By David N. Leff

Just over a century ago, in 1898, geneticist Thomas Hunt Morgan (1866-1945) sliced a small flatworm, or planarian, into 279 bits and pieces. Each tiny fragment regenerated a complete animal.

¿Morgan concluded,¿ observed molecular embryologist Alejandro Sanchez Alvarado, ¿that these planarians were so mysterious that he decided to abandon them. Morgan moved on to Drosophila melanogaster, the fruit fly, because he thought their genetics offered a more tractable problem. And that was a good move,¿ he added. ¿Thanks to Morgan we have modern genetics, Drosophila genes and the drosophilosophers who study them.¿

Sanchez Alvarado is a staff scientist at the Carnegie Institution of Washington, at its department of embryology in Baltimore. His laboratory focuses on that mysterious mechanism by which planaria duplicate themselves.

¿The impetus for this research,¿ he told BioWorld Today, ¿was to find or devise a model system to study regeneration where one could manipulate gene expression. The available models that people use for complex regeneration, such as limb or tail replacement in amphibians, are fairly intractable to molecular manipulation. They are very reticent to the kinds of things that people would do, say, in Drosophila or the C. elegans roundworm nematode. In those models you can knock out a gene, or its expression, then find a phenotype and see what happens.¿

So Sanchez Alvarado and his colleagues searched the animal kingdom¿s family tree of multicellular organisms, looking for creatures capable of regenerating. ¿We chose planaria,¿ he recounted, ¿because they have all three embryonic germ layers that we humans have ¿ endoderm, mesoderm and ectoderm. It¿s from these three layers that most of the structures in the human body form during embryogenesis. And it is from these same three layers that limbs or tails regenerate in salamanders and so forth.¿

Because the genes for these structures show up in fruit flies and roundworms, he went on, ¿we figured that planarians probably have the same genes we have. So we did a series of screens, and pulled out a bunch of genes from these flat worms. We were surprised to find that most of them have a very high degree of homology to human genes.¿

Armed with this knowledge, the group cast about for a method that would allow them to test the function of those planarian genes in regeneration. They hit upon double-stranded RNA to silence those genes, essentially to make knockout flatworms during the process of their regeneration.

Another Carnegie embryologist, Andrew Fire, had recently stumbled on this property of double-stranded RNA (dsRNA) in C. elegans.

¿Those two molecules of RNA are complementary to each other,¿ Sanchez Alvarado explained. ¿So they form a helix that looks more or less like a DNA helix. Fire was trying to knock out roundworm genes using antisense technology. One of his controls was to inject both sense and antisense strands together. To his surprise, he saw that when he did that, the gene-silencing effect was many-fold higher than the individual antisense strand. And it turned out to work not only in C. elegans but in Drosophila and other organisms.¿

Whereupon, Sanchez Alvarado tried dsRNA to knock out a muscle-building gene in his planarians.

Tapping Into Knockout Flatworm Model

¿What we did,¿ he recounted, ¿was to look at the sequence of the gene we wanted to knock out, namely, the myosin muscle protein, which is about 2 or 3 kilobases long. So we selected a region of about 1 kilobase for which we knew the sequence, nucleotide by nucleotide. We subcloned that little fragment and synthesized RNA from one strand and the other strand, then mixed them together.

¿We injected this mixture into the planarians,¿ he went on, ¿inside their body. Somehow the planarian cells took up this mixture, and those cells that were expressing myosin were affected by the dsRNA. They no longer made myosin and their musculature was gone.

¿But the other cells that were not expressing myosin were fine. The animal survived and regenerated just fine. So that indicates to us that the dsRNA is specific, only knocking out the particular target genes that we have in our sights.¿

Sanchez Alvarado is lead author of a paper in the current Proceedings of the National Academy of Sciences, dated April 27, 1999. It¿s titled: ¿Double-stranded RNA specifically disrupts gene expression during planarian regeneration.¿

¿There are 5,000 to 10,000 species of planaria,¿ he pointed out. ¿Ours are of the strain Schmidtea mediterranea, 8 to 10 millimeters long.¿ Those freshwater, free-living flatworms, the team¿s new animal models of regeneration, he continued, ¿have eight chromosomes and come in two biotypes. One, the fissiparous, reproduces by fissioning. These asexual animals fission themselves in half, rupturing their bodies transversally. The tail part will regenerate a head part, and the head part a tail.

¿But they also have a cousin of the same species,¿ he went on, ¿that reproduces sexually. We wanted a species that was able to do both, so we could research their regeneration and then see whether or not the same processes are taking place in embryogenesis. So the next leg of our project will be to characterize the regeneration of specific genes in the normal developmental stages of the sexually reproduced animals.¿

Genes Turned On In Flatworms, Off In People

Sanchez Alvarado made the point that, ¿What our lab wants to understand is why regeneration exists. And why some animals can do it, and others can¿t. We still have to characterize all the genes that are responsible for this process. We know that this organism has these genes. They are also present in us humans. Planarians have them; they regenerate. We have them and don¿t.

¿Humans don¿t regenerate our head or our hand, but we have those same genes. So eventually what needs to be identified is the choreography by which these genes are deployed in time and space. Once this is understood, then one could in principle activate those particular genes in that particular order in those particular places and at those particular times, to mimic the initial events that you see in a planarian.¿

He allowed, ¿This is oversimplified, of course. But if that is true, then you could in principle be able to regenerate a finger or a hand, or something like that. I think that basically we are going to find out ¿ not just my lab, but people working on regeneration in general ¿ the factors that allow regeneration to take place in some organisms, and the factors that do not allow it to take place in other organisms. (See BioWorld Today, Oct. 6, 1998, p. 1.)

¿Because I really don¿t think,¿ Sanchez Alvarado concluded, ¿that there is going to be a regeneration gene. It's just the way the genes are deployed. It¿s too hard for me to imagine that we humans lost the regeneration gene. You usually don¿t lose genes through evolution; you gain more. So it¿s very unlikely that this planarian has jealously guarded its regeneration gene and didn¿t share it with the rest of evolution.¿ n