Functional Diversity of Robo Receptor Immunoglobulin Domains Promotes Distinct Axon Guidance Decisions

SummaryRecognition molecules of the immunoglobulin (Ig) superfamily control axon guidance in the developing nervous system. Ig-like domains are among the most widely represented protein domains in the human genome, and the number of Ig superfamily proteins is strongly correlated with cellular complexity [1]. In Drosophila, three Roundabout (Robo) Ig superfamily receptors respond to their common Slit ligand to regulate axon guidance at the midline: Robo and Robo2 mediate midline repulsion, Robo2 and Robo3 control longitudinal pathway selection, and Robo2 can promote midline crossing [2–5]. How these closely related receptors mediate distinct guidance functions is not understood. We report that the differential functions of Robo2 and Robo3 are specified by their ectodomains and do not reflect differences in cytoplasmic signaling. Functional modularity of Robo2's ectodomain facilitates multiple guidance decisions: Ig1 and Ig3 of Robo2 confer lateral positioning activity, whereas Ig2 confers promidline crossing activity. Robo2's distinct functions are not dependent on greater Slit affinity but are instead due in part to differences in multimerization and receptor-ligand stoichiometry conferred by Robo2's Ig domains. Together, our findings suggest that diverse responses to the Slit guidance cue are imparted by intrinsic structural differences encoded in the extracellular Ig domains of the Robo receptors

Repulsive Axon Guidance Abelson and Enabled Play Opposing Roles Downstream of the Roundabout Receptor

AbstractDrosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Little is known about the signaling mechanisms which function downstream of Robo to mediate repulsion. Here, we present genetic and biochemical evidence that the Abelson (Abl) tyrosine kinase and its substrate Enabled (Ena) play direct and opposing roles in Robo signal transduction. Genetic interactions support a model in which Abl functions to antagonize Robo signaling, while Ena is required in part for Robo's repulsive output. Both Abl and Ena can directly bind to Robo's cytoplasmic domain. A mutant form of Robo that interferes with Ena binding is partially impaired in Robo function, while a mutation in a conserved cytoplasmic tyrosine that can be phosphorylated by Abl generates a hyperactive Robo receptor

Islet Coordinately Regulates Motor Axon Guidance and Dendrite Targeting through the Frazzled/DCC Receptor

Motor neuron axon targeting in the periphery is correlated with the positions of motor neuron inputs in the CNS, but how these processes are coordinated to form a myotopic map remains poorly understood. We show that the LIM homeodomain factor Islet (Isl) controls targeting of both axons and dendrites in Drosophila motor neurons through regulation of the Frazzled (Fra)/DCC receptor. Isl is required for fra expression in ventrally projecting motor neurons, and isl and fra mutants have similar axon guidance defects. Single-cell labeling indicates that isl and fra are also required for dendrite targeting in a subset of motor neurons. Finally, overexpression of Fra rescues axon and dendrite targeting defects in isl mutants. These results indicate that Fra acts downstream of Isl in both the periphery and the CNS, demonstrating how a single regulatory relationship is used in multiple cellular compartments to coordinate neural circuit wiring

Signaling from Axon Guidance Receptors

Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote axon regeneration after injury or disease. Signaling mechanisms that act downstream of four prominent families of axon guidance cues—netrins, semaphorins, ephrins, and slits—have been extensively studied in both invertebrate and vertebrate model systems. Although details of these signaling mechanisms are still fragmentary and there appears to be considerable diversity in how different guidance receptors regulate the motility of the axonal growth cone, a number of common themes have emerged. Here, we review recent insights into how specific receptors for each of these guidance cues engage downstream regulators of the growth cone cytoskeleton to control axon guidance

Brain Tumor promotes axon growth across the midline through interactions with the microtubule stabilizing protein Apc2

