Unravelling How Planaria Regenerate

Planarian flatworms are one of nature's little wonders. Although their 'cross-eyed' appearance is endearing, their real claim to fame comes from their regenerative ability. Split a planarian down the middle and you'll soon have two cross-eyed critters staring back at you; cut one up and each piece will regenerate an entire flatworm. How do they pull of such an incredible feat? In 2011, researchers discovered that planarian regeneration depends on the activity of stem cells ('neoblasts') distributed throughout the flatworm's body, but important questions about the process have remained unanswered. Are certain stem cells responsible for each organ? What activates the stem cells when regeneration is needed? An enterprising team of scientists at the Stowers Institute for Medical Research has brought us closer to answering these questions by developing a new technique to study planarian regeneration and using it to discover some of they genes involved.

Regeneration isn't a uniquely planarian trait; starfish are well-known for growing back lost body parts, and even humans can regenerate to some extent (think of a wound healing). Planarians certainly excel at it, though; a flatworm can recover from being cut up into a staggering 279 tiny pieces, each of which regenerates into a new worm! Here's a fun conundrum for those inclined to such things: which worm, if any, can claim to be the 'original worm'? What if it were only two pieces instead of over 200? Would it make a difference if the two pieces were different sizes?

Undeterred by such philosophical considerations, the researchers used custom microarrays to identify genes which are activated when a planarian regenerates. The crucial innovation was a new way of inducing regeneration in a reproducible context — in other words, finding an easy way to injure many of the little creatures in the same way. Since planaria don't have clear anatomical landmarks, researchers can't use surgery to create identical injuries in many individuals; the resulting variability makes it hard to study the regeneration process. Likewise, surgery introduces secondary damage to other tissues, further complicating analysis. Ideally, researchers would like to be able to cleanly damage or destroy just a single organ and then watch it regenerate -- and that's exactly what the team managed. Carrie Adler, the post-doc who led the study, discovered that exposing the flatworm Schmidtea mediterranea to the chemical sodium azide caused them to lose their pharynx — essentially, their mouth — without damaging other tissues or affecting the regenerative process.

Using this technique, which they termed 'chemical amputation', the team induced lesions in planaria and investigated which genes were activated over the course of the regeneration process. The pharynx lacks neoblasts, but cells near the wound quickly start dividing and regenerate the amputated organ. To identify genes which were interesting, the team combined two screening approaches. First, a microarray picked out genes which were active during regeneration, providing a list of 356 candidates. Next, the team used RNAi to block the activity of each gene in amputated flatworms and checked whether the pharynx still regenerated. This narrowed the list down to twenty genes, which the team divided into different sets. Some genes affected stem cells in general, other affected feeding behaviour, and a handful directly affected the development of the pharynx. Of these, the transcription factor FoxA seemed to play the greatest role in regenerating the pharynx.

The team next looked at how regeneration went wrong in planaria with FoxA knocked down. They found that stem cells still migrated to the wound site and multiplied there, but the resulting outgrowth failed to become a pharynx. They also tried amputating the tails or heads of FoxA knock-downs, which then successfully regenerated. "Targeting FoxA completely blocked pharynx regeneration but had no effect on the regeneration of other organs," said Adler in a press release. “Currently, we think that FoxA triggers a cascade of gene expression that drives stem cells to produce all of the different cells of the pharynx, including muscle, neurons, and epithelial cells.” FoxA is known to play a role in specifying the pharynx in the sea anemone and in the nematode Caenorhabditis elegans, as well regulating the development of the intestine in vertebrates, so it makes sense that it's a central player in pharynx regeneration in planaria. More importantly, its identification can serve as a wedge to pry apart the details of regeneration; coupled with the other genes picked up in this study, it offers an exciting opportunity to expand our understanding of this important process.

Refs
Adler C, et al. Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria. eLife 3:e02238. (2014) doi:10.7554/eLife.02238
Rossant J. Genes for Regeneration. eLife 3:e02517. (2014) doi:10.7554/eLife.02517

Image credits
The Schmidtea mediterranea image is by Alejandro Sánchez Alvarado (one of the paper's authors!) and is distributed under a CC-BY-SA license via Wikimedia Commons. The planarian with fluorescent neoblasts, by Alex Lin and Bret Pearson, is a reproduction of Figure 1 from Rossant (2014) and is distributed under a CC-BY license.