Howard Hughes Medical Institute Investigator; Stowers Institute for Medical Research, Kansas City, MOJay Casillas, Stowers Institute for Medical Research
JAY CASILLAS, STOWERS INSTITUTE FOR MEDICAL RESEARCH
In the fall of 1998, Alejandro Sánchez Alvarado was celebrating the discovery that he could use the newly developed technique of RNA interference (RNAi) to manipulate gene activity in planarians—a crucial step in turning the flatworm into a model organism for studying regeneration. Then the animals started to die.
“Something happened to our water source,” says Sánchez Alvarado, who was a staff associate at the Carnegie Institution of Washington at the time. “All of a sudden, we didn’t have any more animals. It was an experimental kiss of death.” But rather than throw in the towel, Sánchez Alvarado decided to build a new colony. So he started watching the weather—in Spain. “We knew it had to rain for us to be able to collect planarians from this park in Barcelona,” says Sánchez Alvarado. So when the forecast was suitably wet, Sánchez Alvarado and his postdoc, Philip Newmark, jumped on a plane. “When we landed, we hopped in a cab and told the driver, ‘Take us to a butcher. We need to buy some liver.’ ” In their hotel room, the pair minced meat and baited dozens of homemade planaria traps, which they then plopped into every fountain they could find in Montjuïc Park. “We knew our animals had come from one of those fountains, but we didn’t know which,” says Sánchez Alvarado.
The next day they checked their traps and found—one animal. Discouraged, the two abandoned that trap and went to find something to eat. “Barcelona is a nice place to be depressed,” says Sánchez Alvarado. “It’s a beautiful city and the food is great.” The next morning, they returned to the fountain and found the trap teeming with planarians. “Those are the animals we use to this day.”
About losing the original colony Sánchez Alvarado says: “Every year, there’s not a single science fair that does not have a planarian project. Middle-school and high-school students can keep these animals—and we couldn’t! How embarrassing is that?”
But the lab has made up for the rocky start with a series of surprising discoveries that have begun to shed light on the fundamental principles of animal regeneration—revealing the remarkable developmental plasticity that allows these animals to regrow their heads, their tails, and everything in between. Here Sánchez Alvarado waxes philosophical about planarian taste preferences, life beyond the standard models, and learning the King’s English.
SÁNCHEZ ALVARADO SETS OUT
All of a sudden I went from having a system that had phenomenal biology to having a method that allowed me to perturb
Tails you lose. As a postdoctoral fellow in Don Brown’s lab at the Carnegie Institution in 1994, Sánchez Alvarado was exploring why tadpoles that have had a tail amputated mistakenly regenerate an extra set of hind limbs when exposed to retinoic acid. “I identified a bunch of molecules involved in the process, but I couldn’t test them,” he says. The phenomenon didn’t occur in the well-studied Xenopus, but in a genetically intractable European species. “So I was all dressed up with no place to go because I had no way to perturb the function of these molecules. Turns out that was the case for the entire field of regeneration. So I thought, if you really want to do this right, you should identify a model system for regeneration that would be amenable to molecular manipulation.” Which is what he set out to do.
RNA to the rescue. It was Carnegie colleague Andrew Fire who demonstrated that he could silence specific genes in C. elegans by feeding them double-stranded RNA. But Sánchez Alvarado was at first skeptical of the technique. “Andy would come by my little office and say, ‘Hey, Alejandro, you’ve got to try this stuff in your worms’—because he wanted to see if it was universal. And I would say, ‘Oh, Andy, it’s an artifact.’ That was egg on my face! Because of course it worked. All of a sudden I went from having a system that had phenomenal biology to having a method that allowed me to perturb that biology.” Of course, Sánchez Alvarado did have to play with the protocol a bit. “Phil and I became planarian chefs. Because they hate the bacteria you use to feed them the RNA. Hate it. You put the pellet in front of them and they just run in the opposite direction. Eventually we turned to low-melting-point agarose. We hide the bacteria in there. It’s like soft-serve. That seems to work.”
