Sometimes in science it pays to turn over a new leaf or an old laboratory animal. Stephen M. King at the University of Connecticut Health Center recently turned over planarian Schmidtea mediterranea, the nonparasitic flatworm justly renowned for its incredible regenerative powers, and saw on its underside a new way into a old problem. King, who is an ASCB member, believes that planaria could be an alternate model system for studying ciliary motility and its associated diseases now known as ciliopathies.
King's planarian prototype work was recommended by Peter Satir of the Albert Einstein School of Medicine and a longtime leading light in ciliary biology. "Of course it's a classic model for regeneration studies," said Satir, describing how planaria can regrow an entire worm from tiny cut-off fragments, but Satir credits King for looking at the underside of the flatworm and seeing a new model system. "That's the novelty here, that it's used in ciliary motility studies," said Satir.
Planaria live in freshwater ponds and rivers, moved by waves of cilia on their ventral surface beating against a secreted mucus. In humans, a similar mucociliary epithelium lines the respiratory tract, and ciliated epithelia also occur, for example, in the ependyma of the brain, and the fallopian tubes. Synchrony in ciliary beating is essential whether in flatworms or humans. Driven by the molecular motor, dynein, the cilia generate specific waveforms in response to different stimuli. In humans, disturbance of those beating patterns is linked to a variety of diseases or disorders including infertility, bronchiectasis, and situs inversus.
King, with collaborators Pantelis Rompolas, a former student/postdoc in the King lab; Wallace Marshall at the University of California, San Francisco (UCSF); and Marshall's UCSF postdoc Juliette Azimzadeh, laid out the case last year for using planaria as the "ideal system for cilia gene loss-of-function studies."
Recently King spoke by phone from his lab at UCHC in Farmington, CT, with the ASCB Post's Christina Szalinski.
How did you end up working with planaria?
My graduate student at the time, Pantelis Rompolas, and I were sitting in journal club one day and a student was talking about a paper on stem cells where they used planaria and described what a nice system it was for RNAi. We were looking at pictures of the planaria and realized they have an exposed ventral ciliated epithelium and we thought it would be a great idea for us to try it. There was a colleague at the university here who actually had some animals. So we got a founding colony from him and grew them up and started playing around with them and found out that it worked really well.
Why use planaria instead of other established animal model systems for ciliary study such as Chlamydomonas?
The one thing that's really easy to do in planaria is RNAi knockdown, like in Caenorhabditus elegans. So you basically just feed the animals with bacteria in which you have induced a gene-specific double stranded RNA and you feed them every two or three days for maybe three weeks. You get incredibly robust knockdowns. It's really cool.
We have been generating knockdowns of cilia genes and you get very robust phenotypes. Planaria use their ventral cilia to beat against secreted mucus and this causes the animals to glide across the petri dish or whatever substrate they're on. If you disrupt their cilia, whether they don't have any or they don't beat with high frequency, they actually switch their mode of motility. Instead of this nice smooth gliding motion, they use their body muscles to squeeze themselves across the surface—almost like squeezing a tube of toothpaste. So if you're looking for ciliary phenotypes it's really obvious, you can just watch them.
You can do knockdowns in Chlamydomonas but it's not quite as simple. The other issue with the Chlamydomonas is that although there are lots of mutants available, targeted gene disruptions aren't so easy to achieve. My lab now routinely uses a combination of these two model systems.
Can working with planaria answer questions about human diseases?
Absolutely. They will help answer many questions to do with ciliary activity. Defects in cilia have all sorts of effects in humans. For example, if you had defects in motile cilia, you would have primary ciliary dyskinesia (PCD), which has many phenotypes including infertility. People with PCD tend to have bad bronchial problems because the cilia in their lungs can no longer move the mucus that's secreted. There are also developmental defects like situs inversus, where the organs and viscera are in the wrong places because cilia are thought to be involved in setting up a fluid flow in the developing embryo that determines the left-right body axis.
What are the challenges in working with them?
We have yet to come up with a way of easily detaching the cilia, so trying to do any biochemistry is pretty much a nonstarter. The other major challenge in working with them is they are actually rather strong animals. They don't like to be confined under a coverslip. They'll squeeze themselves around it, so it can be quite a frustrating exercise to image the cilia beating. They also don't like the light and, of course, you're illuminating them a lot under the microscope. We still work on Chlamydomonas so we basically use the two systems to look at different things. If we come across a new protein in cilia that we're interested in, one of the first things we do is knock it down in planaria, assuming there is an ortholog, to see what kind of phenotype we're going to get. Then we do biochemical studies in Chlamydomonas. So you can play off the two model systems where you have easy RNAi on the one hand and great biochemistry on the other.
What are the advantages of working with planaria?
Not only can you do the nice RNAi knockdowns, you can also see the cilia. The place where we look at the cilia beating to understand the waveform and beat frequency is right at the head of the dorsal/ventral margin—you can see a nice array of the cilia there.
Is their genome sequenced?
It's sequenced but not completely assembled. Things are in pieces. Sometimes if you look at a small protein, you'll probably find the whole sequence is there, but for large proteins it will come out in chunks.
Are ciliary proteins conserved between planaria and humans?
Many of them are. There have been a few that we haven't been able to find in planaria, but for the most part they are pretty highly conserved and whether we couldn't find them because they don't exist or because the genome isn't completed is an open question.