Corey S. Goodman

Developmental mechanisms that generate precise patterns of neuronal connectivity

The functioning of the nervous system depends upon the underlying detailed and highly stereotyped patterns of neuronal connectivity. How these precise patterns of synaptic connections form during development is the subject of this review. The specificity of synaptic connections unfolds in three major steps: pathway selection, target selection, and address selection. First, the growing tips of neurons, the growth cones, traverse long distances to find their correct target region. En route, they are confronted by a series of choice points and yet correctly navigate these pathways in a remarkably unerring way. Once they reach the correct neighborhood, they contact and recognize their correct target, typically a regionally localized set of neurons. In this way, the overall scaffold of projections and synapses is initially established. But these initial patterns of connections are then refined, as axonal terminals retract and expand to select a specific subset of cells from. within the overall target. This remodeling, called address selection, relies on the context of and competition with surrounding inputs and is capable of transforming a coarse-grained and overlapping projection into a refined and highly tuned pattern of connections. Experiments over the last few decades have clarified the issue of how neural specificity is generated by suggesting that two broad mechanisms work in concert to orchestrate the formation of precise patterns of neural connections during development: those that require neuronal activity (activity dependent), and those that do not (activity independent). The initial steps of growth cone guidance typically occur before neurons become functionally active and rely on molecular mechanisms of pathway and target recognition that are largely activity independent. These mechanisms bring together multiple inputs with appropriate targets to form initial patterns of connections. From this point on, the patterns of neuronal activity within these emerging patterns of connections take over as the predominant mechanism that drives the refinement and remodeling of these initial projections into highly tuned and functioning circuits. This process of activity-dependent synaptic plasticity does not stop at birth but continues throughout the lifetime of the organism as the patterns of neural activity driven by input from the external world continue to modify the strength and structure of dendrites, axonal arbors, and synapses (e.g., Purves et al., 1986; Bailey and Chen, 1989). The growing evidence that adults and embryos may use common molecules and mechanisms to modify their synapses (Cline and ConstantinePaton, 1989; Mayford et al., 1992) has linked the once separate fields of developmental neurobiology and learning and memory. In this review, we consider the range of activityindependent and activity-dependent mechanisms that generate precision of neuronal connections. We then focus on two examples at opposite ends of the spectrum-the connections between motoneurons and muscles and between the retina and higher visual centers--to highlight the extent to which different parts of the nervous system use the same mechanisms but in different proportions to achieve the final specificity.

Plexins Are a Large Family of Receptors for Transmembrane, Secreted, and GPI-Anchored Semaphorins in Vertebrates

In Drosophila, plexin A is a functional receptor for semaphorin-1a. Here we show that the human plexin gene family comprises at least nine members in four subfamilies. Plexin-B1 is a receptor for the transmembrane semaphorin Sema4D (CD100), and plexin-C1 is a receptor for the GPI-anchored semaphorin Sema7A (Sema-K1). Secreted (class 3) semaphorins do not bind directly to plexins, but rather plexins associate with neuropilins, coreceptors for these semaphorins. Plexins are widely expressed: in neurons, the expression of a truncated plexin-A1 protein blocks axon repulsion by Sema3A. The cytoplasmic domain of plexins associates with a tyrosine kinase activity. Plexins may also act as ligands mediating repulsion in epithelial cells in vitro. We conclude that plexins are receptors for multiple (and perhaps all) classes of semaphorins, either alone or in combination with neuropilins, and trigger a novel signal transduction pathway controlling cell repulsion.

Slit Proteins Bind Robo Receptors and Have an Evolutionarily Conserved Role in Repulsive Axon Guidance

Extending axons in the developing nervous system are guided in part by repulsive cues. Genetic analysis in Drosophila, reported in a companion to this paper, identifies the Slit protein as a candidate ligand for the repulsive guidance receptor Roundabout (Robo). Here we describe the characterization of three mammalian Slit homologs and show that the Drosophila Slit protein and at least one of the mammalian Slit proteins, Slit2, are proteolytically processed and show specific, high-affinity binding to Robo proteins. Furthermore, recombinant Slit2 can repel embryonic spinal motor axons in cell culture. These results support the hypothesis that Slit proteins have an evolutionarily conserved role in axon guidance as repulsive ligands for Robo receptors.

