Analysis of the function of the nodal gene during embryo

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，在原肠形成中发挥重要作用。我们最近在小鼠胚胎发育中节点信号方面的工作主要集中在分析一种功能降低的靶向突变等位基因(低晶型)。这种亚型等位基因和完全零等位基因的复合杂合子的胚胎能够在原肠形成时发育通过零等位基因纯合子所见的原肠形成障碍，但随后表现出各种发育异常。这使得我们能够确定结节在原肠形成开始后发生的发育过程中的关键作用，包括前/后和左/右身体轴的图案化，以及中线中胚层和肠道内胚层的发育。然而，由于在节点表达的许多区域中的每一个区域都存在普遍的活性降低，我们的分析没有提供这些发育事件所需的节点表达的洞察。为了进一步剖析节点功能的这些不同的时间和空间域，我们产生了一个条件突变(花)等位基因。携带该等位基因的小鼠与表达Cre重组酶的转基因菌株一起用于发育中的胚胎的不同区域，这是我们从其他研究人员那里开发或获得的。用一种在原肠胚前胚性上皮细胞中表达Cre的菌株饲养小鼠获得的胚胎的初步分析显示，这种亚型等位基因的所有表型都与先前观察到的相同。早期结节功能可能不需要在胚外谱系中表达，现在正在通过使用一种在胚外内脏内胚层特异表达Cre的菌株来解决这一问题。其他正在进行的工作是对具有其他组织特异性的Cre菌株进行的。

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, playing a major role in gastrulation. Our recent work on nodal signaling in mouse embryonic development has focused on the analysis of a targeted mutant allele with reduced function (a hypomorph). Embryos that are compound heterozygotes for this hypomorphic allele and a completely null allele are capable of developing past the block at gastrulation seen in homozygotes for the null allele, but then show a variety of developmental abnormalities. This has allowed us to establish a critical role for nodal in developmental processes occurring after gastrulation begins, including patterning of the anterior/posterior and left/right body axes, and the development of the midline mesendoderm and gut endoderm. However, because there is a generalized reduction of activity in each of the many domains of nodal expression our analysis did not provide insight into where nodal expression is required for these developmental events. To dissect these various temporal and spatial domains of nodal function further, we generated a conditional mutant (floxed) allele. Mice carrying this allele are being used in conjunction with transgenic strains expressing Cre recombinase in various regions of the developing embryo, which we have developed or obtained from other investigators. Preliminary analysis of embryos obtained from breeding floxed mice with a strain expressing Cre in the pregastrulation embryonic epiblast, the cells that will give rise to all the germ layers, has revealed all of the same phenotypes observed earlier for the hypomorphic allele. The possibility that expression in extraembryonic lineages may not be required for early nodal function is now being addressed by the use of a strain expressing Cre specifically in the extraembryonic visceral endoderm. Additional ongoing work is being performed with Cre strains with other tissue specificities.