Look Ma, no cells. In what he calls “a hyper-low-tech experiment” by today's standards, Joshua Sanes was in 1978 able to show that regenerating nerve axons take their cues for new synapse formation from the extracellular matrix (ECM) of muscle cells and not from the cells themselves (Sanes et al., 1978). The beautiful simplicity of the experiment gave scientists studying synapse formation a clear trail to follow to find the signals for axon guidance and differentiation.

From the late 19th century studies by a student of Santiago Ramón y Cajal, it was known that a cut nerve would form a new synapse at the same exact spot on the muscle fiber where the old synapse had been.

“Somehow, the axon is ignoring 99.9% of the muscle fiber and contacting the 0.1%,” says Sanes. “It had to be recognizing something on that spot—a surface component or a chemical.” He and his colleagues reasoned that the regrowing axon first contacts the muscle cell's extracellular matrix sheath, which was called the basement membrane because it was thought to be the support foundation for muscle cells.

The group devised a way to observe damaged frog muscles where axons, but not muscle cells, would grow back. The team cut muscle and nerves in a patch of tissue and then irradiated it so that damaged muscle would not grow back, but damaged nerves with cell bodies outside of the irradiated patch could regenerate into the damaged area. All that remained of the damaged muscle cells were the basement membrane sheaths, made of insoluble glycoproteins that persisted up to four weeks later.

graphic

The ECM sheath left behind after muscle degeneration (top, lining cavity) can direct development of a new synapse (bottom, dark stain).

SANES

These “ghost” sheaths could be stained for a synapse marker, cholinesterase, showing where previous synapses of the neuromuscular junction had existed. The team's EM pictures unmistakably show new nerve terminals forming next to the darkly stained cholinesterase.

Sanes says that although “no one ever thought Nobel, we did feel it was cool and would be helpful in this field.” Indeed, the work showed the ECM to be more than an inert structural support. It was instead playing a clear developmental role—all this at a time when ECM components were just coming to light. Practically, the study also gave Sanes, his coauthor Jack McMahan, and others a narrowly defined location for their search for molecules that direct synapse formation in wound healing and embryonic development.

McMahan's group pursued and identified the ECM molecule agrin that was deposited by motoneurons and “spoke” to the postsynaptic muscle cells (McMahan, 1990). And Sanes's lab discovered that ECM-localized laminin β2 directed differentiation of synapses during both regrowth and development (Hunter et al., 1989). Later knock-out studies showed that both components are critical during normal synaptic development in vivo (Noakes et al., 1995; Gautam et al., 1996).

“This has been one main thread in my lab from work started in that paper,” he says. “We've continued to work in synapse formation at the neuromuscular junction synapse” for the last 25 years. The team has also applied lessons from that system in understanding brain synapses. Their latest contribution has a familiar theme but with a twist: only the correct ECM components, not the nerve cell, is required for the muscle cell to set up its own synapse architecture (Kummer et al., 2004).

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