Hedgehog and the Primary Cilium

A plant poison that gave lambs a single eye and a drug now used against a common skin cancer block the same protein. That protein, Smoothened, sits in the middle of a pathway that tells the cells of an embryo where they are. Most pathways you have met work by adding something, such as a phosphate or a second messenger. This one works by releasing brakes. To follow it you need one piece of logic, a double negative, and one piece of geography, a single hair-like projection on the surface of the cell.

The pathway was found by breaking genes and reading the damage

In the autumn of 1979, at the European Molecular Biology Laboratory in Heidelberg, Christiane Nüsslein-Volhard and Eric Wieschaus began asking how many genes it takes to lay out the body of a fruit fly. Cloning genes from flies was only just beginning, so they broke genes at random. Male flies were fed a chemical that damages DNA, and their descendants were bred into nearly 27,000 separate lines, each carrying one damaged chromosome, until some embryos in a line had two broken copies of the same gene.

Then they looked at skin. On its underside a healthy larva carries belts of tiny spines with smooth cuticle between them, one belt to a segment. An embryo that dies with belts missing, doubled or reversed has lost a gene needed to build that pattern. One mutant had no smooth cuticle left. It was spiny from end to end, and they named the gene hedgehog.

In his Nobel lecture Wieschaus reported that 580 mutations with a describable defect fell into 139 genes, few enough to study one at a time. The first paper, in 1980, described fifteen of them. Grouped with hedgehog in that paper were patched and cubitus interruptus, along with wingless, the fly Wnt gene. Nobody yet knew that hedgehog encoded a signal, patched its receptor and cubitus interruptus the transcription factor at the end of its pathway.

The signal: a protein that carries two lipids and spreads in a gradient

Hedgehog is a secreted protein, and the newly made chain cuts itself in two. Its back part is an enzyme that works once: it breaks the chain and, in the same reaction, joins a molecule of cholesterol to the new end of the front part. A second enzyme adds palmitate, a 16-carbon fatty acid, to the other end. The front part, with a lipid at each end, is the signal.

As with Ras in the lesson on lipid anchors, the lipids hold the protein on a membrane. That creates a problem, because a signal stuck to the cell that made it cannot act at a distance. Cells that make Hedgehog need a dedicated membrane protein, Dispatched, to let it go. How the freed protein then travels is not settled. Proposals include lipoprotein particles, clusters of many Hedgehog molecules, and thin cell extensions that carry it outward. The outcome is agreed: Hedgehog reaches cells many cell diameters away, and its concentration falls with distance from the source.

That makes Hedgehog a morphogen: a signal that spreads from a source and draws different answers from cells at different concentrations. Mammals have three Hedgehog genes, and the best studied is Sonic hedgehog, named after the video-game character. In the limb bud it is made along one edge, the future little-finger side, and which digit forms depends on how much of it the cells saw and for how long. In the neural tube, the embryonic spinal cord, it comes from the midline at the bottom, and cells at increasing distances from it become different kinds of neuron. Each cell reads its position from the dose.

The idea: Hedgehog works by switching off an off-switch

Two membrane proteins receive the signal. Patched crosses the membrane twelve times and looks like a transporter. Its relatives include bacterial pumps and NPC1, a human protein that moves cholesterol out of lysosomes. Smoothened crosses seven times, like the G-protein-coupled receptors of Unit 3.

Hedgehog binds Patched, and Patched is not the activator. Patched is a brake.

No Hedgehog: Patched is active, so Smoothened is off.

Hedgehog present: Patched is switched off, so Smoothened is on.

Picture a car parked on a hill with the handbrake on. Hedgehog does not press the accelerator. It releases the handbrake. That is why a cell that has lost Patched behaves as if Hedgehog were always there.

How does Patched restrain Smoothened? Not by holding it: one Patched can keep many Smoothened molecules quiet. The working model is that Smoothened needs a sterol, most likely cholesterol itself, to switch on, and that Patched uses its transporter-like body to keep that sterol out of reach. Structures show sterol-shaped molecules inside both proteins, and the fatty acid of Hedgehog pushed into Patched like a plug. What exactly Patched moves, and what powers it, are still being worked out.

What Smoothened controls: a transcription factor that is cut in two

The pathway ends at a transcription factor called Gli in vertebrates and Cubitus interruptus (Ci) in flies. With no Hedgehog, PKA adds phosphates to full-length Gli, and two more kinases, CK1 and GSK3, add more. The cluster of phosphates is recognised by an E3 ligase, and the ubiquitin-tagged protein goes to the proteasome. There it is only partly destroyed. The back part, which holds the activating region, is degraded. The front part, which still binds DNA, is released. That fragment is a repressor, and it keeps Hedgehog target genes off.

