Search

Analysis of anteroposterior (AP) axis specification in regenerating planarian flatworms has shown that Wnt/β-catenin signaling is required for posterior specification and that the FGF-like receptor molecule nou-darake (ndk) may be involved in restricting brain regeneration to anterior regions. The relationship between re-establishment of AP identity and correct morphogenesis of the brain is, however, still poorly understood. Here we report the characterization of two axin paralogs in the planarian Schmidtea mediterranea. Although Axins are well known negative regulators of Wnt/β-catenin signaling, no role in AP specification has previously been reported for axin genes in planarians. We show that silencing of Smed-axin genes by RNA interference (RNAi) results in two-tailed planarians, a phenotype previously reported after silencing of Smed-APC-1, another β-catenin inhibitor. More strikingly, we show for the first time that while early brain formation at anterior wounds remains unaffected, subsequent development of the brain is blocked in the two-tailed planarians generated after silencing of Smed-axin genes and Smed-APC-1. These findings suggest that the mechanisms underlying early brain formation can be uncoupled from the specification of AP identity by the Wnt/β-catenin pathway. Finally, the posterior expansion of the brain observed following Smed-ndk RNAi is enhanced by silencing Smed-APC-1, revealing an indirect relationship between the FGFR/Ndk and Wnt/β-catenin signaling systems in establishing the posterior limits of brain differentiation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Noggin genes are mainly known as inhibitors of the Bone Morphogenetic Protein (BMP) signalling pathway. Noggin genes play an important role in various developmental processes such as axis formation and neural differentiation. In vertebrates, inhibition of the BMP pathway is usually carried out together with other inhibitory molecules: chordin and follistatin. Recently, it has been shown in planarians that the BMP pathway has a conserved function in the maintenance and re-establishment of the dorsoventral axis during homeostasis and regeneration. In an attempt to further characterize the BMP pathway in this model we have undertaken an in silico search of noggin genes in the genome of Schmidtea mediterranea. In contrast to other systems in which between one and four noggin genes have been reported, ten genes containing a noggin domain are present in S. mediterranea. These genes have been classified into two groups: noggin genes (two genes) and noggin-like genes (eight genes). Noggin-like genes are characterized by the presence of an insertion of 50-60 amino acids in the middle of the noggin domain. Here, we report the characterization of this expanded family of noggin genes in planarians as well as their expression patterns in both intact and regenerating animals. In situ hybridizations show that planarian noggin genes are expressed in a variety of cell types located in different regions of the planarian body.

The bone morphogenetic protein (BMP) pathway has been shown to play an important role in the establishment of the dorsoventral axis during development in both vertebrate and invertebrate species. In an attempt to unravel the role of BMPs in pattern formation during planarian regeneration, we studied this signaling pathway in Schmidtea mediterranea. Here, we functionally characterize planarian homologues of two key elements of the pathway: Smed-BMP and Smed-Smad1. Whole-mount in situ hybridization showed that Smed-BMP is expressed at the planarian dorsal midline, suggesting a role in dorsoventral patterning, while Smed-Smad1 is widely expressed throughout the mesenchyme and in the central nervous system. RNA interference (RNAi) knockdowns of Smed-BMP or Smed-Smad1 led to the disappearance of dorsal markers along with the ectopic expression of ventral markers on the dorsal side of the treated animals. In almost all cases, a duplicated central nervous system differentiated dorsally after Smed-BMP or Smed-Smad1 RNAi. These defects were observed not only during regeneration but also in intact non-regenerating animals. Our results suggest that the BMP signaling pathway is conserved in planarians and that it plays a key role in the regeneration and maintenance of the dorsoventral axis.

A conserved network of nuclear proteins is crucial to eye formation in both vertebrates and invertebrates. The finding that freshwater planarians can regenerate eyes without the contribution of Pax6 suggests that alternative combinations of regulatory elements may control the morphogenesis of the prototypic planarian eye. To further dissect the molecular events controlling eye regeneration in planarians, we investigated the role of eyes absent (Djeya) and six-1 (Djsix-1) genes in Dugesia japonica. These genes are expressed in both regenerating eyes and in differentiated photoreceptors of intact adults. Through RNAi studies, we show that Djsix-1 and Djeya are both critical for the regeneration of normal eyes in planarians and genetically cooperate in vivo to establish correct eye cell differentiation. We further demonstrate that the genetic interaction is mediated by physical interaction between the evolutionarily conserved domains of these two proteins. These data indicate that planarians use cooperatively Djsix-1 and Djeya for the proper specification of photoreceptors, implicating that the mechanism involving their evolutionarily conserved domains can be very ancient. Finally, both Djsix-1 and Djeya double-stranded RNA are substantially more effective at producing no-eye phenotypes in the second round of regeneration. This is probably due to the significant plasticity of the planarian model system, based on the presence of a stable population of totipotent stem cells, which ensure the rapid cell turnover of all differentiated cell types.

