Kenny Alan P, Oleksyn David W, Newman Laurel A, Angerer Robert C, Angerer Lynne M
Department of Biology, University of Rochester, Rochester, NY 14627, USA.
Dev Biol. 2003 Sep 15;261(2):412-25. doi: 10.1016/s0012-1606(03)00331-2.
Previous studies in sea urchin embryos have demonstrated that nuclearization of beta-catenin is essential for initial steps in the specification of endoderm and mesenchyme, which are derived from vegetal blastomeres. This process begins at the 4th and extends through the 9th cleavage stage, an interval in which the SpSoxB1 transcription regulator is downregulated by beta-catenin-dependent gene products that include the transcription repressor SpKrl. These observations raise the possibility that SpSoxB1 removal is required to allow vegetal development to proceed. Here we show that elevated and ectopic expression of this factor suppresses differentiation of all vegetal cell types, a phenotype that is very similar to that caused by the suppression of beta-catenin nuclear function by cadherin overexpression. Suppression of vegetal fates involves interference at the protein-protein level because a mutation of SpSoxB1 that prevents its binding to DNA does not significantly reduce this activity. Reduction in SpSoxB1 level results in elevated TCF/Lef-beta-catenin-dependent expression of a luciferase reporter gene in vivo, indicating that in the normal embryo this protein suppresses the primary vegetal signaling mechanism that is required for specification of mesenchyme and endoderm. Surprisingly, normal expression of SpSoxB1 is required for gastrulation and endoderm differentiation, as shown by both morpholino-mediated translational interference and expression of a dominant negative protein. Similar gain-of-function and loss-of-function assays of a closely related factor, SpSoxB2, demonstrate that it, too, is required for gastrulation and that its overexpression can suppress vegetal development. However, significant phenotypic differences are apparent in the two perturbations, indicating that SpSoxB1 and SpSoxB2 have at least some distinct developmental functions. The results of all these studies support a model in which the concentration of SpSoxB factors must be tightly regulated along the animal-vegetal axis of the early sea urchin embryo to allow beta-catenin-dependent specification of endoderm and mesenchyme cell fates as well as to activate target genes required for gastrulation.
先前对海胆胚胎的研究表明，β-连环蛋白的核化对于内胚层和间充质（均源自植物性卵裂球）特化的初始步骤至关重要。这个过程从第4次卵裂开始，一直持续到第9次卵裂阶段，在此期间，SpSoxB1转录调节因子被包括转录抑制因子SpKrl在内的β-连环蛋白依赖性基因产物下调。这些观察结果提出了一种可能性，即需要去除SpSoxB1才能使植物性发育继续进行。在这里，我们表明该因子的升高和异位表达会抑制所有植物性细胞类型的分化，这种表型与钙黏蛋白过表达抑制β-连环蛋白核功能所导致的表型非常相似。对植物性命运的抑制涉及蛋白质-蛋白质水平的干扰，因为阻止SpSoxB1与DNA结合的突变并不会显著降低这种活性。SpSoxB1水平的降低会导致体内荧光素酶报告基因的TCF/Lef-β-连环蛋白依赖性表达升高，这表明在正常胚胎中，这种蛋白质会抑制间充质和内胚层特化所需的主要植物性信号传导机制。令人惊讶的是，正如吗啉代介导的翻译干扰和显性负性蛋白的表达所显示的那样，原肠胚形成和内胚层分化需要SpSoxB1的正常表达。对一个密切相关的因子SpSoxB2进行的类似功能获得和功能丧失分析表明，它对于原肠胚形成也是必需的，并且其过表达可以抑制植物性发育。然而，在这两种干扰中明显存在显著的表型差异，这表明SpSoxB1和SpSoxB2至少具有一些不同的发育功能。所有这些研究结果支持了一个模型，即在早期海胆胚胎的动物性-植物性轴上，必须严格调节SpSoxB因子的浓度，以允许β-连环蛋白依赖性地指定内胚层和间充质细胞命运，并激活原肠胚形成所需的靶基因。
