Finding a Signal: NGF, EGF and the Bioassay

In 1952, a scrap of mouse tumor sat near, but not touching, a chick nerve ganglion in a dish, and within a day the ganglion had thrown out a dense halo of fibers. The tumor was releasing something. How do you find a molecule you cannot see, and how do you know when you have caught it?

This lesson follows three scientists and one method. The method is the bioassay: a test on living tissue that tells you how much of an unknown substance a sample holds. It found the first two growth factors, nerve growth factor (NGF) and epidermal growth factor (EGF). Along the way you will see the order in which most signaling molecules get found: a puzzling effect in an animal, a test that reproduces it in a dish, a rich source of material, purification, and last, the receptor.

What does this tell us? A tumor that reaches nerves without touching them

The story starts with Viktor Hamburger, an embryologist who spent most of his career at Washington University in St. Louis. In 1934 he showed that if you cut the limb bud off a chick embryo, far fewer of the nerve cells that would have supplied that limb are left. He first read this as a failure of nerve cells to multiply and mature; later work showed that they die. Something in the tissue at the far end of a nerve fiber was deciding whether the nerve cell lived. Nobody knew what.

In 1947 Hamburger invited Rita Levi-Montalcini to join him. Waiting for her was an observation by his former student Elmer Bueker. A fragment of a mouse tumor called Sarcoma 180, grafted into a three-day-old chick embryo, was invaded by the embryo’s own nerve fibers. Levi-Montalcini and Hamburger repeated it and pushed it further. The ganglia that served the graft swelled. The sympathetic ganglia (clusters of nerve cells outside the brain and spinal cord that control glands, blood vessels and internal organs) grew to about six times the size of the same ganglia in control embryos, and organs were crowded with extra fibers, a condition called hyperinnervation.

What does this tell us? First rule out the simplest explanation, contact. The tumor sat inside the embryo, pressed against its tissues, and Bueker had read his own result as the tumor offering a favorable field for fibers to grow into. So they moved the tumor out. The chorioallantoic membrane is a thin, blood-vessel-rich sheet that lines the inside of the eggshell. It is plumbed into the embryo’s circulation and touches none of its internal tissue. They grafted the tumor there. The ganglia deep inside the embryo swelled just as before. Only blood connected the two tissues, so the tumor had to be putting something soluble into it.

Three inferences follow:

1. Neurons are overproduced. A ganglion’s size depends largely on how many neurons survive, though cell size counts too. One that swells six-fold with a tumor nearby points, at least in part, to a reserve of neurons that would normally have died.
2. A target-derived factor exists. Hamburger’s limb bud and the tumor both act as targets that release a diffusible substance the neurons depend on. The tumor is simply a very generous target.
3. Competition is implied. If a target’s supply is limited, neurons must compete for it. Picture musical chairs, with the supply of factor as the chairs. A neuron that gets no chair dies, and the tumor added chairs.

The graft showed that a molecule must exist. It could not say what the molecule was.

How would you go about identifying the factor?

You cannot purify what you cannot detect, and a tumor in an embryo is a slow, messy detector. Hamburger’s department at Washington University had no tissue-culture facility, so in 1952 Levi-Montalcini traveled to Rio de Janeiro, carrying two mice bearing the tumors in her handbag. There she grew chick ganglia in a hanging-drop culture, with the ganglion in a clot of blood plasma, and a tumor fragment placed near them but not touching. Within 24 hours each ganglion had put out a dense halo of fibers, thickest toward the tumor. The whole experiment now fit in a dish, with no embryo.

That halo is a bioassay. It uses a living response as the measuring device, which means it works before you know what the molecule is. It can also be made quantitative. Dilute a sample step by step, and the highest dilution that still gives a halo tells you how much factor the sample holds. Divide by the milligrams of protein in the sample and you have the specific activity, the activity per milligram of protein. Every purification step is then judged by one question: did the activity per milligram go up?