When Smoothened is on, the marking stops. Gli stays whole and becomes an activator. Flies do both jobs with the one Ci protein. Mammals have three Gli proteins, with Gli3 supplying most of the repressor and Gli2 most of the activator. Recent work indicates that active Smoothened stops the marking by using a decoy sequence within itself to block the active site of the catalytic subunit of PKA, so that Gli escapes the first phosphates; how that step is controlled is still being worked out.

In Unit 3, PKA carried a hormone’s message forward; here its resting activity keeps a pathway silent. Wnt uses the same pair of kinases, CK1 and GSK3, to mark β-catenin for complete destruction, and Notch cuts the receptor itself. In Hedgehog the cut falls on the transcription factor, and the signal works by preventing it.

Design problem

Hedgehog switches off Patched, and Patched switches off Smoothened. Predict whether the pathway is on or off, with and without Hedgehog, in four cells: one without Patched, one without Smoothened, one without both, and one with a Smoothened that Patched cannot restrain. Then say which of two tumours a drug that blocks Smoothened can treat: one that lost Patched, or one that lost a brake that acts after Smoothened.

In vertebrates the pathway runs inside an antenna

Most of your cells carry one primary cilium: a single hair-like projection that does not beat, built on a core of microtubules and covered by its own stretch of membrane. It is assembled by intraflagellar transport, a shuttle of motor proteins that carries parts up and down the shaft.

In 2003 Kathryn Anderson’s laboratory at the Sloan-Kettering Institute was studying mouse mutants from a chemical mutagenesis screen. Two had defects in the neural tube and limbs that pointed to broken Sonic hedgehog signalling. The damaged genes turned out to encode intraflagellar transport proteins. Crosses placed their action after Patched and before the target genes, but a position in a sequence is not a place in the cell.

The place came in 2005. Kevin Corbit, in Jeremy Reiter’s laboratory at the University of California, San Francisco, followed a tagged Smoothened and found that it collected in the primary cilium when the pathway was switched on. Then the group changed the short stretch of Smoothened that sends it there. That protein stayed in the body of the cell and could no longer pass the signal on.

Later work showed that without Hedgehog, Patched sits at the cilium and Smoothened stays out. When Hedgehog binds, Patched leaves, Smoothened moves in, and Gli proteins gathered at the tip of the cilium are sent to the nucleus as activators. Cutting Gli to its repressor also needs the cilium, which is why a mouse without cilia shows too little Hedgehog response in some tissues and signs of too much in others.

Flies largely manage without: most fly cells have no cilium, and the fly pathway was worked out in cells that lack one. A cilium-based branch of Hedgehog signalling was reported in 2014 in fly olfactory neurons, which are ciliated. Why vertebrates send the same brake and the same switch through this compartment is not known.

Blocking Smoothened deforms an embryo and treats a tumour

In the 1950s, sheep ranchers in Idaho found lambs born with a single eye in the middle of the face. Investigators from the US Department of Agriculture traced the defect to the corn lily, Veratrum californicum, eaten by the ewe on about the fourteenth day of pregnancy. The alkaloid later purified from the plant was named cyclopamine.

In 1996 mouse embryos lacking Sonic hedgehog were found to have the same single eye. In 1998 two laboratories, those of Philip Beachy and Henk Roelink, showed that cyclopamine stops cells responding to Sonic hedgehog, and in 2002 Beachy’s laboratory showed that it binds Smoothened. The early forebrain starts with one eye field, and Sonic hedgehog from the midline tissue beneath it divides that field and the forebrain into left and right. In people the failure of this division is called holoprosencephaly, and inheriting one faulty copy of the Sonic hedgehog gene is one known cause.

The opposite fault, a pathway stuck on, gives a tumour. Basal cell carcinoma is the most common human cancer, and the large majority of these tumours run the Hedgehog pathway with no Hedgehog present. Most have lost both copies of PTCH1, the human Patched gene. A smaller share carry a Smoothened that Patched cannot restrain. People with Gorlin syndrome are born with one faulty copy of PTCH1 in every cell, so any skin cell that loses the other copy has no brake. They develop many basal cell carcinomas, often starting young.

Vismodegib is a drug that binds among the seven helices of Smoothened and holds it off. It was approved in the United States in 2012 for basal cell carcinomas that have spread, and for locally advanced ones that have come back after surgery or that cannot be treated with either surgery or radiation. Tumours can escape it through a change in Smoothened that stops the drug binding, or through a change further down the pathway. And the drug’s label repeats the lesson of the lambs: it must not be taken during pregnancy.