Best supporting actors. When Sánchez Alvarado began his work on planarians, he initially focused his attention on their stem cells, also known as neoblasts. “These cells are remarkable. They’re adult stem cells that are capable of giving rise to tissues from all three germ layers. But what we have begun to discover in these past few years is that these differentiated tissues are just as remarkable.” It’s these differentiated cells that launch the regeneration program. Take, for example, the establishment of the animal’s dorsoventral axis, which is triggered by the expression of BMP along the midline. “We asked, ‘If we remove a tiny fragment from an animal’s side—which is not expressing BMP—can we then watch the midline regenerate?’ When we do that experiment, we see that the little piece of tissue that we removed begins to express BMP. We initially thought that maybe the neoblasts, the stem cells in the tissue, were making progeny that begin to express BMP so they can form a new midline. But it happened too quickly—within less than 24 hours, which is faster than these cells can divide.” That made them think that maybe it was the differentiated tissue itself that was responding. So the researchers irradiated the flatworms to kill off the dividing stem cells. And when they amputated a small piece of tissue, they saw the same pattern of expression. “That means that an amputation has instructed the postmitotic differentiated tissues to change their genomic output”—a finding published in Development in 2007.
This unexpected plasticity is not limited to BMP expression. The same is true for cell death. Lop off a planarian’s tail, and cells sprinkled throughout that amputated end will undergo apoptosis. “That’s probably because the tissue is remodeling itself to allow regeneration to proceed and to reset scale and proportions. So we asked: Does this happen in the absence of neoblasts?” Again, they irradiated animals and found that the patterns of cell death were identical, with and without stem cells—work published in Developmental Biology in 2010. “That suggests that not only are the preexisting tissues plastic in their genomic output, but that the processes that allow for all this remodeling seem to be taking place independent of the neoblasts themselves. So it seems that the differentiated tissues are as important as, if not more important than, the resident stem cells. They are not just passive bystanders providing scaffolding for new tissue to form. They’re actually probably responsible for creating the context in which these stem cells can produce the right cells in the right numbers to allow regeneration to proceed. To me that’s one of the most interesting things we’ve found so far.”
SÁNCHEZ ALVARADO SPEAKS OUT
I love finding an article and then pulling out the tome next to it and thumbing through, just to see what was being published the year before.
Into the unknown.“‘I think grants are now being given to hunters and not explorers. There are all these pejorative terms for exploration—fishing expedition—but it’s a valid way to do science. Hypothesis-driven research is great if you have a body of knowledge that’s sufficiently strong to allow you to propose testable hypotheses. But to suggest that we have a thorough comprehension of all the fundamental principles that make life possible is fanciful,” says Sánchez Alvarado. Of course, exploration is not for the faint of heart. “If you’re prepared to stomach failure, you can fund as many risk takers as you want—in the hopes that one or two will actually find something. But if you start funding fewer and fewer, it’s a self-fulfilling prophecy. ‘We’re wasting money and we’re not getting results.’ That’s because you’re funding fewer and fewer explorers. There’s no obvious method for selecting the ones that will be successful. If there were, risk-taking would be an institution.”
Casting a wider net. “Transcriptome analysis is really changing the face of what we do in the lab. Now you can do quite well with very, very small amounts of material and still produce complex cDNA libraries that can be analyzed for the transcripts that are represented. So if you want to work on some organism that’s really rare—or that was beyond the purview of investigation because it’s difficult to keep in the lab or the tools weren’t there—all you need are a few samples, a few specimens, captured at a time when the biological activity you’re interested in is being displayed, and you can purify the RNA and get the transcriptome. So at the very least you know what genes are associated with the biological phenomenon you hope to understand. As the technologies continue to get cheaper and cheaper, there’ll be no excuse not to look beyond the usual suspects, beyond mice and Drosophila and C. elegans. I think we’ve deluded ourselves into thinking that we have enough model systems—that these few animals encompass the entire gamut of physiological activity on the face of this planet, including our own. But they don’t have a monopoly on interesting biological phenomena. If we want to understand regeneration, if we want to understand aging, if we want to understand any of these attributes that emerge after birth, why not go out and see if there are organisms that would be much better models to study these questions than the ones we already have.”
