Greg J. Bashaw

Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo’s cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo’s ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment. The formation of sterotyped neuronal connections during embryonic development is essential for animal survival and behavior. In particular, establishing proper connectivity at the midline is critical for the orchestration of rhythmic behaviors. Conserved genetic programs that instruct axon guidance at the midline have been characterized in multiple model systems, but the signaling mechanisms underlying axon guidance are not well understood. Slits and Robos comprise conserved families of axon guidance cues and receptors that control midline guidance by preventing inappropriate midline crossing. Here, we identify a novel mechanism that is required for Robo receptor activation and Robo-dependent axon repulsion in vivo. Using a combination of molecular genetic and cell biological approaches, we define a role for Slit-dependent trafficking of Robo from the plasma membrane to the early and late endosomes that contributes to Robo activation and signaling. In previous work, endocytic trafficking has been shown to modulate axon guidance responses by regulating surface levels of guidance receptors. In contrast, our observations indicate that endocytosis of the Robo receptor itself is required for receptor activation and precedes the recruitment of a key downstream signaling effector to the Robo receptor cytoplasmic domain.

During nervous system development, commissural axons cross the midline despite the presence of repellant ligands. In Drosophila, commissural axons avoid premature responsiveness to the midline repellant Slit by expressing the endosomal sorting receptor Commissureless, which reduces surface expression of the Slit receptor Roundabout1 (Robo1). In this study, we describe a distinct mechanism to inhibit Robo1 repulsion and promote midline crossing, in which Roundabout2 (Robo2) binds to and prevents Robo1 signaling. Unexpectedly, we find that Robo2 is expressed in midline cells during the early stages of commissural axon guidance, and that over-expression of Robo2 can rescue robo2-dependent midline crossing defects non-cell autonomously. We show that the extracellular domains required for binding to Robo1 are also required for Robo2's ability to promote midline crossing, in both gain-of-function and rescue assays. These findings indicate that at least two independent mechanisms to overcome Slit-Robo1 repulsion in pre-crossing commissural axons have evolved in Drosophila.

In bilaterally symmetric animals, the precise assembly of neural circuitry at the midline is essential for coordination of the left and right sides of the body. Commissural axons must first be directed across the midline and then be prevented from re‐crossing in order to ensure proper midline connectivity. Here, we review the attractants and repellents that direct axonal navigation at the ventral midline and the receptors on commissural neurons through which they signal. In addition, we discuss the mechanisms that commissural axons use to switch their responsiveness to midline‐derived cues, so that they are initially responsive to midline attractants and subsequently responsive to midline repellents. WIREs Dev Biol 2015, 4:377–389. doi: 10.1002/wdev.185This article is categorized under: Nervous System Development > Flies Nervous System Development > Vertebrates: General Principles

Transcription factors establish the tremendous diversity of cell types in the nervous system by regulating the expression of genes that give a cell its morphological and functional properties. Although many studies have identified requirements for specific transcription factors during the different steps of neural circuit assembly, few have identified the downstream effectors by which they control neuronal morphology. In this Review, we highlight recent work that has elucidated the functional relationships between transcription factors and the downstream effectors through which they regulate neural connectivity in multiple model systems, with a focus on axon guidance and dendrite morphogenesis.

To develop a functional nervous system, axons must initially navigate through a complex environment, directed by guidance ligands and receptors. These receptors must link to intracellular signaling cascades to direct axon pathfinding decisions. The Abelson tyrosine kinase (Abl) plays a crucial role in multiple Drosophila axon guidance pathways during development, though the mechanism by which Abl elicits a diverse set of guidance outputs is currently unknown. We identified Abl in a genetic screen for genes that contribute to Netrin-dependent axon guidance in midline-crossing (commissural) neurons. We find that Abl interacts both physically and genetically with the Netrin receptor Frazzled, and that disrupting this interaction prevents Abl from promoting midline axon crossing. Moreover, we find that Abl exerts its diverse activities through at least two different mechanisms: (1) a partly kinase-independent, structural function in midline attraction through its C-terminal F-actin binding domain (FABD) and (2) a kinase-dependent inhibition of repulsive guidance pathways that does not require the Abl C terminus. Abl also regulates motor axon pathfinding through a non-overlapping set of functional domains. These results highlight how a multifunctional kinase can trigger diverse axon guidance outcomes through the use of distinct structural motifs.

The phylogenetically conserved Netrin family of chemoattractants signal outgrowth and attractive turning of commissural axons through the Deleted in Colorectal Carcinoma (DCC) family of receptors. Src family kinases are thought to be major signaling effectors of Netrin/DCC. In vertebrates, Src and the closely related Fyn kinases phosphorylate DCC and form a receptor-bound signaling complex leading to activation of downstream effectors. Here we show that, in theDrosophilaembryonic CNS,Srckinases are dispensable for midline attraction of commissural axons. Consistent with this observation, tyrosine phosphorylation of the Netrin receptor DCC or itsDrosophilaortholog, Frazzled, is not necessary for attraction to Netrin. Moreover, we uncover an unexpected function of Src kinases: inhibition of midline axon crossing through a novel mechanism. We propose that distinct signaling outputs must exist for midline axon crossing independent of Src kinases in commissural neurons.