<div><p>Commissural axons must cross the midline to establish reciprocal connections between the two sides of the body. This process is highly conserved between invertebrates and vertebrates and depends on guidance cues and their receptors to instruct axon trajectories. The DCC family receptor Frazzled (Fra) signals chemoattraction and promotes midline crossing in response to its ligand Netrin. However, in Netrin or <i>fra</i> mutants, the loss of crossing is incomplete, suggesting the existence of additional pathways. Here, we identify Brain Tumor (Brat), a tripartite motif protein, as a new regulator of midline crossing in the <i>Drosophila</i> CNS. Genetic analysis indicates that Brat acts independently of the Netrin/Fra pathway. In addition, we show that through its B-Box domains, Brat acts cell autonomously to regulate the expression and localization of Adenomatous polyposis coli-2 (Apc2), a key component of the Wnt canonical signaling pathway, to promote axon growth across the midline. Genetic evidence indicates that the role of Brat and Apc2 to promote axon growth across the midline is independent of Wnt and Beta-catenin-mediated transcriptional regulation. Instead, we propose that Brat promotes midline crossing through directing the localization or stability of Apc2 at the plus ends of microtubules in navigating commissural axons. These findings define a new mechanism in the coordination of axon growth and guidance at the midline.</p></div

The Homeodomain Transcription Factor Hb9 Controls Axon Guidance in Drosophila through the Regulation of Robo Receptors

Transcription factors establish neural diversity and wiring specificity; however, how they orchestrate changes in cell morphology remains poorly understood. The Drosophila Roundabout (Robo) receptors regulate connectivity in the CNS, but how their precise expression domains are established is unknown. Here, we show that the homeodomain transcription factor Hb9 acts upstream of Robo2 and Robo3 to regulate axon guidance in the Drosophila embryo. In ventrally projecting motor neurons, hb9 is required for robo2 expression, and restoring Robo2 activity in hb9 mutants rescues motor axon defects. Hb9 requires its conserved repressor domain and functions in parallel with Nkx6 to regulate robo2. Moreover, hb9 can regulate the medio-lateral position of axons through robo2 and robo3, and restoring robo3 expression in hb9 mutants rescues the lateral position defects of a subset of neurons. Altogether, these data identify Robo2 and Robo3 as key effectors of Hb9 in regulating nervous system development

Slit-Dependent Endocytic Trafficking of the Robo Receptor Is Required for Son of Sevenless Recruitment and Midline Axon Repulsion

<div><p>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 <i>in vitro</i> 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 <i>in vitro</i>; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity <i>in vivo</i>. 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.</p></div

Brat acts in parallel to the <i>Netrin-Fra</i> pathway.

<p>(A-D) Stage 15–16 embryos of the indicated genotypes carrying eg-GAL4 and UAS-tauMycGFP transgenes, stained with anti-GFP (green) (A-D) or anti-HRP (magenta) (A’-D’) antibodies. Anti-GFP labels cell bodies and axons of the eagle neurons (EG and EW), Anti-HRP reveals all of the CNS axons. Scale bar represents 10μm (A). Arrowheads indicate segments with non-crossing EW axons (A-D) or thin commissures (A’-D’). (A) EW neurons cross in the posterior commissure in 100% of segments in wild-type embryos. (A’) In every segment thick anterior and posterior commissures are formed as axons cross the midline. (B) In <i>fra</i> mutants EW neurons fail to cross in 36% of segments. (B’) <i>fra</i> mutants show thinner commissures. (C) and (C’) <i>brat</i> homozygous mutants show no obvious signs of commissural guidance defects: EW neurons fail to cross in only 4% of segments. (D) In <i>fra</i>, <i>brat</i> double mutants EW axons fail to cross the midline in 56% of segments. (D’) <i>fra</i>, <i>brat</i> double mutants also show thinner commissures. (E) Quantification of EW midline crossing defects in the genotypes shown in (B-D). Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed by multiple comparisons using ANOVA (^∗∗∗∗p < 0.0001). (F) Schematic diagrams of the EW axon trajectories observed in each genotype; the EW axons can cross the midline (Cross), grow ipsilaterally (Ipsi) or stall (Stall). (G, I, K) In <i>fra</i> mutants, 55% of the EW axons cross the midline (G), 29% grow ipsilaterally (I) and 16% remain stalled (K). (H, J, L) In <i>fra</i>, <i>brat</i> double mutants, 38% of the EW axons cross the midline (H), 28% grow ipsilaterally (J) and 34% stall (L). (M) Quantification of the distribution of the EW axon trajectories in the genotypes shown in (G-L). Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed using Chi-squared test (****p < 0.0001).</p

