Comm sorts robo to control axon guidance at the Drosophila midline

Axon growth across the Drosophila midline requires Comm to downregulate Robo, the receptor for the midline repellent Slit. We show here that comm is required in neurons, not in midline cells as previously thought, and that it is expressed specifically and transiently in commissural neurons. Comm acts as a sorting receptor for Robo, diverting it from the synthetic to the late endocytic pathway. A conserved cytoplasmic LPSY motif is required for endosomal sorting of Comm in vitro and for Comm to downregulate Robo and promote midline crossing in vivo. Axon traffic at the CNS midline is thus controlled by the intracellular trafficking of the Robo guidance receptor, which in turn depends on the precisely regulated expression of the Comm sorting receptor.     

Gliolectin-mediated carbohydrate binding at the Drosophila midline ensures the fidelity of axon pathfinding.

Gliolectin is a carbohydrate-binding protein (lectin) that mediates cell adhesion in vitro and is expressed by midline glial cells in the Drosophila melanogaster embryo. Gliolectin expression is maximal during early pathfinding of commissural axons across the midline (stages 12-13), a process that requires extensive signaling and cell-cell interactions between the midline glia and extending axons. Deletion of the gliolectin locus disrupts the formation of commissural pathways and also delays the completion of longitudinal pathfinding. The disruption in commissure formation is accompanied by reduced axon-glial contact, such that extending axons grow on other axons and form a tightly fasciculated bundle that arches over the midline. By contrast, pioneering commissural axons normally cross the midline as a distributed array of fibers that interdigitate among the midline glia, maximizing contact and, therefor, communication between axon and glia. Restoration of Gliolectin protein expression in the midline glia rescues the observed pathfinding defects of null mutants in a dose-dependent manner. Hypomorphic alleles generated by ethylmethanesulfonate mutagenesis exhibit a similar phenotype in combination with a deletion and these defects are also rescued by transgenic expression of Gliolectin protein. The observed phenotypes indicate that carbohydrate-lectin interactions at the Drosophila midline provide the necessary surface contact to capture extending axons, thereby ensuring that combinatorial codes of positive and negative growth signals are interpreted appropriately.     

VEGF shows its attractive side at the midline

Vascular endothelial growth factor (VEGF) family members are best known for their powerful mitotic and angiogenic activities toward endothelial cells. Two independent studies in this issue of Neuron now provide compelling evidence that VEGF-A secreted at the CNS midline functions as an attractant for developing axons of spinal commissural neurons and contralaterally projecting retinal ganglion cells.     

Receptor tyrosine phosphatases regulate axon guidance across the midline of the Drosophila embryo

Neural receptor-linked protein tyrosine phosphatases (RPTPs) are required for guidance of motoneuron and photoreceptor growth cones in Drosophila. These phosphatases have not been implicated in growth cone responses to specific guidance cues, however, so it is unknown which aspects of axonal pathfinding are controlled by their activities. Three RPTPs, known as DLAR, DPTP69D, and DPTP99A, have been genetically characterized thus far. Here we report the isolation of mutations in the fourth neural RPTP, DPTP10D. The analysis of double mutant phenotypes shows that DPTP10D and DPTP69D are necessary for repulsion of growth cones from the midline of the embryonic central nervous system. Repulsion is thought to be triggered by binding of the secreted protein Slit, which is expressed by midline glia, to Roundabout (Robo) receptors on growth cones. Robo repulsion is downregulated by the Commissureless (Comm) protein, allowing axons to cross the midline. Here we show that the Rptp mutations genetically interact with robo, slit and comm. The nature of these interactions suggests that DPTP10D and DPTP69D are positive regulators of Slit/Roundabout repulsive signaling. We also show that elimination of all four neural RPTPs converts most noncrossing longitudinal pathways into commissures that cross the midline, indicating that tyrosine phosphorylation controls the manner in which growth cones respond to midline signals.     

