Tace/ADAM17 is a bi-directional regulator of axon guidance that coordinates distinct Frazzled and Dcc receptor signaling outputs.

Frazzled (Fra) and deleted in colorectal cancer (Dcc) are homologous receptors that promote axon attraction in response to netrin. In Drosophila, Fra also acts independently of netrin by releasing an intracellular domain (ICD) that activates gene transcription. How neurons coordinate these pathways to make accurate guidance decisions is unclear. Here we show that the ADAM metalloprotease Tace cleaves Fra, and this instructs the switch between the two pathways. Genetic manipulations that either increase or decrease Tace levels disrupt midline crossing of commissural axons. These conflicting phenotypes reflect Tace's function as a bi-directional regulator of axon guidance, a function conserved in its vertebrate homolog ADAM17: while Tace induces the formation of the Fra ICD to activate transcription, excessive Tace cleavage of Fra and Dcc suppresses the response to netrin. We propose that Tace and ADAM17 are key regulators of midline axon guidance by establishing the balance between netrin-dependent and netrin-independent signaling.

Frazzled/Dcc acts independently of Netrin to promote germline survival during Drosophila oogenesis.

The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. In this study, we report that Fra is required in the Drosophila female germline for the progression of egg chambers through mid-oogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, our observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis.

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

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 fra 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 Drosophila 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.

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.

The Intracellular Domain of the Frazzled/DCC Receptor Is a Transcription Factor Required for Commissural Axon Guidance.

In commissural neurons of Drosophila, the conserved Frazzled (Fra)/Deleted in Colorectal Cancer (DCC) receptor promotes midline axon crossing by signaling locally in response to Netrin and by inducing transcription of commissureless (comm), an antagonist of Slit-Roundabout midline repulsion, through an unknown mechanism. Here, we show that Fra is cleaved to release its intracellular domain (ICD), which shuttles between the cytoplasm and the nucleus, where it functions as a transcriptional activator. Rescue and gain-of-function experiments demonstrate that the Fra ICD is sufficient to regulate comm expression and that both γ-secretase proteolysis of Fra and Fra's function as a transcriptional activator are required for its ability to regulate comm in vivo. Our data uncover an unexpected role for the Fra ICD as a transcription factor whose activity regulates the responsiveness of commissural axons at the midline and raise the possibility that nuclear signaling may be a common output of axon guidance receptors.

Src inhibits midline axon crossing independent of Frazzled/Deleted in Colorectal Carcinoma (DCC) receptor tyrosine phosphorylation.

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 the Drosophila embryonic CNS, Src kinases are dispensable for midline attraction of commissural axons. Consistent with this observation, tyrosine phosphorylation of the Netrin receptor DCC or its Drosophila ortholog, 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.

A frazzled/DCC-dependent transcriptional switch regulates midline axon guidance.

Precise wiring of the nervous system depends on coordinating the action of conserved families of proteins that direct axons to their appropriate targets. Slit-roundabout repulsion and netrin-deleted in colorectal cancer (DCC) (frazzled) attraction must be tightly regulated to control midline axon guidance in vertebrates and invertebrates, but the mechanism mediating this regulation is poorly defined. Here, we show that the Fra receptor has two genetically separable functions in regulating midline guidance in Drosophila. First, Fra mediates canonical chemoattraction in response to netrin, and, second, it functions independently of netrin to activate commissureless transcription, allowing attraction to be coupled to the down-regulation of repulsion in precrossing commissural axons.

Cytoplasmic domain requirements for Frazzled-mediated attractive axon turning at the Drosophila midline.

The conserved DCC ligand-receptor pair Netrin and Frazzled (Fra) has a well-established role in axon guidance. However, the specific sequence motifs required for orchestrating downstream signaling events are not well understood. Evidence from vertebrates suggests that P3 is important for transducing Netrin-mediated turning and outgrowth, whereas in C. elegans it was shown that the P1 and P2 conserved sequence motifs are required for a gain-of-function outgrowth response. Here, we demonstrate that Drosophila fra mutant embryos exhibit guidance defects in a specific subset of commissural axons and these defects can be rescued cell-autonomously by expressing wild-type Fra exclusively in these neurons. Furthermore, structure-function studies indicate that the conserved P3 motif (but not P1 or P2) is required for growth cone attraction at the Drosophila midline. Surprisingly, in contrast to vertebrate DCC, P3 does not mediate receptor self-association, and self-association is not sufficient to promote Fra-dependent attraction. We also show that in contrast to previous findings, the cytoplasmic domain of Fra is not required for axonal localization and that neuronal expression of a truncated Fra receptor lacking the entire cytoplasmic domain (Fra delta C) results in dose-dependent defects in commissural axon guidance. These findings represent the first systematic dissection of the cytoplasmic domains required for Fra-mediated axon attraction in the context of full-length receptors in an intact organism and provide important insights into attractive axon guidance at the midline.

Independent functions of Slit-Robo repulsion and Netrin-Frazzled attraction regulate axon crossing at the midline in Drosophila.

Slit and Netrin and their respective neuronal receptors play critical roles in patterning axonal connections in the developing nervous system by regulating the decision of whether or not to cross the midline. Studies of both invertebrate and vertebrate systems support the idea that Netrin, secreted by midline cells, signals through DCC (Deleted in Colorectal Carcinoma)/UNC40/Frazzled receptors to attract commissural axons toward and across the midline, whereas Slit signals through Robo family receptors to prevent commissural axons from recrossing the midline, as well as to prevent ipsilateral axons from ever crossing. Recent evidence from both Xenopus neuronal cell culture and Drosophila genetics have suggested that these signals may interact more directly in a hierarchical relationship, such that one response extinguishes the other. Here we present loss- and gain-of-function genetic evidence showing that the influence of Slit and Netrin on midline axon crossing is dictated by both independent and interdependent signaling functions of the Robo and Frazzled (Fra) receptors. Our results are not consistent with the proposal based on genetic analysis in Drosophila that the sole function of Slit and Robo during midline guidance is to repress Netrin attraction.

Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline.

Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases and thus is uniquely poised to integrate signals that affect both gene expression and cytoskeletal reorganization. Here, using genetics, biochemistry, and cell biology, we demonstrate that Sos is recruited to the plasma membrane, where it forms a ternary complex with the Roundabout receptor and the SH3-SH2 adaptor protein Dreadlocks (Dock) to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand. Intriguingly, the Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity. These results provide in vivo evidence that the Ras and RhoGEF domains of Sos are separable signaling modules and support a model in which Robo recruits Sos to the membrane via Dock to activate Rac during midline repulsion.