In this week’s post just let me present to you some of our own recent results on planarian regeneration. After amputation different events must take place in the right order and at the exact time to achieve a successful regeneration. In the case of planarian regeneration one of the first event after wound healing is the establishment of polarity, that is, the animal must know whether to regenerate a head or a tail. Then, once the polarity of the forming blastema is determined other events occur; these include patterning, growth and differentiation of those blastemas. In recent years several genes have been shown to play an important role in the re-establishment of polarity and pole formation in planaria. However, less is known about how for example, a new brain differentiates within an anterior blastema. Now, in a paper from my laboratory, we have characterized a gene that is important for head regeneration in planarians, mainly through its role on the differentiation of the brain primordia (http://www.ncbi.nlm.nih.gov/pubmed/24700819).
In a previous work we had reported that Smed-egfr-3, a homologue of the epidermal growth factor receptor family was important for blastema growth most probably by affecting cell differentiation. Then, we decided to do some DGE analyses to identify putative downstream targets of Smed-egfr-3. One of the isolated genes was Smed-egr-4 a homologue of the early growth response gene family of zinc finger transcription factors.
In situ hybridizations showed that egr-4 was expressed in the CNS, especially the brain, and the mesenchyme in irradiation-insensitive cells (that is, in post mitotic differentiated cells). As expected, the expression of egr-4 was downregulated after egfr-3 RNAi. However, we found out that egr-4 went through two phases of expression, an early expression immediately after amputation or wounding and up to two days, that was independent of egfr-3 and a second phase from the second day of regeneration that depends on egfr-3. The silencing of egr-4 blocked specifically anterior regeneration without affecting the regeneration of posterior regions. In egr-4 RNAi animals, anterior blastemas were very small compared to controls and they failed to regenerate a normal brain or the proper pattern of several other anterior markers. On the other side, the silencing of egr-4 did not seem to largely affect the number of neoblasts (planarian pluripotent stem cells) and although there was a slight decrease in the number of mitotic cells, this did not seem to explain by itself the lack of regeneration. Moreover, other markers suggested that the silencing of egr-4 could be affecting the differentiation of neoblast late progeny.
Recently, other genes have been reported to show a similar phenotype. In those cases, the inhibition of regeneration has been associated to the failure of re-establishing a proper polarity or pole as the expression of polarity determinants is inhibited. Therefore, we wanted to determine whether the impairment of head regeneration observed after egr-4 RNAi was due to defects in the re-establishment of the anterior polarity. However, that did not seem to be the case, after analyzing the expression of several polarity determinants, which suggests that egr-4 might be affecting an event downstream of anterior polarity re-establishment.
As egr-4 was expressed in the mature and differentiated cephalic ganglia we next investigated if egr-4 was required for CNS regeneration. By performing combinatorial RNAi of egr-4 together with an APC homologue we found out that egr-4 appeared to be required for the differentiation of the brain primordia. Given the fact that egr-4 RNAi inhibited anterior but not posterior regeneration and the Wnt/b-catenin pathway mediates the specification of head versus tail regeneration (the silencing of b-catenin1 transforms any blastema into an anterior blastema that will differentiate into a head) we further investigated the relationship between egr-4 and this pathway. Surprisingly double RNAi of egr-4 and b-catenin1 resulted in normal anterior regeneration, which suggested that in a normal situation egr-4 may antagonize b-catenin activity to allow head regeneration. Finally, experiments in which egr-4 was silenced at different time points after amputation and other experiments in which some brain tissues were left after amputation indicated that: (1) the action of egr-4 would be required during the first 2-3 days of regeneration, being dispensable for regeneration after that stage, and (2) the presence of brain tissues was able to rescue the defects observed after the silencing of egr-4.
Overall our results suggest that egr-4 plays a key role in the differentiation of the brain primordia by antagonizing b-catenin function downstream of polarity determinants.
An interesting question raised from these results is that if egr-4 is required for the early differentiation of the cephalic ganglia, how does inhibition of CNS differentiation blocks head regeneration? What we hypothesize is that the brain primordia could send some signal(s) to promote the proliferation, migration and/or differentiation of the neoblasts to allow blastema growth and head regeneration. In the absence of proper brain primordia after egr-4 RNAi, the lack of such putative inducing signal would explain the inhibition of head regeneration. Further experiments are needed to explore this putative relationship between brain differentiation and head regeneration in planarians, which would support the view of an evolutionarily conserved role of the nervous system in animal regeneration.