Everyone’s a critic. “It’s a part of our culture to criticize. And because we tend to hear more about the negatives than the positives, we assume that the job of a reviewer is to find something wrong. Also, busy PIs may farm out the papers to review to younger people in the lab—who then think, ‘I want to show my PI that I can be a critical thinker, so I have to find flaws.’ I think we need to take the time to train the next generation of reviewers, for grants as well as papers. After all, at some point one of your students is going to be a reviewer for one of your grants. So it’s important to say, ‘Look, you don’t have to find some error just to show how clever you are. There’s no crime in saying that you think something is actually pretty good.’”
Money-back guarantee. “When you and I went to college, most of what we learned was valid—and was going to be valid for a few years. So that was money well spent. Today, by the time you graduate, a big chunk of what you learned, particularly in the sciences, is obsolete. Information is accumulating and turning over at such a rapid rate. It’s likely that my kids will have to molt and metamorphose their knowledge base multiple times during their lives. I don’t think this has ever happened at this scale in the history of humankind. You could live through the entire 18th century just reading a couple of books and you’d be okay. You can’t do that now. Don’t get me wrong. I think that this is what we should be doing as a species—trying to understand the universe. But I think universities should start giving back money for the information that’s obsolete by the time you graduate. If one-third of what you learned is no longer true, it seems fair to me that they should consider giving one-third of your money back.”
SÁNCHEZ ALVARADO SAVORS
Listen and learn. When Sánchez Alvarado left Venezuela to become an undergraduate at Vanderbilt University, he spoke no English. “I figured I would learn one way or the other. And I did.” His method: “I listened to the news on the radio. That’s when I realized I was beginning to distinguish words from each other. Before that, everything sounded like one incredibly long word.” And at the advice of his freshman English professor, Sánchez Alvarado lent his ear to the Bard. “He suggested I go to the library and pull out the LPs from the Royal Shakespeare Company and listen as I read the play. That was absolutely phenomenal, because I could read a sentence and hear where there was supposed to be irony or where it was supposed to be funny. Then I could begin to identify in my mind these higher-level relationships of meaning. I’m a big Shakespeare fan because of that.”
Bibliophilia. “I love going to libraries. Every time I go to Woods Hole, I make it a rule to spend at least several hours every evening just combing through the stacks. I love finding an article and then pulling out the tome next to it and thumbing through, just to see what was being published the year before. Sometimes I tell students to go look at the first issue of PNAS or Science or Nature—just to see what people were up to at the time. I think it gives them a sense of wonder and discovery, an appreciation of the sheer pleasure of finding things out.”
No hay brevity en español. “I love short stories. Maybe because of my schedule. I like the new Latin American authors who are writing short stories and relatively short books.” Although that ‘relatively’ is relative. “As beautiful a language as Spanish is, and as much as I love it, there are some things that are very difficult to do in Spanish with the economy of words you can do in English. If I were to write my papers in Spanish, they’d be at least three times as long.”
When to mentor. Sánchez Alvarado’s PhD advisor, Jeff Robbins (now at the Cincinnati Children’s Hospital Medical Center), was a tough editor—except when it came to his thesis. “When I got it back, I was surprised there were no comments. He said to me, ‘Your thesis should reflect the intellectual maturity you had at the time you wrote it. So when you go back, years from now, and look at it, you can see how far—or how not far—you’ve come.’ Isn’t that awesome? That’s what a great mentor does—gives you the opportunity to become a better scientist, a better thinker, a better person.”