A Drosophila homolog of cyclase-associated proteins collaborates with the Abl tyrosine kinase to control midline axon pathfinding

We demonstrate that Drosophila capulet (capt), a homolog of the adenylyl cyclase-associated protein that binds and regulates actin in yeast, associates with Abl in Drosophila cells, suggesting a functional relationship in vivo. We find a robust and specific genetic interaction between capt and Abl at the midline choice point where the growth cone repellent Slit functions to restrict axon crossing. Genetic interactions between capt and slit support a model where Capt and Abl collaborate as part of the repellent response. Further support for this model is provided by genetic interactions that both capt and Abl display with multiple members of the Roundabout receptor family. These studies identify Capulet as part of an emerging pathway linking guidance signals to regulation of cytoskeletal dynamics and suggest that the Abl pathway mediates signals downstream of multiple Roundabout receptors.     

The Drosophila RNA-binding protein ELAV is required for commissural axon midline crossing via control of commissureless mRNA expression in neurons

Drosophila ELAV is the founding member of an evolutionarily conserved family of RNA-binding proteins considered as key inducers of neuronal differentiation. Although several ELAV-specific targets have been identified, little is known about the role of elav during neural development. Here, we report a detailed characterization of the elav mutant commissural phenotype. The reduced number of commissures in elav mutant embryos is not due to loss or misspecification of neural cells but results from defects in commissural axon projections across the midline. We establish a causal relationship between the elav mutant commissural phenotype and a reduction in the expression of commissureless, a key component of the Robo/Slit growth cone repulsive signalling pathway. In the nerve cord of elav mutant embryos, comm mRNA expression is strongly reduced in neurons, but not in midline glial cells. Furthermore, specific expression of an elav transgene in posterior neurons of each segment of an elav mutant nerve cord restores comm mRNA expression in these cells, as well as the formation of posterior commissures. Finally, forced expression of comm in specific commissural neuron subsets rescues the midline crossing defects of these neurons in elav mutant embryos, further indicating that elav acts cell autonomously on comm expression.     

The phosphoinositide phosphatase Sac1 is required for midline axon guidance

Sac1 phosphoinositide (PI) phosphatases are important regulators of PtdIns(4)P turnover at the ER, Golgi, and plasma membrane (PM) and are involved in diverse cellular processes including cytoskeletal organization and vesicular trafficking. Here, we present evidence that Sac1 regulates axon guidance in the embryonic CNS of Drosophila. Sac1 is expressed on three longitudinal axon tracts that are defined by the cell adhesion molecule Fasciclin II (Fas II). Mutations in the sac1 gene cause ectopic midline crossing of Fas II-positive axon tracts. This phenotype is rescued by neuronal expression of wild-type Sac1 but not by a catalytically-inactive mutant. Finally, sac1 displays dosage-sensitive genetic interactions with mutations in the genes that encode the midline repellent Slit and its axonal receptor Robo. Taken together, our results suggest that Sac1-mediated regulation of PIs is critical for Slit/Robo-dependent axon repulsion at the CNS midline.     

Slit is the midline repellent for the robo receptor in Drosophila

Previous studies suggested that Roundabout (Robo) is a repulsive guidance receptor on growth cones that binds to an unknown midline ligand. Here we present genetic evidence that Slit is the midline Robo ligand; a companion paper presents biochemical evidence that Slit binds Robo. Slit is a large extracellular matrix protein expressed by midline glia. In slit mutants, growth cones enter the midline but never leave it; they abnormally continue to express high levels of Robo while at the midline. slit and robo display dosage-sensitive genetic interactions, indicating that they function in the same pathway. slit is also required for migration of muscle precursors away from the midline. Slit appears to function as a short-range repellent controlling axon crossing of the midline and as a long-range chemorepellent controlling mesoderm migration away from the midline.     

Conserved roles for Slit and Robo proteins in midline commissural axon guidance

In Drosophila, Slit at the midline activates Robo receptors on commissural axons, thereby repelling them out of the midline into distinct longitudinal tracts on the contralateral side of the central nervous system. In the vertebrate spinal cord, Robo1 and Robo2 are expressed by commissural neurons, whereas all three Slit homologs are expressed at the ventral midline. Previous analysis of Slit1;Slit2 double mutant spinal cords failed to reveal a defect in commissural axon guidance. We report here that when all six Slit alleles are removed, many commissural axons fail to leave the midline, while others recross it. In addition, Robo1 and Robo2 single mutants show guidance defects that reveal a role for these two receptors in guiding commissural axons to different positions within the ventral and lateral funiculi. These results demonstrate a key role for Slit/Robo signaling in midline commissural axon guidance in vertebrates.     

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.     

