Within the planarian field a hot topic in the last years has been the discussion about which factors control the re-establishment of polarity and whether or not signalling centers functioning as organizers exist during regeneration. Different genes are required to trigger anterior regeneration in terms of providing identity and the proper patterning cues. Among them, wnt1, notum, pbx, prep and follistatin play key roles.
Now, a recent paper from the laboratory of Kerstin Bartscherer report on the central role of the transcription factors foxD (Forkhead) and zicA (zinc finger transcription factor) (http://www.ncbi.nlm.nih.gov/pubmed/24704339). The results presented here complement and expand previous reports on foxD function during planarian regeneration from the laboratories of Peter Reddien (http://www.ncbi.nlm.nih.gov/pubmed/24415944) and Phil Newmark (http://www.ncbi.nlm.nih.gov/pubmed/23297191).
FoxD is expressed at the tip of the head where it co-localizes with other markers of this anterior pole, such as notum and follistatin. Upon its silencing, anterior regeneration was severely impaired as some animals developed small blastemas with cyclopic eyes (eyes fused at the midline) and aberrant small brains, and other worms regenerated extremely small blastemas with no eyes or cephalic ganglia. Next, the authors decided to search for putative downstream targets of foxD by sequencing the transcriptomes of control and foxD(RNAi) regenerating tail pieces. One of the identified candidates was a zinc finger transcription factor that was named Smed-zicA. Remarkably, zicA was also expressed at the anterior tip of the head similarly to foxD, notum and follistatin. In fact, zicA co-localizes with foxD, notum and follistatin-expressing cells. Upon RNAi silencing of zicA, the treated planarians displayed very similar defects on anterior regeneration as those shown after foxD RNAi. To validate that zicA was a target of foxD the authors show how the expression of zicA disappeared after foxD RNAi. However, they also found that foxD expression depended on zicA, which suggests that these two genes may regulate each other’s expression. Also, double silencing of foxD and zicA resulted in more severe phenotypes, further supporting that both genes act together during anterior regeneration.
After either foxD or zicA RNAi the expression of several genes normally expressed at the anterior region was significantly reduced. On the contrary, the expression of genes normally expressed at the posterior pole was unaffected. In planarians notum has been shown to play a key role in the establishment of anterior polarity at very early stages of regeneration. Even though the coalesced notum expression at the anterior tip was inhibited after foxD or zicA RNAi at 3 days of regeneration, the silencing of any of these 2 genes did not affect the early expression of notum. This suggests that foxD and zicA would be required for head regeneration downstream of early polarity determinants. Also, the expression of follistatin at the anterior tip was lost after foxD or zicA RNAi at 3 days of regeneration. Previously, follistatin has been suggested to function together with notum to determine a putative anterior signalling center. In addition, the silencing of foxD or zicA lead to a rapid decrease of the expression of notum and follistatin at the anterior pole of intact non-regenerating animals, just after 3-7 days. After irradiation, however, cells expressing notum and follistatin at the anterior pole were maintained at least until day 8 after treatment. Thus, these results suggest that the loss of notum and follistatin-expressing cells would not be caused by stem cell-based turnover, but would support a role for foxD and zicA in the regulation of the expression of notum and follistatin at those anterior pole cells.
Finally, the authors analyzed whether or not foxD and zicA were expressed in differentiating anterior pole cells, as foxD and zicA-positive cells were lost after irradiation in 3 day-regenerating fragments. They found that foxD and zicA co-localize with the stem cell marker Smedwi-1. But they also co-localize with cells that do not express Smedwi-1 anymore but still are positive for SMEDWI-1 protein. These results indicate that foxD and zicA are expressed in the progenitors of the cells that will give rise to the anterior pole, and somehow required for their differentiation and the formation of this pole. Terminal differentiated cells at the anterior pole express foxD and zicA, whereas progenitor cells closer to the stump co-express these to factors together with SMEDWI-1.
Further experiments are necessary to define the exact relationships between the different genes that appear to be important for the development of this anterior pole (pbx, follistatin, foxD, prep and zicA), as well to see up to which extend this anterior pole works as an organizing signalling center.