Vegfc/Vegfr3 signaling is critical for lymphangiogenesis, the sprouting of lymphatic vessels. In zebrafish, cells sprouting from the posterior cardinal vein can either form lymphatic precursor cells or contribute to intersegmental vein formation. Both, the Vegfc-dependent differential induction of Prox1a in sprouting cells as well as a Notch-mediated pre-pattern within intersegmental vessels have been associated with the regulation of secondary sprout behavior. However, how exactly a differential lymphatic versus venous sprout cell behavior is achieved is not fully understood. Here, we characterize a zebrafish mutant in the adaptor protein Grb2b, and demonstrate through genetic interaction studies that Grb2b acts within the Vegfr3 pathway. Mutant embryos exhibit phenotypes that are consistent with reduced Vegfr3 signaling outputs prior to the sprouting of endothelial cells from the vein. During secondary sprouting stages, loss of grb2b leads to defective cell behaviors resulting in a loss of parachordal lymphangioblasts, while only partially affecting the number of intersegmental veins. A second GRB2 zebrafish ortholog, grb2a, contributes to the development of lymphatic structures in the meninges and in the head, but not in the trunk. Our results illustrate an essential role of Grb2b in vivo for cell migration to the horizontal myoseptum and for the correct formation of the lymphatic vasculature, while being less critically required in intersegmental vein formation. Thus, there appear to be higher requirements for Grb2b and therefore Vegfr3 downstream signaling levels in lymphatic versus vein precursor-generating sprouts.

Vegfc/Vegfr3 信号传导对于淋巴管生成（淋巴管的萌芽）至关重要。在斑马鱼中，从后主静脉发芽的细胞既可以形成淋巴前体细胞，也可以促进节间静脉的形成。两者，发芽细胞中 Prox1a 的 Vegfc 依赖性差异诱导以及节间血管内 Notch 介导的预模式都与二次发芽行为的调节有关。然而，如何准确地实现不同的淋巴与静脉芽细胞行为尚不完全清楚。在这里，我们描述了接头蛋白 Grb2b 中的斑马鱼突变体，并通过遗传相互作用研究证明 Grb2b 在 Vegfr3 通路中起作用。突变胚胎表现出的表型与血管内皮细胞萌发之前减少的 Vegfr3 信号输出一致。在二次发芽阶段，损失grb2b导致有缺陷的细胞行为，导致腱旁淋巴母细胞丢失，而仅部分影响节间静脉的数量。第二个 GRB2 斑马鱼直系同源物grb2a有助于脑膜和头部淋巴结构的发育，但不影响躯干。我们的结果说明了 Grb2b 在体内对细胞迁移到水平肌隔膜和淋巴脉管系统的正确形成的重要作用，同时在节间静脉形成中的要求较低。因此，似乎对 Grb2b 和 Vegfr3 下游信号水平在淋巴与静脉前体产生的芽中的要求更高。