Analysis of the function of the nodal gene during embryo

Nodal是一种转化生长因子（TGF）-β样蛋白，我们是在逆转录病毒插入突变的基础上发现的，并显示出对早期发育至关重要。我们的研究表明，该突变在原肠胚形成中起主要作用。在过去的几年里，我们的主要重点一直是在发展后期的节点信号及其在左/右轴发展的作用。这项工作源于我们的关键观察，即nodal在中线节点周围不对称地表达，并且仅在早期体节阶段脊椎动物胚胎的左侧板中胚层中表达。这种不对称的表达模式使我们研究nodal是否调节器官的发育，如心脏和肺，这些器官在体内的结构或位置是不对称的。由于nodal插入突变在左/右发育之前破坏原肠胚形成时的发育，我们产生了侧接loxP位点（floxed）的nodal等位基因，以允许我们通过与在特定阶段和/或组织表达Cre重组酶的转基因小鼠杂交来有条件地删除nodal。出乎意料，但偶然的是，我们发现，floxed节点等位基因是亚型的。胚胎复合杂合的floxed和一个nodal无效等位基因经历原肠胚形成，但随后显示异常，在后期阶段，分为三个不同的表型类。这些结果是在不存在任何Cre介导的重组的情况下发现的。一个突变类的表型的综合分析，现在提供了确凿的证据，节点信号在适当的左/右不对称发展的心脏，肺，血管和胃中发挥的重要作用。这种亚型节点等位基因的功能降低也为我们进一步了解节点在其他发育过程中的功能提供了非常丰富的信息。我们对nodal hypomorph的研究已经确立了nodal在前/后体轴发育和前脑正确模式形成中的重要功能。我们的分析表明，节点在这些过程中起着两个作用。首先，原肠胚形成前的淋巴结信号对于特定的胚外细胞移动到邻近未来头部区域的位置是必不可少的。这些胚外细胞提供了最初的神经诱导信号，胚胎细胞将成为前脑。然后，在稍后阶段需要节点信令来建立节点。淋巴结是迁移到头部区域的细胞的来源，取代原始的胚外细胞，并提供第二个神经诱导信号。因此，我们对结节亚形的分析为两个重要的发育问题提供了关键的见解。 目前对原肠胚形成开始时中胚层形成过程中节点信号传导的最终结果的了解仍然有限。为了进一步理解，我们利用了P19多能胚胎癌细胞，其为早期发育期间发生的分化事件提供了细胞培养模型系统。我们发现过表达P19的细胞可向中胚层分化。我们利用P19细胞的反应性和重组nodal蛋白的可用性来剖析nodal信号通路的细胞内组分。我们的研究表明，结信号通过Smad2和Smad3。这些Smads也被TGF-β和激活素（另一种TGF-β样因子）使用。然而，我们也已经表明，nodal信号传导不同于TGF-β和激活素信号传导，需要EGF-CFC家族的细胞外蛋白的功能，无论是Cripto还是隐藏蛋白。在我们的实验室中开发了产生重组nodal蛋白的能力，我们现在正在解决nodal与候选受体和EGF-CFC蛋白的物理相互作用。我们还利用P19系统来确定早期胚胎细胞的基因表达谱如何响应nodal。我们在发育胚胎中表达的20，000个小鼠基因的微阵列筛选中比较了过表达nodal的P19细胞与正常P19细胞。这项研究揭示了许多基因的表达在这两个细胞群体中显着不同。我们正在确定是否有任何这些是直接节点的靶基因，通过分析表达在P19细胞简单处理蛋白质和表达在发展中的野生型和节点突变胚胎。例如，我们确定HoxB1是一个在过度表达nodal的P19细胞中下调的基因。早期HoxB1在正常胚胎中的表达完全重叠的节点，是异常低的节点亚型突变体。这是一个例子，说明微阵列方法将如何帮助我们对节点信号的生物学后果产生新的见解。

Nodal is a transforming growth factor (TGF)-beta like protein that we discovered on the basis of a retroviral insertional mutation and showed to be essential for early development. Our studies on the nodalnull mutation revealed a major role in gastrulation. Our primary emphasis in the past few years has been on nodal signaling later in development and its role in left/right axial development. This work stems from our key observation that nodal is expressed asymmetrically around the midline node and only in the left lateral plate mesoderm of early somite stage vertebrate embryos. This asymmetric expression pattern has led us to investigate whether nodal regulates the development of organs such as the heart and lungs that are asymmetric in structure or position within the body. Because the nodal insertional mutation disrupts development at gastrulation prior to left/right development, we generated a nodal allele flanked by loxP sites (floxed) to allow us to conditionally delete nodal by crossing to transgenic mice expressing Cre recombinase at specific stages and/or tissues. Unexpectedly, but fortuitously, we discovered that the floxed nodal allele is hypomorphic. Embryos compound heterozygous for the floxed and a nodal null allele undergo gastrulation but then display abnormalities at later stages that fall into three distinct phenotypic classes. These results were found in the absence of any Cre mediated recombination. Comprehensive analysis of the phenotype of one mutant class has now provided conclusive evidence of the essential role nodal signaling plays in the proper left/right asymmetric development of the heart, lungs, vasculature and stomach. The reduced function of this hypomorphic nodal allele also has been extremely informative for furthering our understanding of nodal function in other developmental processes. Our study of the nodal hypomorph has established an essential function for nodal in the development of the anterior/posterior body axis and correct patterning of the forebrain. Our analysis has shown that nodal plays two roles in these processes. First, nodal signaling prior to gastrulation is essential for the movement of specific extraembryonic cells to a position adjacent to the future head region. These extraembryonic cells provide the initial neural inducing signal to embryonic cells that will become the forebrain. Nodal signaling then is required at a later stage to establish the node. The node is the source of cells that migrate to the head region, replacing the original extraembryonic cells, and provide a second neural inducing signal. Thus our analysis of the nodal hypomorph has provided key insight into two important developmental problems. Current knowledge of the ultimate outcome of nodal signaling in the process of mesoderm formation at the start of gastrulation is still limited. To further our understanding we have utilized P19 pluripotent embryonal carcinoma cells, which provide a cell culture model system for differentiation events occurring during early development. We found that P19 cells overexpressing nodal differentiate into mesoderm. We exploited the responsiveness of P19 cells and the availability of recombinant nodal protein to dissect the intracellular components of the nodal signaling pathway. Our studies show that nodal signals through Smad2 and Smad3. These Smads are also used by TGF-beta and activin, another TGF-beta like factor. However, we also have shown that nodal signaling differs from TGF-beta and activin signaling in requiring the function of an extracellular protein of the EGF-CFC family, either Cripto or Cryptic. Having developed the capacity to generate recombinant nodal protein in our laboratory we are now addressing the physical interaction of nodal with candidate receptors and with EGF-CFC proteins. We are also exploiting the P19 system to determine how the gene expression profiles of early embryonic cells change in response to nodal. We have compared the P19 cells that overexpress nodal with normal P19 cells in a screen of micro-arrays of 20,000 mouse genes expressed in the developing embryo. This study has revealed a number of genes whose expression differs significantly in these two cell populations. We are determining whether any of these are direct nodal target genes by analyzing expression in P19 cells briefly treated with protein and expression in the developing wild-type and nodal mutant embryo. For instance, we identified HoxB1 as a gene downregulated in P19 cells overexpressing nodal. Early HoxB1 expression in the normal embryo completely overlaps that of nodal and is abnormally low in the nodal hypomorphic mutant. This is one example of how the microarray approach will help us to develop novel insights into the biological consequences of nodal signaling.