Analysis of the function of the nodal gene during embryonic development

Nodeal是我们在逆转录病毒插入突变的基础上发现的一种转化生长因子(TGF)-β样蛋白，被证明是早期发育所必需的。我们对结节零突变的研究揭示了它在原肠胚形成中的重要作用。结节在发育后期、结节周围和左侧板中胚层也有表达。然而，零突变体的早期致命性阻碍了在这些其他时间和地点对节点功能的遗传分析。因此，我们利用Cre/loxP策略制造了一个条件突变等位基因。在描述这种“花状”等位基因的过程中，我们发现它是亚型的。携带亚形等位基因和空等位基因的胚胎通过原肠胚发育，但在后期表现出异常的表型。在胚胎第7.5天(E)，一个子集显示一个错位的原始条纹和结节，这为结节在前后轴发育中的意外功能提供了证据。到E8.5时，有三种不同的突变类型明显，每一种都显示出不同程度的前部图案缺陷。一类也完全缺乏节点的左LPM表达。这些胚胎也缺乏正常的Pitx2、Lefty-1和Lefty-2的不对称表达，表现为心脏随机的左右不对称、大血管转位、肺的右异构性、中线或反胃。这一详细的分析为Node在内脏器官左右手不对称的发展中所起的关键作用提供了确凿的遗传学证据。第二类缺乏结节周围的表达，并显示严重的中线缺陷，包括融合的体节。这是一种新的表型，提供了对节点在中线的作用的第一次洞察。然而，这一类也缺乏侧板表达。为了确认是结节周围的结节丢失导致了中线缺陷，我们利用了在胚胎这一区域表达Cre的转基因小鼠和小鼠的结节。结节最早的关键功能之一是在原肠形成开始时形成中胚层。为了剖析原肠形成过程中结节信号的生化成分，我们利用P19多能胚胎癌细胞和重组结节蛋白建立了细胞培养模型系统。我们发现，节点信号激活了Par3-Lux，这是一种荧光素酶报告，以前被证明专门对激活素和转化生长因子-β做出反应。然而，Node不能诱导pTlx2-Lux，这是一个对骨形态发生蛋白有特异性反应的报告基因。我们还证明了Node诱导p(CagA)12，这是一个先前被SMAD3特异性激活的报告。显性负性Smad2的表达显著降低了根瘤处理诱导的荧光素酶报告活性水平。最后，我们发现节点信号迅速导致Smad2的磷酸化。这些结果提供了第一个直接的生化证据，证明节点信号是由激活素/转化生长因子-β途径Smads、Smad2和Smad3共同介导的。我们还发现，EGF/CFC蛋白Cripto和CRYPTIC是节点信号所必需的，这一途径与激活素或转化生长因子-β信号通路不同。我们现在正在使用这个系统来鉴定和分离结节靶基因。我们正在筛选代表15,000个在发育中的胚胎中表达的小鼠基因的微阵列，以识别在P19细胞中表达的基因，这些基因在结节治疗后表达水平发生变化。

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 nodal null mutation revealed a major role in gastrulation. Nodal also is expressed later in development, around the node and in the left lateral plate mesoderm. However, the early lethality of the null mutant has prevented genetic analysis of nodal function at these other times and places. Therefore we made a conditional mutant allele, using a Cre/loxP strategy. In the course of characterizing this "floxed" allele we discovered that it is hypomorphic. Embryos carrying the hypomorphic allele and a null allele progress through gastrulation but then display abnormal phenotypes at later stages. At embryonic day (E) 7.5 a subset show a mis-positioned primitive streak and node, providing evidence for an unsuspected function for nodal in the development of the anterior-posterior axis. By E8.5 three distinct mutant classes are evident, each showing anterior patterning defects of varying degrees of severity. One class also completely lacks left LPM expression of nodal. These embryos also lack normal asymmetric expression of Pitx2,lefty-1and lefty-2,and exhibit random left-right asymmetry of the heart, transpositions of the great vessels, right isomerism of the lungs, and midline or reversed stomachs. This detailed analysis has provided conclusive genetic evidence for nodal's essential role in the development of left-right handed asymmetry of the visceral organs. A second class lacks nodal expression around the node and shows severe midline defects, including fused somites. This is a novel phenotype providing the first insight into nodal's role in the midline. However, this class also lacks lateral plate expression. To confirm that it is the loss of nodal from around the node that leads to the midline defects, we are utilizing the floxed nodal mice in conjunction with transgenic mice that express Cre in this region of the embryo. One of the earliest critical functions of nodal is in the formation of mesoderm at the initiation of gastrulation. To dissect the biochemical components of nodal signaling during gastrulation, we established a cell culture model system utilizing P19 pluripotent embryonal carcinoma cells and recombinant nodal protein. We found that nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and TGF-beta. However, nodal was unable to induce pTlx2-Lux, a reporter specifically responsive to bone morphogenetic proteins. We also demonstrated that nodal induces p(CAGA)12, a reporter previously shown to be specifically activated by Smad3. Expression of a dominant negative Smad2 significantly reduced the level of luciferase reporter activity induced by nodal treatment. Finally, we showed that nodal signaling rapidly leads to the phosphorylation of Smad2. These results provide the first direct biochemical evidence that nodal signaling is mediated by both activin/TGF-beta pathway Smads, Smad2 and Smad3. We also showed that the EGF/CFC proteins cripto and cryptic are required for nodal signaling, distinguishing this pathway from activin or TGF-beta signaling. We are now using this system to identify and isolate nodal target genes. We are screening microarrays representing 15,000 mouse genes expressed in the developing embryo to identify genes expressed in P19 cells that undergo changes in expression levels following nodal treatment.