Midline crossing is sensitive to reduced Apc2 and Arm function and does not require Arm transcriptional activity.

<p>(A-D) Stage 15–16 embryos of the indicated genotype carrying eg-GAL4 and UAS-tauMycGFP transgenes, stained with anti-GFP (grey or green) (A-C) or anti-HRP (magenta) (D) antibodies. Anti-GFP labels cell bodies and axons of the eagle neurons (EG and EW), Anti-HRP reveals all of the CNS axons. Scale bar represents 10μm (A). Arrowheads indicate segments with non-crossing EW axons (A-C) or thin commissures (D). (A) In a FraΔC background the heterozygosity for <i>Apc2</i> enhances the EW crossing defects to 59%. (B) In the embryos double heterozygous for <i>Apc2</i> and <i>brat</i> expressing UAS-FraΔC selectively in eagle neurons, EW axons fail to cross in the posterior commissure in 72% of segments. (C) In <i>Apc2</i> and <i>brat</i> double mutant embryos, EW axons fail to cross in the posterior commissure in 20% of segments and show thinner commissures in some segments (D). (E) Quantification of EW midline crossing defects in the genotypes shown in (A-D). Df (2L) Exel6168 is a chromosomal deficiency containing <i>Apc2</i>. Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed by multiple comparisons using ANOVA (^∗∗∗∗p< 0.0001). (F) Quantification of EW midline crossing defects in the indicated genotypes. Data are presented as mean ± SEM. 20 embryos were scored for each genotype. Significance was assessed by multiple comparisons using ANOVA (^∗∗∗p < 0.001).</p

Apc2 expression co-localizes with EB1 in growing axon and cell bodies of Eagle neurons and is reduced in <i>brat</i> mutant embryos.

<p>(A-F’) Stage 13 and 16 embryos carrying eg-GAL4, UAS-Apc2GFP and UAS-EB1RFP transgenes, stained with anti-GFP (green) and anti-RFP (red) antibodies. Anti-GFP and anti-RFP label cell bodies and axons of the eagle neurons (EG and EW). Scale bar represents 10μm (A) or 2μm (C’ and F’). (A-C’) At stage 13, Apc2 and EB1 expression co-localize in the growing axon and the cell body of the Eagle neurons. (D-F’) At stage 16, Apc2 and EB1 expression co-localize in the elongated axon of the Eagle neurons. (G-H’) Stage 15–16 embryos of the indicated genotype carrying eg-GAL4 and UAS-Apc2GFP transgenes, stained with anti-GFP antibodies. Anti-GFP labels cell bodies and axons of the eagle neurons (EG and EW). Scale bar represents 10μm (G) or 5 μm (G’). (G) and (G’) In control embryos the average of the GFP signal intensity reflecting the Apc2 transgene expression, corresponds to 89% in cell bodies and 79% in axons. (H) and (H’) <i>brat</i> homozygous mutant embryos, show a decrease of the GFP signal intensity to 50% in cell bodies and 25% in axons, reflecting a reduction of the Apc2 transgene expression. (I) Quantification of the GFP staining signal intensity shown in (G-H’). Data are presented as mean ± SEM. 10 embryos were scored for each genotype. Significance was assessed using the Student’s t-test (^∗∗∗∗p < 0.0001).</p