Crossing the Midline Once: Slit, Robo and Comm

A commissural axon is drawn toward the midline of the nervous system, crosses it, and then treats that same midline as forbidden ground for the rest of its life.

The midline did not change while the axon crossed it. In the fly it is a row of glial cells; in the vertebrate spinal cord it is the floor plate, a strip of cells along the ventral midline. The same cells present the same molecules before, during and after any one axon passes. So the change has to be in the axon. The question is the one Chris Deppmann puts on the board: Switch between Attraction and Repulsion? What does a growth cone change at the midline, and how does it make the change stick?

The problem: one crossing, and only one

Your left and right sides have to talk. Sensory information from one side of the body ends up in the opposite half of the brain, and the two halves of the spinal cord coordinate walking. Both need commissural axons, axons that cross the midline to the other side. Their neighbours, ipsilateral axons, stay on the side where they started and must never cross.

A commissural axon has three jobs. It must grow toward the midline, which in vertebrates it does partly by following netrin released by the floor plate (the mechanism is in that lesson). It must enter the midline and leave it. And it must never come back. Each job fails in a different way. If the axon kept its attraction to the midline after crossing, it would stall there. If it were repelled too early, it would never cross at all. If it lost its repulsion after crossing, it might wander back.

Think of a one-way turnstile. It lets you through from one side and locks behind you, and the lock is part of you, not the gate. The real mechanism is close to that: the axon carries the lock, and it turns the lock on at the right moment by changing what is on its own surface.

Reading the fly ladder

The answer was found in the fly embryo. Stained with an antibody that marks its axons, the fly’s central nerve cord looks like a ladder: two long rails run the length of the body, and in each segment two rungs, the commissures, cross between them. Most commissural axons cross once, turn, and run in a rail on the far side.

In 1993 Michael Seeger, Guy Tear, David Ferres-Marco and Corey Goodman reported a screen for mutations that disturb this ladder. Among the genes recovered were two named for what the ladder looked like. Each mutant broke the ladder in a different way:

• roundabout (robo): too many crossings. Ipsilateral axons cross, and commissural axons cross and then cross back. Chris’s shorthand is “Roundabout: KO of Robo”, KO meaning knockout, a loss of the gene.
• commissureless (comm): almost no axon crosses. The rungs nearly vanish. In his words, “Commissureless: KO of Comm.”
• slit: axons enter the midline and do not leave. Over time the two rails collapse into a single bundle running down the middle.

In 1998 Thomas Kidd, Tear, Goodman and colleagues cloned robo. It encodes a receptor of the immunoglobulin superfamily that spans the membrane once, and vertebrates have relatives of it. Its timing was the telling part. Axons that never cross carry Robo on their growth cones from the start. Axons that do cross carry high levels of Robo only after they have crossed.

A receptor that keeps axons off the midline should have a repellent at the midline. In 1999 two back-to-back papers in Cell named it. Kidd, Kimberly Bland and Goodman showed by genetics that Slit, a large protein secreted by the midline glia, acts with Robo: flies missing one copy of each gene had crossing errors that flies missing one copy of either gene rarely showed. Katja Brose, Bland and colleagues from Goodman’s laboratory and Marc Tessier-Lavigne’s showed that Slit proteins bind Robo receptors and that recombinant Slit2 repels embryonic spinal motor axons in culture. Slit is the midline’s “Repulsive Cues: Don’t Go There.” On the growth cone, Robo turns that signal into retreat partly through proteins that switch off Rho-family GTPases, the cytoskeleton regulators you met in that lesson.

Why do slit and robo mutants look different, if one is the ligand of the other? Flies have more than one Robo receptor. Losing Robo removes much of the repulsion, so axons cross freely, but they still drift out of the midline. Losing Slit removes the cue for all of the receptors, and axons that reach the midline have nothing pushing them out.

