February 15, 2002 – Two research teams have converged on a novel gene that appears to regulate key aspects of communication between nerve and muscle cells. Knowing the identity and function of these regulatory signals, which have remained largely mysterious until now, will allow researchers to better understand how the nervous system forges important connections during development.
The two research teams – one led by Howard Hughes Medical Institute investigator Michael O' Connor and his colleagues -- reported the discovery and characterization of the gene in fruit flies in articles in the February 15, 2002, issue of Neuron. The other team, led by former HHMI investigator Corey Goodman, discovered the same gene via a different route.
Both research teams identified the gene, wishful thinking (wit), by studying the larval neuromuscular junction (NMJ) in the fruit fly Drosophila. The Drosophila NMJ consists of 30 muscle fibers that are attached to 35 neurons. The well-characterized system is a prime model for exploring how muscle growth triggers the growth of its innervating motor neurons that drive muscle contraction.
One of the central features of this increase in neuronal growth is the parallel increase in the number of synapses -- the junctions between the neurons and muscle cells that trigger muscle contraction. Synaptic development also occurs in the brain and elsewhere within the nervous system in both invertebrates and vertebrates.
"It's been pretty clear from a number of experiments that there is some kind of signaling that happens at the neuromuscular junction that coordinates the muscle growth with synapse growth," said O'Connor, who is at the University of Minnesota. "Otherwise, the fly ends up with muscle cells that don't receive enough neurotransmitters to contract properly, or they receive too much and overcontract."
O'Connor and his colleagues were not specifically searching for genes involved in synaptic development when they began their screen of the Drosophila genome. Rather, they were looking for new members of the "bone morphogenetic protein" (BMP) receptor family. BMPs have previously been implicated in mediating many different aspects of development.
"Before we began the search, we didn't know that there was any BMP signaling in neurons," said O'Connor. "It has been known for some time that BMPs can affect neuronal cell fate, but that's very different from synaptic growth. So, we set out to identify genes for receptors, isolate mutants and just go where the resulting phenotypes took us."
Taking a different approach, Goodman and his colleagues at the University of California, Berkeley, did a direct screen for mutations in Drosophila genes that affect synaptic growth in Drosophila larvae. According to Goodman, synaptic growth likely involves proteins on either side of the synapse between neurons and muscle cells -- that is, the transmitting "presynaptic" side on the neuron and the receiving "postsynaptic" side on the muscle cell.
"From many experiments we had done over the past five years, we had accumulated evidence that the two sides of the synapse had a complex conversation with each other to regulate size and strength," said Goodman, who is now President and Chief Executive Officer at Renovis, Inc., a biotechnology company in San Francisco. "The genetic evidence clearly suggested the existence of several different retrograde factors -- signaling molecules that originated from the postsynaptic side of the synapse that influenced the growth or amount of transmitter release by the presynaptic side."
Researchers in Goodman’s laboratory mounted a large-scale effort to create fly mutants that exhibited abnormalities in various parts of the synaptic machinery, which they detected using microscopic examination.
The two research teams found one BMP receptor gene -- which they dubbed wishful thinking -- that seemed to be involved in synaptic growth and development.
"The first hint that wit probably had a neuronal aspect to its function came when we looked at the expression pattern for the receptor and found it to be heavily expressed in the nervous system," said O’Connor. Other researchers had already screened the chromosomal region containing wit for mutations, and O'Connor and his colleagues identified fly mutations in which wit function was eliminated. Although the mutant flies died before they emerged from their pupal case, initial examination of those flies revealed no obvious abnormalities. At that point, the researchers believed it was only wishful thinking to hope for a more dramatic effect of the mutation -- hence the gene’s name, said O’Connor. But when lead author of the Neuron paper, Guillermo Marques, studied the mutants more closely, he discovered something surprising.
"Guillermo saw that the flies were moving inside the pupal case, but they couldn't get out -- and if he pulled them out, they had very spastic movements. This suggested to us that they had a neuronal defect of some sort but the animals' nerve cells appeared normal," said O’Connor.
Two of O’Connor’s co-authors, Bing Zhang and Hong Bao of the University of Texas, Austin, discovered that even though the general architecture of the mutant flies' nervous systems appeared normal, these animals exhibited severe defects in synaptic transmission. Their additional studies revealed that the mutants were also defective in releasing neurotransmitters from tiny sacs called synaptic vesicles. Examination of the synapses of the mutant flies' neurons also revealed that they had fewer synaptic "boutons" -- the small bulbs from which the neuron sends signals to the muscle cell.
O'Connor and his colleagues also began to trace how the receptors produced by the wit gene in motor neurons were receiving signals from muscle cells. They showed that signals sent to the motor neurons activated transcription factors, molecules that switch on genes involved in developing synapses.
O'Connor and his colleagues plan to trace how the signal sent from the muscle cell to the motor neuron moves down the axon to the main cell body of the neuron, where it triggers more signaling. Both O’Connor and Goodman are also examining how the Wit protein signal regulates synaptic growth.
"While until now there has been only circumstantial evidence that there is signaling between muscle cells and neurons that coordinate their formation, we didn't know what those molecules might be," said O'Connor. "So, this is the first signaling cascade that's been identified that must be involved in this coordination."
"This paper is just the beginning," said Goodman. "All of the ongoing work points to the discovery of a retrograde signaling mechanism, in which the postsynaptic side of the synapse sends a signal that is received by Wit and another receptor subunit on the presynaptic side, and this regulates synaptic growth." Goodman termed the discovery of the wit gene "a major breakthrough in the field" that would certainly lead to searches in vertebrates for similar genes that control synaptic size and strength.
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