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Along with Sánchez Alvarado, Phil Newmark (left) and Peter Reddien brought planarians into the molecular era.
A Chance Meeting
Amid antique locomotive engines at the National Railway Museum in York, England, Sánchez Alvarado sat next to Newmark at the 1996 annual meeting banquet of the British Society for Developmental Biology. At the time, Newmark was in Jaume Baguñà’s lab at University of Barcelona as a Damon Runyon Cancer Research Foundation postdoctoral fellow, learning everything he could about fundamental planarian biology to eventually set up a line of research in the United States.
“Here I found another member of my tribe,” recalls Sánchez Alvarado. The two contemporaries, just one year apart in age, hatched a plan to convince the Carnegie Institution and Damon Runyon to let Newmark be a postdoc with Sánchez Alvarado. The two would attempt to turn planarians into a cultured lab animal that could serve as a system for deconstructing the molecular pathways that control regeneration.
Both Carnegie and Damon Runyon “really took a risk and supported us,” says Newmark. “There were plenty of people [along the way] who thought this was craziness.”
The two personified dogged persistence. Early on, they spent a sleepless weekend cutting heads and tails off of about 1,000 animals. These experiments would tell them in the coming months which genes were expressed in intact heads and tails compared with regenerating heads and tails. Newmark also worked on modifying an in situ hybridization protocol, which is used to mark specific genes with a dye to see where the gene is turned on in the whole animal. It wasn’t easy. The animals’ mucus covering tended to suck up the dye and turn everything dark blue.
With almost any method they attempted for tracking the planarians’ stem cells—which are called neoblasts and make up roughly 30 percent of the animal’s cells—the team had to work through trial and error. They figured out how to label the neoblasts and began making libraries of the genes expressed during various stages of regeneration.
During his time at Carnegie, Newmark also created a genetically identical line of worms by cutting and growing up pieces from one individual collected from the Barcelona park. That line has been going for almost 12 years and is used by all the planarian laboratories in the United States. No more mucking through dormant fountains.
In 1998, Andrew Fire, working downstairs from the two at Carnegie, had discovered a way to silence specific genes in C. elegans. The method, called RNA interference, or RNAi, works by introducing a double-stranded RNA that matches the message of the gene a researcher wants to knock down. The RNAi sets off a process in the cell that destroys the mRNA message. The method, for which Fire shared the 2006 Nobel Prize in Physiology or Medicine with HHMI investigator Craig Mello, effectively turns off the gene.
Fire asked Newmark and Sánchez Alvarado to try RNAi in planarians to see if the method would work beyond C. elegans. Sánchez Alvarado first tried silencing the planarian tubulin and myosin genes since the resulting proteins are found in every cell and he had the dyes to track them. He recalls looking at the treated worms under the microscope at 1:00 or 2:00 a.m.: their regeneration stumps, or blastemas, had no tubulin or myosin.
Now they had a loss-of-function assay for the planarians—a tool with which to ask, if this gene is missing, how does it affect the regeneration mechanism? All they needed was some regeneration-specific genes to test.
“We needed to clone genes like there was no tomorrow so that we could do RNAi screening. That kept us occupied for eight years or so,” says Sánchez Alvarado. Newmark calls the RNAi work a crucial breakthrough from their years at Carnegie: 1997 to 2001.
“Now we could do functional biology. We could ask whether inhibiting the expression of a given gene could disrupt the regenerative process. We could really learn something from these animals. It was a fantastic time,” says Newmark, now at the University of Illinois at Urbana–Champaign.
Photos: Newark: Darrell Hoemann / AP ©HHMI, Reddien: Jason Grow