Expression of engrailed proteins in arthropods, annelids, and chordates

Abstract engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated sub-set of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.

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.

The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules

In addition to its expression on subsets of axons, grasshopper Semaphorin I (Sema I, previously called Fasciclin [Fas] IV) is expressed on an epithelial stripe in the limb bud, where it functions in the guidance of two sensory growth cones as they abruptly turn upon encountering this sema I boundary. We report here on the cloning and characterization of two sema genes in Drosophila, one in human, and the identification of two related viral sequences, all of which encode proteins with conserved Semaphorin domains. Drosophila sema (D-Sema) I is a transmembrane protein, while D-Sema II and human Sema III are putative secreted proteins that are similar to the recently reported chick collapsin. D-Sema I and D-Sema II are expressed by subsets of neurons and muscles. Genetic analysis in Drosophila reveals that semall is an essential gene that is required for both proper adult behavior and survival.

Roundabout Controls Axon Crossing of the CNS Midline and Defines a Novel Subfamily of Evolutionarily Conserved Guidance Receptors

The robo gene in Drosophila was identified in a large-scale mutant screen for genes that control the decision by axons to cross the CNS midline. In robo mutants, too many axons cross and recross the midline. Here we show that robo encodes an axon guidance receptor that defines a novel subfamily of immunoglobulin superfamily proteins that is highly conserved from fruit flies to mammals. For those axons that never cross the midline, Robo is expressed on their growth cones from the outset; for the majority of axons that do cross the midline, Robo is expressed at high levels on their growth cones only after they cross the midline. Transgenic rescue experiments reveal that Robo can function in a cell-autonomous fashion. Robo appears to function as the gatekeeper controlling midline crossing.

Mutations affecting growth cone guidance in drosophila: Genes necessary for guidance toward or away from the midline

We performed a large-scale screen for mutations that affect the development of CNS axon pathways in the Drosophila embryo. We screened embryos from over 13,500 balanced lines and saved over 250 mutant lines whose phenotypes suggest possible defects in growth cone guidance. Here we focus on two new genes: commissureless (comm) and roundabout (robo). Mutations in comm lead to an absence of nearly all CNS axon commissures, such that growth cones that normally project across the midline instead now extend only on their own side. Mutations in robo lead to the opposite misrouting, such that some growth cones that normally extend only on their own side instead now project across the midline. The phenotypes of these two genes suggest that they may encode components of attractive and repulsive signaling systems at the midline that either guide growth cones across the midline or keep them on their own side.

Neuropilin-2, a Novel Member of the Neuropilin Family, Is a High Affinity Receptor for the Semaphorins Sema E and Sema IV but Not Sema III

Semaphorins are a large family of secreted and transmembrane proteins, several of which are implicated in repulsive axon guidance. Neuropilin (neuropilin-1) was recently identified as a receptor for Collapsin-1/Semaphorin III/D (Sema III). We report the identification of a related protein, neuropilin-2, whose mRNA is expressed by developing neurons in a pattern largely, though not completely, nonoverlapping with that of neuropilin-1. Unlike neuropilin-1, which binds with high affinity to the three structurally related semaphorins Sema III, Sema E, and Sema IV, neuropilin-2 shows high affinity binding only to Sema E and Sema IV, not Sema III. These results identify neuropilins as a family of receptors (or components of receptors) for at least one semaphorin subfamily. They also suggest that the specificity of action of different members of this subfamily may be determined by the complement of neuropilins expressed by responsive cells.

Characterization and cloning of fasciclin III: A glycoprotein expressed on a subset of neurons and axon pathways in Drosophila

To identify candidates for neuronal recognition molecules in Drosophila, we used monoclonal antibodies to search for surface glycoproteins expressed on subsets of axon bundles (or fascicles) during development. Here we report on the characterization and cloning of fasciclin III, which is expressed on a subset of neurons and axon pathways in the Drosophila embryo. Fasciclin III is also expressed at other times and places including transient segmentally repeated patches in the neuroepithelium and segmentally repeated stripes in the body epidermis. Antisera generated against each of four highly related forms of the protein were used for cDNA expression cloning to identify a single gene, which was confirmed to encode fasciclin III by tissue in situ hybridization and genetic deficiency analysis.