Drosophila Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors. Robo receptors signal midline repulsion in response to Slit ligands, which bind to the N-terminal Ig1 domain in most family members. In the Drosophila embryonic ventral nerve cord, Robo1 and Robo2 signal Slit-dependent midline repulsion, while Robo2 also regulates the medial-lateral position of longitudinal axon pathways and acts non-autonomously to promote midline crossing of commissural axons. While Robo2 signals midline repulsion in response to Slit, it is less clear whether Robo2's other activities are also Slit-dependent. To determine which of Robo2's axon guidance roles depend on its Slit-binding Ig1 domain, we used a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategy to replace the endogenous robo2 gene with a robo2 variant lacking the Ig1 domain (robo2∆Ig1). We compare the expression and localization of Robo2∆Ig1 protein with full-length Robo2 in embryonic neurons in vivo and examine its ability to substitute for Robo2 to mediate midline repulsion and lateral axon pathway formation. We find that the removal of the Ig1 domain from Robo2∆Ig1 disrupts both of these axon guidance activities. In addition, we find that the Ig1 domain of Robo2 is required for its proper subcellular localization in embryonic neurons, a role that is not shared by the Ig1 domain of Robo1. Finally, we report that although FasII-positive lateral axons are misguided in embryos expressing Robo2∆Ig1, the axons that normally express Robo2 are correctly guided to the lateral zone, suggesting that Robo2 may guide lateral longitudinal axons through a cell non-autonomous mechanism.

中文翻译：

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果蝇 Robo2 的多个轴突引导活动需要狭缝结合 Ig1 域

果蝇Robo2 是进化上保守的 Roundabout (Robo) 轴突引导受体家族的成员。Robo 受体响应 Slit 配体发出中线排斥信号，Slit 配体与大多数家族成员的 N 末端 Ig1 结构域结合。在果蝇胚胎腹神经索、Robo1 和 Robo2 发出 Slit 依赖的中线排斥信号，而 Robo2 还调节纵向轴突通路的内侧-外侧位置，并非自主地促进连合轴突的中线交叉。虽然 Robo2 对 Slit 发出中线排斥的信号，但 Robo2 的其他活动是否也依赖于 Slit 尚不清楚。为了确定 Robo2 的哪些轴突引导作用取决于其 Slit 结合 Ig1 结构域，我们使用基于聚集的规则间隔短回文重复 (CRISPR)/Cas9 的策略将内源性robo2基因替换为缺乏 Ig1 结构域的robo2变体(robo2 ΔIg1). 我们比较了 Robo2ΔIg1 蛋白与全长 Robo2 在体内胚胎神经元中的表达和定位，并检查了其替代 Robo2 介导中线排斥和侧轴突通路形成的能力。我们发现从 Robo2ΔIg1 中去除 Ig1 域会破坏这两种轴突引导活动。此外，我们发现 Robo2 的 Ig1 结构域是其在胚胎神经元中正确亚细胞定位所必需的，而 Robo1 的 Ig1 结构域不具有这种作用。最后，我们报告说，尽管 FasII 阳性侧轴突在表达 Robo2ΔIg1 的胚胎中被误导，通常表达 Robo2 的轴突被正确引导到外侧区域，这表明 Robo2 可能通过细胞非自主机制引导横向纵向轴突。