ANALYSIS OF THE FUNCTION OF THE NODAL GENE DURING EMBRYONIC DEVELOPMENT

我们对小鼠在原肠胚形成前引起胚胎停滞的突变进行了分析，鉴定并分离出了nodal基因，这是一种编码tgf - β分泌信号分子超家族成员的基因。我们发现，将体外合成的淋巴结mRNA注射到青蛙或鱼类胚胎中，或用重组淋巴结蛋白处理青蛙胚胎，可将细胞重定向到中胚层，这表明在正常小鼠发育过程中，淋巴结是一种中胚层诱导因子。我们还表明，在胚胎发育后期，在左右（L/R）身体轴的建立过程中，淋巴结具有重要作用。所有脊椎动物都有典型的、保守的左/右内脏不对称，例如左心。最近，来自几个不同实验室的研究已经开始解释胚胎左/右轴是如何建立的。结果表明，在正常形态的L/R不对称出现之前，nodal是鸡胚中沿L/R轴不对称表达的少数基因之一。这种不对称表达在进化上是保守的：在相似阶段和位置的小鼠胚胎中，nodal是不对称表达的，就像Xenopus nodal相关-1 （Xnr-1）在青蛙胚胎中一样。在对小鸡进行的进一步实验中，对胚胎进行了操作，使nodal在身体的左右两侧对称地表达，或者使nodal完全不表达。在两组实验中，大约一半的胚胎发育为右侧心脏。这些结果表明，节点不是心脏形成所必需的，相反，它的不对称表达是心脏发育具有特征极性的必要条件。我们检查了内脏逆位（iv）小鼠突变体中淋巴结的表达，其中L/R发育是随机的（大约一半的iv小鼠心脏位置正常，而一半的心脏位置相反）。在单个iv胚胎中，发现淋巴结表达要么对称（两侧或两侧均不对称），要么不对称（仅左侧或仅右侧）。因此，iv基因的突变通过随机化淋巴结表达的位置来干扰L/R的发展。最近，我们一直在研究另外4种显示L/R发育改变的突变体。这些突变包括Ft、Mgat、nt和SIL位点的突变。我们分析了所有四个菌株的淋巴结表达，再次发现了改变的模式：Mgat和nt像iv一样随机，而所有SIL和Ft胚胎都在两侧表达淋巴结。目前，我们正在更详细地分析iv、SIL和Mgat突变体，以了解为什么它们对淋巴结表达的影响如此不同。与iv相比，我们在Mgat和SIL突变体中发现沿胚胎中部的胚胎结构存在缺陷，但缺陷发生的时间和性质不同。在SIL突变体中一个独特的发现是缺少sonic hedgehog （shh）信号转导通路的活性，这是许多发育过程的重要组成部分。我们目前正在研究SIL在shh通路中的作用，以及淋巴结表达的双侧对称性是由于shh信号中断、中线结构缺陷还是两者兼而有之。这些结果进一步支持了节点在解释和传递脊椎动物的L/R模式信息中所起的关键作用。-动物模型，细胞增殖，发育，胚胎干细胞，遗传学，生长因子，原位杂交，蛋白质折叠，受体，-既不是人类受试者，也不是人类组织

Our analysis of a mouse mutation causing embryonic arrest prior to gastrulation led to the identification and isolation of nodal, a gene encoding a member of the TGF-beta superfamily of secreted signaling molecules. We found that injection of in vitro synthesized nodal mRNA into frog or fish embryos, or treatment of frog embryos with recombinant nodal protein, redirected cells to a mesodermal fate, suggesting that nodal acts as a mesoderm inducing factor during normal mouse development. We have also shown that nodal has an important role later in embryonic development, during the establishment of the left- right (L/R) body axis. All vertebrates have characteristic and conserved L/R visceral asymmetries, for example the left-sided heart. Recently, studies from several different labs have begun to explain how the embryonic L/R axis is established. We showed that nodal is one of a small number of genes expressed asymmetrically along the L/R axis in the chick embryo before the appearance of normal morphological L/R asymmetry. This asymmetric expression has been evolutionarily conserved: nodal is asymmetrically expressed in mouse embryos at similar stages and locations, as is Xenopus nodal related-1 (Xnr-1) in frog embryos. In further experiments in the chick, embryos were manipulated so that nodal was expressed symmetrically on both the left and right sides of the body, or so that nodal was not expressed at all. In both sets of experiments, approximately half the embryos developed a right-sided heart. These results have shown that nodal is not required for the heart to form, rather its asymmetric expression is necessary for the heart to develop with characteristic polarity. We examined nodal expression in the situs inversus viscerum (iv) mouse mutant in which L/R development is randomized (approximately half of iv mice have normal heart placement while half have reversed heart location). In individual iv embryos, nodal expression was found to be either symmetric (on both sides or on neither side), or asymmetric (on the left side only or on the right side only). Thus, mutations in the iv gene disturb L/R development by randomizing the location of nodal expression.More recently, we have been studying 4 additional mutants that show altered L/R development. These include mutations at the Ft, Mgat, nt and SIL loci. We analyzed nodal expression in all four strains and again found altered patterns: Mgat and nt were random like iv whereas all SIL and Ft embryos expressed nodal on both sides. Currently, we are analyzing iv, SIL and Mgat mutants in greater detail to understand why each affects nodal expression so differently. In contrast to iv, we found defects in embryonic structures along the middle of the embryo in Mgat and SIL mutants, but the timing and nature of the defects were different. A unique finding in SIL mutants was the lack of activity of the sonic hedgehog (shh) signal transduction pathway, an essential component of a number of developmental processes. We are now studying the role of SIL in the shh pathway and whether bilateral symmetry of nodal expression results from disrupted shh signaling, defective midline structures or both. These results further support the critical role nodal plays in interpreting and relaying L/R patterning information in vertebrates. - Animal models, cell proliferation, Development, embryonic stem cells, Genetics, growth factors, in situ hybridization, protein folding, receptors, - Neither Human Subjects nor Human Tissues