在胚胎早期发育过程中，背部决定与体轴建立同时受到母源与合子期多个信号通路的协同调控。伴随着胚胎发育早期剧烈的细胞增殖、分化与运动，胚胎逐渐形成一个具备背腹轴线、前后轴线以及左右轴线，可以行使正常功能的个体。胚胎的背部决定与体轴建立对胚胎的正常发育与图式形成具有决定性意义，因此，对该过程的研究具有重要意义。 斑马鱼是一种适合进行遗传筛选的模式生物，早期大量的突变与筛选工作为了解背腹轴线确立的机制提供了重要信息。很多重要的合子信号通路，包括Wnt、Bmp、Nodal和Fgf信号通路，共同参与调控脊椎动物背腹轴线的建立。但是，由于母源因子的研究有诸多需要克服的困难，至今对母源因子是如何调控背部决定与体轴建立的过程知之甚少，仍有很多亟待解决的问题。 本研究发现了一个斑马鱼母源自发突变体Mhuluwatsu01sm，其中si:dkey-121h17.7基因的上游调控区自发产生了一个约7.3 kb的大片段插入突变。该突变导致其纯合突变体雌鱼产生的后代均为严重腹部化表型胚胎，缺乏背部与头部组织。母源突变体Mhuluwatsu01sm胚胎没有斑马鱼的组织中心（胚盾）形成，且检测不到背部特异基因表达。通过体外mRNA合成注射的挽救实验以及CRISPR/Cas9介导的si:dkey-121h17.7突变体，确认了该表型是由于si:dkey-121h17.7基因表达缺失引起的。胚胎免疫荧光结果显示，内源Huluwa蛋白在囊胚早期定位于胚盘边缘区未来背侧的部分细胞的细胞膜上。同时，研究表明过表达huluwa可以激活背部基因表达，并可高效地诱导体轴形成。在野生型胚胎中注射huluwa mRNA可以诱导第二体轴形成。进一步研究显示 Huluwa可以促进β-catenin的入核，进而激活背部特异基因的表达。最后，本研究探讨了Huluwa与经典的Wnt/β-catenin信号通路中Wnt受体与配体的关系。研究表明，Huluwa通过β-catenin影响背部决定，且此过程可能不依赖于Wnt配体与受体。 综上所述，本研究报道了一个母源突变体Mhuluwa，其si:dkey-121h17.7基因突变导致胚胎完全腹部化表型。Huluwa蛋白在囊胚早期定位于胚胎未来背侧并可在没有Wnt配体激活受体的情况下促进β-catenin入核，激活背部特异基因在胚胎背侧表达，最终决定背部命运与体轴形成。
During early embryogenesis, dorsal determination and axis formation are controlled by both maternal and zygotic signaling pathways. Three major body axes, including dorsoventral axis, anteroposterior axis and left-right axis, are formed during embryogenesis through cell proliferation, differentiation and movement. Dorsal determination and body axis formation are essential for embryo development. Therefore, it is meaningful to investigate how dorsal side is determined. Zebrafish is a model organism suitable for genetic screenings, which have provided abundant information on axis formation. Marternal and zygotic signaling pathways, including Wnt, Bmp, Nodal and Fgf signaling, coordinate vertebrate dorsoventral axis formation. However, it remains largely unknown how maternal factors regulate dorsal determination. Here we demonstrate a zebrafish maternal-effect huluwatsu01sm mutant, which carries a 7.3 kilobase insertion upstream of si:dkey-121h17.7 gene. All offspring of homozygous mutant female fish exhibit severe ventralized phenotype. Maternal huluwatsu01sm mutant embryos lack organizer formation and dorsal marker gene expression. The mutated gene was confirmed by both si:dkey-121h17.7 mRNA injection and CRISPR/Cas9-mediated mutant generation. Huluwa protein is located on cell membrane of dorsal margin cell at blastula stage. huluwa overexpression can induce dorsal marker expression and body axis formation. Further research revealed that Huluwa affects nuclear localization of β-catenin independent of Wnt ligands and receptors. Together, we studied a previously uncharacterized maternal-effect mutant huluwa. The mutation disrupts maternal expression of si:dkey-121h17.7 and leads to severe ventralizion. Huluwa protein is located in the future dorsal region at early blastula stage and can promote dorsal determination in a Wnt ligand/receptor-independent way. It can induce β-catenin nuclear translocation and therefore induce dorsal-specific gene expression, dorsal determination and dorsoventrol axis formation.