The assay needed a biochemist. Hamburger and Levi-Montalcini went around the university looking for one willing to take on the problem, and in 1953 Stanley Cohen joined the department. When Cohen described his results at the journal club run by Arthur Kornberg, Kornberg suggested that a virus might be responsible. A tumor is a place where viruses live, and one riding in the extract could be causing the growth. Kornberg also suggested a control: treat the extract with an enzyme that destroys nucleic acid, and if fibers still sprout, no virus is needed. Snake venom is a source of such an enzyme.

The control refused to behave. The treated extract still worked, and venom by itself, with no tumor in the dish, worked far better than the tumor ever had. Venom is a modified salivary secretion, which led Cohen to test the salivary gland of a male mouse. It was just as potent. The mouse submaxillary gland became the main source of NGF. A control can turn out to be the richest source you have, so look at what each control does, beyond whether it passed.

Design problem

You have a crude extract that makes nerve fibers grow from a ganglion in a dish. Design the assay you would use to purify the active factor, and say what three controls you need before you believe a fraction is “it”.

Next experiment: a crude extract with two factors in it

If NGF controls sympathetic neurons, removing it should remove them. Cohen raised an antiserum against purified NGF, and Levi-Montalcini showed in 1960 that injecting it into young animals destroyed most of their sympathetic ganglia. Put the two results side by side. Add extra target-derived factor and ganglia swell. Take the factor away and ganglia are lost. In short, targets decide, through how much factor they supply, how many neurons of the peripheral nervous system survive. Adding the factor shows it is enough to expand a ganglion. Removing it shows it is needed.

Then came a stranger result. Cohen injected newborn mice daily with crude male mouse salivary gland extract. As expected, their sympathetic ganglia were enlarged. But the mice also opened their eyelids about 7 days after birth instead of 12 to 14, and their teeth came through early. Purified NGF given alone did none of that. The crude extract must have carried a second protein with its own activity. It acts on skin, so it was named epidermal growth factor.

The lesson sits in the method. The halo assay counted nerve fibers and was blind to EGF. A whole newborn mouse responds to everything in a crude extract. When you start from a mixture, the assay you choose decides which molecule you find.

What a growth factor does, and why the receptor came next

“Growth factor” is a misleading name. The mature neurons NGF acts on have stopped dividing, and NGF does not restart them. It keeps them alive and drives their fibers to grow. EGF thickens the epidermis, the outer layer of skin, which made it a candidate burn therapy, and it does make cells divide. It also affects survival and differentiation, and its pathway is among the most commonly mutated or overactive in cancer. A growth factor is best defined as a secreted protein that tells a target cell how to behave. The instruction can be to divide. It can also be to stay alive, or to mature into a specialized cell.

A message does nothing until something on the target cell hears it. NGF binds a receptor called TrkA, and EGF binds the EGF receptor, EGFR. Cohen approached the receptor as a biochemist. A growth factor, he reasoned, should control some chemical reaction that you could show in a cell-free system, as Sutherland had when glucagon added to membranes produced cyclic AMP. At the end of the 1970s his laboratory at Vanderbilt University used membranes from A-431 cells, a human tumor cell line with many EGF receptors. The preparation held no living cells. When EGF was added, phosphate began to move onto protein, work in which Graham Carpenter did much of the biochemistry. Hirota Ushiro then found that the phosphate was landing on tyrosine, after the first analysis had mistaken it for threonine. Tyrosine had only just been recognized as a site for a regulatory phosphate, through the Rous sarcoma virus kinase, and phosphotyrosine is less than 1% of the phosphate on proteins in a normal cell, so almost nobody had looked.

The receptor had been pictured as an antenna, with the hormone docking outside and the business happening further in. Purifying the receptor from these membranes showed one protein with two halves. EGF binds the half outside the cell, and the half inside is an enzyme. TrkA, the receptor for NGF, is also a receptor tyrosine kinase. How that enzyme switches on is the subject of a later lesson, and so is how a signal picked up at a nerve ending reaches the cell body.

Many growth factors, NGF among them, are dimers, while their receptors sit in the membrane as single chains. How might a ligand switch such a receptor on?