Wnt proteins are a family of highly conserved secreted glycoproteins that regulate cell-to-cell interactions during embryogenesis. They act as signaling molecules and take part in many crucial decisions throughout the development of organisms ranging from Hydra to human. We have isolated and characterized the expression of a member of the Wnt family, Gtwnt-5 gene in the planarian Girardia tigrina. Planarians are free-living members (Class Turbellaria) of the Phylum Platyhelminthes. They are best known for their high regenerative capabilities. These organisms have an apparently simple central nervous system (CNS) from a morphological perspective, with cephalic ganglia in the dorsal anterior region and two ventral main nerve cords along the body. However, a large number of planarian neural genes have recently been identified and therefore it is possible to define different molecular and functional domains in the planarian brain. The present study shows expression of Gtwnt-5 in a subpopulation of the whole CNS of intact organisms, being activated during regeneration. Gtwnt-5 reveals a differential spatial pattern: the expression is preferentially found in the most external region of the CNS. In addition, a kind of iterative pattern has been observed at the ganglia level, suggesting that the planarian brain might not be a continuous structure but compartmented or regionalized. Gtwnt-5 signal is also detected at the sensors of the worm: at the auricle level and all around the cephalic periphery. All these data provide us with a new neural marker for the planarian brain, and can be used to follow regeneration of the CNS.

Six/sine oculis (Six/so) class genes, with representatives in vertebrates and invertebrates, include members with key developmental roles in the anterior part of the central nervous system (CNS) and eye. Having characterized the role of the first planarian gene of the Six/so family in eye development, we attempted to identify novel genes of this family related to the platyhelminth eye genetic network. We isolated a new Six/so gene in the planarian Girardia tigrina, Gtsix-3, which belongs to the Six3/6 class. Whole mount in situ hybridization revealed Gtsix3 expression in a stripe surrounding the cephalic ganglia in adults. This spatial pattern corresponds to the cephalic branches, the nerve cells that connect the CNS with the marginal sensory organs located continuously at the edge of the head. During head regeneration, Gtsix-3 shows delayed activation compared to other head genes, with an initial two spot pattern that later evolves to a continuous lateral expression in the new regenerated cephalic ganglia with a final reduction to the adult pattern. However, Gtsix-3 is not activated in tail regeneration and no eye expression is observed at any regenerative stage. These findings provide a new marker for the developing anterior nervous system and evidence the complexity of planarian brain.

Six/sine oculis (Six/so) class genes, with representatives in vertebrates and invertebrates, include members with key developmental roles in the anterior part of the central nervous system (CNS) and eye. Having characterized the role of the first planarian gene of the Six/so family in eye development, we attempted to identify novel genes of this family related to the platyhelminth eye genetic network. We isolated a new Six/so gene in the planarian Girardia tigrina, Gtsix-3, which belongs to the Six3/6 class. Whole mount in situ hybridization revealed Gtsix3 expression in a stripe surrounding the cephalic ganglia in adults. This spatial pattern corresponds to the cephalic branches, the nerve cells that connect the CNS with the marginal sensory organs located continuously at the edge of the head. During head regeneration, Gtsix-3 shows delayed activation compared to other head genes, with an initial two spot pattern that later evolves to a continuous lateral expression in the new regenerated cephalic ganglia with a final reduction to the adult pattern. However, Gtsix-3 is not activated in tail regeneration and no eye expression is observed at any regenerative stage. These findings provide a new marker for the developing anterior nervous system and evidence the complexity of planarian brain.

POU domain proteins are a large family of transcription factors that have been identified in a variety of metazoans, from freshwater sponges, planarians and nematodes to arthropods, echinoderms and vertebrates. Many of these proteins are implicated in the development and establishment of the nervous system. In this paper we describe the identification of the planarian genes GtPOU-1, GtPOU-3 and GtPOU-4, which belong to the subclasses III and IV of POU-domain genes. Their similarity with other members of the POU family is restricted to the POU and homeo domains, plus some peptide sequences scattered in the linker and flanking regions. As with other subclass III POU genes, GtPOU-1 is devoid of introns. Axial transcript distribution by RT-PCR and immunohistochemical assays, performed with a polyclonal antibody raised against the GtPOU-1 fusion protein, indicate that both the GtPOU-1 transcript and protein are continuously expressed along the antero-posterior axis. A monoclonal antibody raised against the same fusion protein indicates that a GtPOU-1-specific epitope, probably obtained by post-translational modification, is present in neural cells from both the central and peripheral nerve systems of the adult planarian's anterior third. Moreover, the GtPOU-1-specific epitope shows a dynamic expression pattern during regeneration, always marking the most anterior region of the planarian nervous system. Both the rapid and general GtPOU-1-specific epitope modification, during posterior regeneration, indicate that regeneration is a global process involving all planarian regions, including those that are far from the wound, by a combination of morphallactic and epimorphic mechanisms., (Copyright 1998 Elsevier Science Ireland Ltd. All Rights Reserved.)