Transcription profiling of mouse Dll3 mutant vs. wild-type 9.5 dpc somite-level tissue

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rphn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a; regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function. Experiment Overall Design: To enrich for genes in the somite level tissues that are specifically disrupted by Dll3 mutation, we compared microdissected tissues from wild-type and Dll3 mutant embryos. We generated biological replicate pools from Dll3+/+ (wild-type) or Dll3neo/neo embryos for a total of six pools. Microarray analysis using Affymetrix MOE430A arrays was carried out on the biological pool triplicates for both wild-type and mutant genotypes.Delta样蛋白3（Delta-like 3, Dll3）是一种仅在哺乳动物中被鉴定到的Notch信号通路（Notch signaling pathway）偏离型配体与调节因子。Dll3的无效突变会扰乱Notch靶基因Hes1、Hes5及Lfng的周期性表达，但不会影响Hes7的周期性表达。相较于Dll1或Notch1，Dll3突变对前体节中胚层（presomitic mesoderm）内基因表达的影响程度更轻，但在人类与小鼠中均会引发严重的分节表型与脊椎缺陷。鉴于Dll3可特异性扰乱体节周期性的关键调控因子，我们开展了功能分析以鉴定与该分节表型相关的靶基因。我们利用显微切割获取的体节与前体节中胚层胚胎组织，鉴定出一批在上述组织中富集的新基因，包括Limch1、Rphn2及A130022J15Rik。令人意外的是，我们仅鉴定出少量受Dll3突变影响的基因，其中包括参与肋骨与脊椎模式形成的体节基因Uncx，以及在斑马鱼中为Notch/Wnt信号调节因子、在小鼠中为周期性表达基因的Nrarp。为探究Dll3突变对Nrarp的影响，我们对该基因在体节发生早期（胚胎日龄8.5天，days post coitum, dpc）至晚期（胚胎日龄10.5天，dpc）的周期性表达模式进行了表征。在9.5 dpc时，Nrarp的周期性表达时相与Lfng及Wnt通路基因Axin2存在显著差异，但到10.5 dpc时，其时相似乎与Lfng同步。Nrarp的周期性表达似乎依赖于Dll3，但无需Lfng的调节。在Dll3无效突变胚胎中，Nrarp呈现静态表达模式。然而，在Lfng无效突变胚胎中，Nrarp在8.5 dpc时呈静态表达，至9.5 dpc时恢复周期性表达，并在10.5 dpc时呈现动态表达。与之相反，在Wnt3a无效突变胚胎中，前体节中胚层内完全检测不到Nrarp的表达。为阐明Dll3在体节发生调控中的作用机制，我们发现Nrarp是一类潜在的关键调控因子，其正常功能依赖于Dll3而非Lfng。实验整体设计：为富集受Dll3突变特异性影响的体节水平组织基因，我们对野生型与Dll3突变型胚胎的显微切割组织进行了对比分析。我们分别从Dll3+/+（野生型）或Dll3neo/neo胚胎中构建生物学重复混合样本，共计6组。针对野生型与突变型两种基因型，我们对其生物学重复混合样本进行了三次重复的Affymetrix MOE430A芯片微阵列分析。