感性的多基因系统。在携带Cecr2突变的小鼠中，颅神经管缺陷露脑畸形的外显率存在品系特异性差异：BALB/cCrl品系的外显率为74%，而FVB/N品系为0%。全基因组连锁分析显示，19号染色体的一个区域部分参与了该外显率差异的调控。本研究通过对三个亚区间同源导入系的遗传分析进一步证实，该19号染色体区域包含不止一个修饰基因。胚胎分析结果显示，尽管Cecr2突变会导致两个品系的胎鼠神经管均变宽，但FVB/N胎鼠可克服该异常并完成神经管闭合。通过对两个品系的神经褶闭合期雌性胚胎进行微阵列分析，我们鉴定出了19号染色体区域内的差异表达基因，其中包括Arhgap19——由于该亚系特有的终止密码子突变，其在BALB/cCrl品系中的表达量更低。该区域的修饰基因具有特殊研究价值，因为其与人类10号染色体q25区域存在同线性（syntenic），而该区域正是人类神经管缺陷易感位点所在。【稿件待投稿】 **实验细节**：取泰勒发育阶段13~14（11~14体节）的雌性胚胎，剥离其胚外膜以提取RNA，并进行Affymetrix微阵列杂交。每个芯片对应单个胚胎，BALB/cCrl、Cecr2GT45Bic BALB/cCrl、FVB/N、Cecr2Gt45Bic FVB/N四种基因型各设置4次生物学重复。