Katja Brose

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.

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.

Biochemical Purification of a Mammalian Slit Protein as a Positive Regulator of Sensory Axon Elongation and Branching

Many neurons in both vertebrates and invertebrates innervate multiple targets by sprouting secondary axon collaterals (or branches) from a primary axon shaft. To begin to identify molecular regulators of axon branch initiation or extension, we studied the growth of single sensory axons in an in vitro collagen assay system and identified an activity in extracts of embryonic spinal cord and of postnatal and adult brain that promotes the elongation and formation of extensive branches by these axons. Biochemical purification of the activity from calf brain extracts led to the identification of an amino-terminal fragment of Slit2 as the main active component and to the discovery of a distinct activity that potentiates its effects. These results indicate that Slit proteins may function as positive regulators of axon collateral formation during the establishment or remodeling of neural circuits.

Hepatocyte Growth Factor/Scatter Factor Is an Axonal Chemoattractant and a Neurotrophic Factor for Spinal Motor Neurons

In the embryonic nervous system, developing axons can be guided to their targets by diffusible factors secreted by their intermediate and final cellular targets. To date only one family of chemoattractants for developing axons has been identified. Grafting and ablation experiments in fish, amphibians, and birds have suggested that spinal motor axons are guided to their targets in the limb in part by a succession of chemoattractants made by the sclerotome and by the limb mesenchyme, two intermediate targets that these axons encounter en route to their target muscles. Here we identify the limb mesenchyme-derived chemoattractant as hepatocyte growth factor/scatter factor (HGF/SF), a diffusible ligand for the c-Met receptor tyrosine kinase, and we also implicate HGF/SF at later stages as a muscle-derived survival factor for motoneurons. These results indicate that, in addition to functioning as a mitogen, a motogen, and a morphogen in nonneural systems, HGF/SF can function as a guidance and survival factor in the developing nervous system.

The Divergent Robo Family Protein Rig-1/Robo3 Is a Negative Regulator of Slit Responsiveness Required for Midline Crossing by Commissural Axons

Commissural axons in vertebrates and insects are initially attracted to the nervous system midline, but once they reach this intermediate target they undergo a dramatic switch, becoming responsive to repellent Slit proteins at the midline, which expel them onto the next leg of their trajectory. We have unexpectedly implicated a divergent member of the Robo family, Rig-1 (or Robo3), in preventing premature Slit sensitivity in mammals. Expression of Rig-1 protein by commissural axons is inversely correlated with Slit sensitivity. Removal of Rig-1 results in a total failure of commissural axons to cross. Genetic and in vitro analyses indicate that Rig-1 functions to repress Slit responsiveness similarly to Commissureless (Comm) in Drosophila. Unlike Comm, however, Rig-1 does not produce its effect by downregulating Robo receptors on precrossing commissural axon membranes. These results identify a mechanism for regulating Slit repulsion that helps choreograph the precise switch from attraction to repulsion at a key intermediate axonal target.

Slit proteins: key regulators of axon guidance, axonal branching, and cell migration

In the past year, Slit proteins have been identified as important regulators of axon guidance and cell migration in Drosophila and vertebrates. Remarkably, they were simultaneously identified as negative regulators, repelling various axonal and cell migrations in both invertebrates and vertebrates, and as positive regulators, stimulating branching and extension of at least one class of axons in vertebrates.

SLIT2-MEDIATED CHEMOREPULSION AND COLLAPSE OF DEVELOPING FOREBRAIN AXONS

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptor and its secreted ligand Slit. In rodents, Robo and Slit are expressed in the spinal cord and Slit can repel spinal motor axons in vitro. Here, we extend these findings into higher brain centers by showing that Robo1 and Robo2, as well as Slit1 and Slit2, are often expressed in complementary patterns in the developing forebrain. Furthermore, we show that human Slit2 can repel olfactory and hippocampal axons and collapse their growth cones.

Retinal Ganglion Cell Axon Guidance in the Mouse Optic Chiasm: Expression and Function of Robos and Slits

The ventral midline of the nervous system is an important choice point at which growing axons decide whether to cross and project contralaterally or remain on the same side of the brain. InDrosophila, the decision to cross or avoid the CNS midline is controlled, at least in part, by the Roundabout (Robo) receptor on the axons and its ligand, Slit, an inhibitory extracellular matrix molecule secreted by the midline glia. Vertebrate homologs of these molecules have been cloned and have also been implicated in regulating axon guidance. Using in situ hybridization, we have determined the expression patterns of robo1,2and slit1,2,3 in the mouse retina and in the region of the developing optic chiasm, a ventral midline structure in which retinal ganglion cell (RGC) axons diverge to either side of the brain. The receptors and ligands are expressed at the appropriate time and place, in both the retina and the ventral diencephalon, to be able to influence RGC axon guidance. In vitro,slit2 is inhibitory to RGC axons, with outgrowth of both ipsilaterally and contralaterally projecting axons being strongly affected. Overall, these results indicate that Robos and Slits alone do not directly control RGC axon divergence at the optic chiasm and may additionally function as a general inhibitory guidance system involved in determining the relative position of the optic chiasm at the ventral midline of the developing hypothalamus.

Twenty Years of Exciting Neuroscience

Twenty years ago, Neuron was launched with the aim of providing a forum for the publication of research in cellular and molecular neurobiology. In the late eighties, molecular biology had exploded as a field and was providing powerful new experimental tools for probing cellular function. The founding editors of the journal—Zach Hall, A.J. Hudspeth, Eric Kandel, and Louis Reichardt—envisioned this new journal they called Neuron as a home for the burgeoning new field at the interface of molecular biology and cellular neurobiology.

New Online Access Policy for Neuron

We are very pleased to announce a change in the access policy for Neuron and the entire Cell Press journal collection. Beginning in January of 2005, we will provide free access to the online archive of Neuron and the other Cell Press journals for content that is 12 months old or older. This policy applies to the recent archive that dates from 1995, when Cell Press first published online. Free access to this archive will be available on both ScienceDirect.com and the Cell Press journal sites, including www.neuron.org. From January onward, all Cell Press issues from 1995 through December 2003 will be freely available. Each month, as new issues are published, year-old content will be added to the freely accessible recent archive.In making this change, we are incorporating the principle of a freely accessible archive without adopting the Open Access author-pays business model, which we believe is untested and potentially flawed from both editorial and financial perspectives. Many members of the scientific community, editorial board members, authors, and reviewers have discussed these issues with me and the other Neuron editors, over the phone, at meetings, and during lab visits. I speak for all the editors at Neuron and Cell Press when I say that we are all enormously grateful for all the input and are very pleased that through an open dialogue with the scientific community we were able to arrive at this positive outcome.The editors at Cell Press are committed to making a positive contribution to scientific communication through publishing journals that serve the communitys needs and are widely disseminated. To that end, we are pleased to be able to add our new policy to these existing initiatives:– participation in the HINARI project of the WHO, which distributes journals for free to developing countries (http://www.healthinternetwork.org/index.php);– a copyright policy that gives authors liberal rights;– free advance online publication of selected papers;– online submission and review for the convenience of authors and reviewers;– conference sponsorship and prizes. This year, Neuron will be hosting “Neurons and Memory,” the second annual Neuron satellite meeting at the SFN meeting in San Diego on October 21–22.We are extremely grateful for the support and interest that the community has extended to Neuron over the years, and as always, we look forward to any feedback you may have on these and other editorial issues.