Freshwater planarians are well known by their abilities to regenerate a whole animal from a tiny piece of their bodies. This process is driven by pluripotent adult stem cells, the neoblasts (the only cells with mitotic activity). Recently, several studies have reported different specific transcription factors required to commit neoblasts subpopulations into distinct cell lineages. However, it is still unclear how neoblasts are directed into the different lineages that conform, for example, discrete organs. Now, a paper from the laboratory of Alejandro Sánchez-Alvarado describes a novel method to analyse pharynx regeneration in these animals and identifies FoxA as a key factor to regulate a genetic program underlying the regeneration of this organ (http://www.ncbi.nlm.nih.gov/pubmed/24737865).
The planarian pharynx is a peculiar organ because it does not contain neoblasts within it but still depends on neoblasts for its continuous cell renewal and regeneration upon injury or amputation. Previous results had suggested that neoblasts in the body mesenchyme, at the base and around the pharynx, sustain pharynx cell renewal and regeneration by migrating from their original position to the inside of this organ. A remarkable approach used in this paper is that the authors have found a chemical way to selectively remove the whole pharynx from the rest of the animal. Treatment with sodium azide for a short time causes pharynx extrusion and dislodgement. Other organs and systems, including the gut, do not seem to be affected by this treatment. Seven to ten days after this chemical amputation, a new normal pharynx is regenerated in these animals.
After pharynx amputation there is a transient decrease in neoblast mitotic activity, maybe due to the action of sodium azide. In terms of whole-body mitotic activity, this did not significantly change throughout the regenerative process. However, 24 hour after amputation there is a significant increase of proliferation around the wound site. That is, there is a local mitotic peak at 24 hours that then decreases over time. In order to identify transcripts upregulated during this early event of pharynx regeneration the authors isolated plugs of tissues surrounding the pharynx wound site at different time points: 0h, 24h, 48h and 72h. They identified 718 genes that were upregulated at 24h after amputation and cloned 274 of them. Moreover, they cloned 82 genes that were upregulated at 48h and 72h of regeneration, but not at 24h. Some of these genes were validated by in situ, revealing some cases of genes that were not detected in intact uninjured animals but were strongly upregulated at the wound region after amputation.
Next, the authors performed an RNAi screen of the 356 cloned genes. As a read-out to detect possible defects in pharynx regeneration the authors scored the capacity of feeding of those animals. By setting up a threshold of 50% defect in food uptake they found 20 genes. After RNAi of these genes and chemical amputation of their pharynges the authors analysed three features: % of food uptake, pharynx length and mitotic activity in the whole body. According to this, the 20 genes were classified into 3 distinct categories: 1) general regulators of stem cell function as their silencing significantly inhibited neoblast proliferation and pharynx regeneration; 2) specific effectors, whose silencing did not affect neoblast proliferation but regenerated smaller or abnormal pharynges not completely functional; and 3) others, whose silencing did not affect neoblast proliferation or pharynx regeneration but impaired effective food uptake.
One of the genes identified in the category of specific effectors was a homologue of FoxA, a conserved transcription factor required for foregut development in different animals. Planarian FoxA is expressed in different pharyngeal cell types (epithelium, muscle and neurons) and in cells in the mesenchyme surrounding this organ. The authors showed how these mesenchymal cells disappear upon irradiation indicating that they are either neoblasts or early neoblast progenitors. The silencing of FoxA impaired pharynx regeneration upon chemical amputation. Compared to controls, in which after 3 days they had formed a small well-patterned pharynx rudiment, the FoxA RNAi planarians had a disorganized mass of cells. This suggested that FoxA would be required to produce pharyngeal cells and to pattern them properly. In fact, a percentage of cells co-express FoxA and Smedwi-1 (a neoblast specific marker), and this number significantly increases upon pharynx amputation, further supporting that these cells define pharyngeal progenitors. The fact that neoblast proliferation is not affected after FoxA RNAi suggests that FoxA might play a key role in directing neoblast progeny into their differentiation towards pharyngeal cell types.
Finally, the authors analysed the expression of FoxA after silencing the remaining 19 genes identified in their screen defining a molecular pathway driving pharynx regeneration. In summary, this study presents a novel approach to analyse organ regeneration in planarians and identifies FoxA as a key regulator driving pharyngeal cell types differentiation from pluripotent neoblasts.