Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria

Abstract

Planarian flatworms regenerate every organ after amputation. Adult pluripotent stem cells drive this ability, but how injury activates and directs stem cells into the appropriate lineages is unclear. Here we describe a single-organ regeneration assay in which ejection of the planarian pharynx is selectively induced by brief exposure of animals to sodium azide. To identify genes required for pharynx regeneration, we performed an RNAi screen of 356 genes upregulated after amputation, using successful feeding as a proxy for regeneration. We found that knockdown of 20 genes caused a wide range of regeneration phenotypes and that RNAi of the forkhead transcription factor FoxA, which is expressed in a subpopulation of stem cells, specifically inhibited regrowth of the pharynx. Selective amputation of the pharynx therefore permits the identification of genes required for organ-specific regeneration and suggests an ancient function for FoxA-dependent transcriptional programs in driving regeneration.Some animals can regrow whole limbs or organs after amputation. Flatworms called planaria, for example, can regenerate their whole body from small pieces. This remarkable ability depends on neoblasts—a type of stem cell found in planaria that can detect damaged or lost organs, migrate to the site of damage, produce the required cells, and integrate into the remaining tissues. Researchers hope that studying these animals will reveal ways to use stem cells to regenerate injured limbs or organs in humans.Planaria have been used in many studies of regeneration. However, manually amputating organs from the flatworms is time-consuming and the resulting wounds vary, which makes it hard to compare regeneration between animals treated in different ways.Now, Adler et al. have developed a new technique for studying regeneration in planaria. Placing the flatworms briefly into a solution of sodium azide causes the pharynx—an organ that is used for both eating and excretion–to drop off. Using chemicals in this way means the loss of the pharynx leaves a uniform wound, with no damage to the adjacent digestive system, and that large numbers of planaria with identical wounds can be produced rapidly. To ensure that treatment with sodium azide did not alter normal regeneration processes in planaria, Adler et al. carried out manual amputation of tissue in sodium azide-treated flatworms; regeneration in these flatworms was identical to that seen in untreated planaria.Planaria in which the pharynx had been removed by sodium azide exposure showed rapid recruitment of neoblasts to the wound site, where they formed epidermal, muscle and nerve cells, and organized into a functioning pharynx within a few days. Adler et al. then identified 20 genes that were required for various stages of regeneration. These experiments revealed that a transcription factor (a protein that controls gene expression) called FoxA was specifically required for the regrowth of the pharynx. This is a previously unknown function for FoxA.