Commissureless: keeping the receptor off the surface

Comm is the piece that makes the switch. In 1998 Kidd, Claire Russell, Goodman and Tear combined the mutants. An embryo lacking both robo and comm looked like the robo mutant. That is an epistasis test: when a double mutant looks like one of the single mutants, that gene acts downstream. Comm’s job is to lower Robo, so once Robo is gone, losing Comm changes nothing. They also made Comm in every neuron. Robo protein fell, and the ladder looked like robo.

How Comm lowers Robo has been revised more than once. The early model had Comm made in midline cells and transferred into the axons crossing them. In 2002 Marios Georgiou and Tear reported that comm is needed in the commissural neurons as well as in midline cells. Later that year Krystyna Keleman, Barry Dickson and colleagues proposed the model the evidence best supports today. Comm is made in commissural neurons for a short time before and during crossing. It meets newly made Robo inside the cell and diverts it from the route to the surface into late endosomes and lysosomes, where Robo is destroyed. A short sequence in Comm, LPSY, was needed for this sorting in their assays. Once the axon has crossed, the neuron stops making Comm, Robo reaches the growth cone, and Slit at the midline it just crossed now repels it. Sorting receptors toward lysosomes is covered in that lesson and receptor trafficking in that lesson.

Treat this as a model, not a closed case. Keleman, Carlos Ribeiro and Dickson’s 2005 work in living embryos found that Comm stops Robo-GFP from reaching the axon tip, but did not support a required role for the ubiquitin tagging once proposed for Comm. Other laboratories have since found further brakes on fly Robo before crossing, so sorting may be one of several. And Comm is a fly protein: no vertebrate Comm has been found.

The vertebrate answer: Robo3 silences instead of destroying

The fly has one slit gene. Mice have three, and two conventional Robo receptors, Robo1 and Robo2. In 2004 Hua Long, Tessier-Lavigne and colleagues removed Slit genes in the mouse. Earlier work on mice lacking Slit1 and Slit2 had found no defect in commissural axon guidance. Only when all three Slit genes were removed did spinal commissural axons show the fly phenotype: many stalled in the floor plate and others crossed and then turned back. Redundancy of this kind makes a once-only decision hard to break by one mutation.

Vertebrates still need an axon that ignores Slit before crossing. The same year, Christelle Sabatier, Alain Chédotal, Tessier-Lavigne and colleagues found the vertebrate solution in an unusual member of the Robo family, Robo3, also called Rig-1. Robo3 is made on commissural axons before they cross and largely disappears afterwards. In mice lacking Robo3, commissural axons approach the floor plate and turn away without crossing. Robo3 does not clear Robo1 and Robo2 from the surface the way fly Comm clears Robo. It blunts their response to Slit while they stay in the membrane. Flies destroy the receptor; vertebrates silence it. Both give an axon that is deaf before crossing and responsive after.

The same gene matters in people. Mutations in human ROBO3 cause horizontal gaze palsy with progressive scoliosis, and in patients the motor and sensory pathways that normally cross in the brainstem appear uncrossed. Robo3 has other jobs as well, including binding a second ligand, NELL2, so it does more than mute Robo1.

Design problem

Chris polls his class: “What happens in a Slit knockout mouse?” Use the model to predict what you would see in the spinal cord of a mouse lacking all three Slit genes. Then predict a Robo3-null mouse, and explain why two mutants in one pathway give nearly opposite results.

A decision stays made

What makes the crossing final is physical. The neuron changes the amount or activity of a receptor on its own growth cone, and it keeps the change. The receptor is the same protein throughout, and the midline tissue is the same. In flies the neuron stops making the protein that sent Robo to be destroyed. In vertebrates it stops making the Robo3 that muted Robo1 and Robo2, and Robo1 and Robo2 are left free to respond.

Attraction has to be dealt with too. A crossed axon must stop being drawn to the midline as well as start being repelled by it, and how those two changes are coupled is still being worked out.

You can now predict a midline phenotype from three questions. Is the repellent there? Can the axon hear it? And when does it start hearing it? An axon that hears Slit too early never crosses; one that never hears it crosses again. The same logic, with different cues, decides which retinal axons cross at the optic chiasm, a case taken up in that lesson.