Smad4-dependent pathways control basement membrane deposition and endodermal cell migration at early stages of mouse development.
Smad4-dependent pathways control basement membrane deposition and endodermal cell migration at early stages of mouse development.
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DOI：
10.1186/1471-213x-9-54
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发表时间：
2009-10-22
影响因子：
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通讯作者：
Robertson EJ
中科院分区：
文献类型：
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作者：
Costello I;Biondi CA;Taylor JM;Bikoff EK;Robertson EJ
Smad4突变胚胎在植入后不久停止生长，表现为近远端轴缩短，上胚层显著减少，内脏内胚层增厚。有条件的救援实验表明，绕过胚胎外内胚层对Smad4的主要要求，可以使上胚层形成原肠。因此，不依赖Smad4的转化生长因子-β信号足以促进中胚层的形成和图案化。为了进一步分析胚胎外组织对Smad4活性的贡献，并确定Smad4在早期胚胎中的依赖途径，我们对Smad4缺失的胚胎干细胞(ES)和第4天的类胚体(EBS)进行了转录谱分析。用Illumina阵列分析野生型和Smad4缺失ES细胞和第4天EBS的转录本。除了几个已知的转化生长因子-β/骨形态发生蛋白的靶基因外，我们还鉴定了许多在突变体中错误表达的依赖于Smad4的转录本。正如预期的那样，中胚层细胞标志物显著下调。我们还观察到非规范的效力标记(Pramel7，Tbx3，Zscan4)，生殖细胞标记(Aire，Tuba3a，Dnmt3l)以及早期内胚层标记(DPP4，H19，Dcn)的增加。此外，细胞外基质(ECM)重塑酶Mmp14和Mmp9在Smad4突变ES和EB群体中的表达减少。这些变化，加上层粘连蛋白α1水平的增加，导致基底膜过度沉积。同样，在Smad4缺失的E6.5胚胎中，我们观察到与增厚的内胚层相关的扩张的基底膜(BM)。Smad4功能缺失导致基因表达模式的戏剧性变化，内皮细胞谱系导致BM底层过度沉积或无法分解和重塑。这些结构异常可能扰乱了原肠形成所需的上胚层和上覆内脏内胚层之间的相互信号传递。
Smad4 mutant embryos arrest shortly after implantation and display a characteristic shortened proximodistal axis, a significantly reduced epiblast, as well as a thickened visceral endoderm layer. Conditional rescue experiments demonstrate that bypassing the primary requirement for Smad4 in the extra-embryonic endoderm allows the epiblast to gastrulate. Smad4-independent TGF-β signals are thus sufficient to promote mesoderm formation and patterning. To further analyse essential Smad4 activities contributed by the extra-embryonic tissues, and characterise Smad4 dependent pathways in the early embryo, here we performed transcriptional profiling of Smad4 null embryonic stem (ES) cells and day 4 embryoid bodies (EBs). Transcripts from wild-type versus Smad4 null ES cells and day 4 EBs were analysed using Illumina arrays. In addition to several known TGF-β/BMP target genes, we identified numerous Smad4-dependent transcripts that are mis-expressed in the mutants. As expected, mesodermal cell markers were dramatically down-regulated. We also observed an increase in non-canonical potency markers (Pramel7, Tbx3, Zscan4), germ cell markers (Aire, Tuba3a, Dnmt3l) as well as early endoderm markers (Dpp4, H19, Dcn). Additionally, expression of the extracellular matrix (ECM) remodelling enzymes Mmp14 and Mmp9 was decreased in Smad4 mutant ES and EB populations. These changes, in combination with increased levels of laminin alpha1, cause excessive basement membrane deposition. Similarly, in the context of the Smad4 null E6.5 embryos we observed an expanded basement membrane (BM) associated with the thickened endoderm layer. Smad4 functional loss results in a dramatic shift in gene expression patterns and in the endodermal cell lineage causes an excess deposition of, or an inability to breakdown and remodel, the underlying BM layer. These structural abnormalities probably disrupt reciprocal signalling between the epiblast and overlying visceral endoderm required for gastrulation.