Rhomboid function in the midline of the Drosophila CNS

Rhomboid (Rho), a cell surface, seven-transmembrane domain protein, participates in Spitz-dependent activation of the Drosophila EGF receptor (EGFR). By contrast to transient expression in other embryonic tissues, rho is expressed continuously in the embryonic and larval Midline Glia (MG) lineage and is required upstream of, or in parallel with, S, Spi, and EGFR to establish MG cell number. EGFR signaling is necessary for the expression of rho in the MG and sufficient to stimulate rho expression in additional MG progenitors. rho expression is required continuously from embryonic stage 9-17 to suppress apoptosis in the MG. Although rho misexpression can increase MG number through a non-cell autonomous mechanism, the pattern of normal rho expression suggests that it functions by enhancing autocrine or paracrine signaling among MG cells.     

A novel Dbl family RhoGEF promotes Rho-dependent axon attraction to the central nervous system midline in Drosophila and overcomes Robo repulsion.

The key role of the Rho family GTPases Rac, Rho, and CDC42 in regulating the actin cytoskeleton is well established (Hall, A. 1998. Science. 279:509-514). Increasing evidence suggests that the Rho GTPases and their upstream positive regulators, guanine nucleotide exchange factors (GEFs), also play important roles in the control of growth cone guidance in the developing nervous system (Luo, L. 2000. Nat. Rev. Neurosci. 1:173-180; Dickson, B.J. 2001. Curr. Opin. Neurobiol. 11:103-110). Here, we present the identification and molecular characterization of a novel Dbl family Rho GEF, GEF64C, that promotes axon attraction to the central nervous system midline in the embryonic Drosophila nervous system. In sensitized genetic backgrounds, loss of GEF64C function causes a phenotype where too few axons cross the midline. In contrast, ectopic expression of GEF64C throughout the nervous system results in a phenotype in which far too many axons cross the midline, a phenotype reminiscent of loss of function mutations in the Roundabout (Robo) repulsive guidance receptor. Genetic analysis indicates that GEF64C expression can in fact overcome Robo repulsion. Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42.     

Cytoplasmic dynein and microtubule transport in the axon: The action connection

The neuron uses two families of microtubule-based motors for fast axonal transport, kinesin, and cytoplasmic dynein. Cytoplasmic dynein moves membranous organelles from the distal regions of the axon to the cell body. Because dynein is synthesized in the cell body, it must first be delivered to the axon tip. It has recently been shown that cytoplasmic dynein is moved from the cell body along the axon by two different mechanisms. A small amount is associated with fast anterograde transport, the membranous organelles moved by kinesin. Most of the dynein is transported in slow component b, the actin-based transport compartment. Dynactin, a protein complex that binds dynein, is also transported in slow component b. The dynein in slow component b binds to microtubules in an ATP-dependent manner in vitro, suggesting that this dynein is enzymatically active. The finding that functionally active dynein, and dynactin, are associated with the actin-based transport compartment suggests a mechanism whereby dynein anchored to the actin cytoskeleton via dynactin provides the motive force for microtubule movement in the axon.     

Evolution at the subgene level: domain rearrangements in the Drosophila phylogeny

Although the possibility of gene evolution by domain rearrangements has long been appreciated, current methods for reconstructing and systematically analyzing gene family evolution are limited to events such as duplication, loss, and sometimes, horizontal transfer. However, within the Drosophila clade, we find domain rearrangements occur in 35.9% of gene families, and thus, any comprehensive study of gene evolution in these species will need to account for such events. Here, we present a new computational model and algorithm for reconstructing gene evolution at the domain level. We develop a method for detecting homologous domains between genes and present a phylogenetic algorithm for reconstructing maximum parsimony evolutionary histories that include domain generation, duplication, loss, merge (fusion), and split (fission) events. Using this method, we find that genes involved in fusion and fission are enriched in signaling and development, suggesting that domain rearrangements and reuse may be crucial in these processes. We also find that fusion is more abundant than fission, and that fusion and fission events occur predominantly alongside duplication, with 92.5% and 34.3% of fusion and fission events retaining ancestral architectures in the duplicated copies. We provide a catalog of ～9,000 genes that undergo domain rearrangement across nine sequenced species, along with possible mechanisms for their formation. These results dramatically expand on evolution at the subgene level and offer several insights into how new genes and functions arise between species.