TGF-β/Nodal信号通路在斑马鱼胚胎背腹分化过程中发挥重要作用。为了进一步探究该信号通路的功能及作用机制, 文章采用酵母双杂交的方法, 以斑马鱼Smad2/3a为诱饵蛋白筛选得到一系列Smad2/3a的互作蛋白, 其中之一为Rbb4l (Retinoblastoma binding protein 4, like)。已有的报道表明, Rbb4l的人类同源蛋白RBBP4 (Retinoblastoma binding protein 4) 是染色质修饰相关的复合体的组成成分, 但它在脊椎动物胚胎发育过程中的作用还知之甚少。文章通过体外及体内的一系列实验表明, Rbb4l能直接与Smad3a互作, 增强TGF-β/Nodal信号。在斑马鱼胚胎中过表达rbb4l导致胚胎的背部化, 伴随着背部标记基因表达区域的扩大和腹部标记基因表达区域的缩小。相反, 在胚胎中下调rbb4l的表达, 可以导致胚胎在24 hpf(hours post-fertilization)左右出现腹部化的表型。文章进一步通过一系列的挽救实验, 证明在缺少Nodal信号的情况下, rbb4l的过表达不能引起胚胎的背部化。综上所述, Rbb4l可以增强Nodal/Smad2/3信号, 并且这种正向调节的功能依赖于Nodal信号本身。
The TGF-β/Nodal signaling pathway plays an important role in the zebrafish dorsoventral patterning process. To further explore the function and mechanism of this signaling pathway, we identified a set of Smad2/3a interacting proteins by the yeast two-hybrid screen. Rbb4l (Retinoblastoma binding protein 4, like) is one of the identified proteins. Human RBBP4 (Retinoblastoma binding protein 4), the homolog of zebrafish Rbb4l, has been shown to form complexes with other chromatin modifiers, but its roles in embryonic development remain unknown. In this study, we showed that Rbb4l directly interacted with Smad3a and enhances TGF-β/Nodal signaling. In zebrafish embryos, rbb4l overexpression resulted in an expanded expression of dorsal markers with a reduction of ventral markers expression, suggesting a dorsalizing func-tion. On the contrary, rbb4l knockdown caused ventralized phenotype of the embryos at 24 hours post-fertilization (hpf). Furthermore, a series of rescue experiments showed that rbb4l failed to cause embryonic dorsalization in the absence of Nodal signal. Together, our data suggested that Rbb4l acts as an enhancer of Nodal/Smad2/3 signaling during embryogenesis, and depends on the existence of Nodal signaling.
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