What are the exact mechanisms that control the re-establishment of axial polarity remains as an important question in many regeneration models. In freshwater planarians several studies in the last years have identified the Wnt/β-catenin signalling pathway as pivotal for the establishment and maintenance of the anteroposterior (AP) axis. Thus, several Wnts and Wnt receptors are expressed in the posterior regions whereas different inhibitors of Wnt signalling are expressed at the anterior end. Consequently, the inhibition of this pathway by silencing either β-catenin or some Wnt genes (such as Wnt1) results in the regeneration of heads instead of tails. Even in intact animals the silencing of these genes transforms the tail region into a head with a proper brain. Conversely, the activation of the Wnt/β-catenin pathway in anterior regions leads to the transformation of those heads into tails. Therefore, it seems quite clear that Wnt/β-catenin is required to specify posterior fates in these animals.
Now, a paper from the laboratory of Kerstin Bartscherer has aimed to identify genes regulated by this pathway (http://www.ncbi.nlm.nih.gov/pubmed/25558068) that could be involved in controlling tissue polarity. In order to identify target genes the authors carried out RNAseq experiments after silencing Smed-β-catenin1 in intact animals. Initially they identified 440 downregulated and 348 upregulated transcripts. Among the downregulated genes they found previously described posterior genes and, conversely, upregulated genes included some known anterior genes. Then, they selected 70 genes downregulated after β-catenin silencing and analysed their expression patterns by in situ hybridization. Of them, 35% displayed differential expression along the AP axis with high expression in the tail. Among the other genes, 21 of them were expressed within or around the digestive system. When they analysed the expression of most of these genes after β-catenin RNAi, they found that their expression was strongly inhibited, validating thus the RNAseq data. Also, they found that many of the upregulated genes after β-catenin RNAi were mainly expressed in anterior regions, as expected.
Next, they selected some of these genes that showed a graded expression along the AP axis and analysed in more detail their expression by FISH. These experiments clearly showed this graded expression, high in the tail and lower towards more anterior regions, strengthening the hypothesis that a β-catenin activity gradient may control gene expression. Recently, it has been described that collagen-positive subepidermal muscle cells express several position control genes (PCGs), which has lead to propose that these muscle cells could somehow provide positional information along the AP axis. Here, the authors show that some of the putative novel targets of β-catenin are also expressed in those posterior muscle cells. In addition, some of these genes were expressed in intestinal muscle cells, which suggests that the gut musculature could be also a source of PCGs. Then, and in order to determine whether planarian neoblasts (totipotent stem cells) differentially expressed the candidate genes sp5 and abdBa along the AP axis they isolated different FACS-sorted cell populations from anterior and posterior regions and quantified their expression in those cell fractions. Interestingly, they found that sp5 and abdBa transcripts were higher in the stem cells from posterior regions compared to anterior neoblasts, suggesting that planarian neoblasts may respond to graded β-catenin activity along the AP axis.
Notum is a secreted Wnt inhibitor that, in planarians, is expressed at the anterior tip of the animal and whose silencing leads to the regeneration of heads instead of tails. It has been proposed that notum regulates early polarity decisions through the inhibition of β-catenin activity at anterior-facing wounds. As the authors reasoned that notum inhibition might induce β-catenin-dependent genes they carried out RNAseq experiments after notum RNAi at 18 hours of regeneration, a stage in which polarity decisions are supposed to occur. By comparing the two sets of genes from the two different RNAseq experiments, they found that 38 of the downregulated genes after β-catenin RNAi were induced after notum silencing. Conversely, 16 of the upregulated genes after β-catenin RNAi were inhibited after notum RNAi. Thus, these 54 genes could represent the genes involved in early polarity decisions during planarian regeneration. Remarkably, 33 out of these 54 genes were also present in a previously generated set of Wnt/β-catenin target genes in zebrafish, which suggests that the common requirement of the Wnt/β-catenin pathway during regeneration in planarians and zebrafish may include a shared set of target genes.
One of the novel candidate target genes identified here was teashirt, a Tsh-related zinc finger protein, a gene that appears to act as modulator of Wnt signalling during Drosophila and Xenopus development. In planarians, teashirt (tsh) was expressed all throughout the central nervous system as well as in a graded pattern in the mesenchyme with highest expression in the tail. Tsh expression was induced upon amputation and, as predicted, it was inhibited after β-catenin RNAi and induced after β-catenin overactivation. Remarkably, a tsh homolog in zebrafish was detected in fin regenerates at 3 days after amputation, being this expression also dependent on the Wnt/β-catenin pathway further supporting an evolutionarily conserved role of Wnt/β-catenin in ensuring correct tissue identity and/or patterning during regeneration. Going back to planarians, the silencing of tsh resulted in two-headed planarians, a phenotype reminiscent of β-catenin and Wnt1 RNAi. These results indicated that tsh would be required to suppress anterior fate at a posterior-facing wound. During posterior regeneration Wnt1 is rapidly induced in the stump. Tsh co-localized with wnt1-positive cells both in intact and regenerating tails. Morevover, tsh was also expressed in neoblasts and that expression seemed to be higher in neoblasts from posterior regions.
In summary, the authors show here that tsh is expressed in wnt-positive cells, probably subepidermal muscle cells, as well as in a subpopulation of neoblasts in both cases in a β-catenin-dependent manner. Tsh could be then a downstream transducer of Wnt signalling important to regulate AP polarity.