Teresa Adell

Silencing of Smed-βcatenin1 generates radial-like hypercephalized planarians

Little is known about the molecular mechanisms responsible for axis establishment during non-embryonic processes such as regeneration and homeostasis. To address this issue, we set out to analyze the role of the canonical Wnt pathway in planarians, flatworms renowned for their extraordinary morphological plasticity. Canonical Wnt signalling is an evolutionarily conserved mechanism to confer polarity during embryonic development, specifying the anteroposterior (AP) axis in most bilaterians and the dorsoventral (DV) axis in early vertebrate embryos. β-Catenin is a key element in this pathway, although it is a bifunctional protein that is also involved in cell-cell adhesion. Here, we report the characterization of two β-catenin homologs from Schmidtea mediterranea (Smed-βcatenin1/2). Loss of function of Smed-βcatenin1, but not Smed-βcatenin2, in both regenerating and intact planarians, generates radial-like hypercephalized planarians in which the AP axis disappears but the DV axis remains unaffected, representing a unique example of a striking body symmetry transformation. The radial-like hypercephalized phenotype demonstrates the requirement for Smed-βcatenin1 in AP axis re-establishment and maintenance, and supports a conserved role for canonical Wnt signalling in AP axis specification, whereas the role of β-catenin in DV axis establishment would be a vertebrate innovation. When considered alongside the protein domains present in each S. mediterranea β-catenin and the results of functional assays in Xenopus embryos demonstrating nuclear accumulation and axis induction with Smed-βcatenin1, but not Smed-βcatenin2, these data suggest that S. mediterraneaβ -catenins could be functionally specialized and that only Smed-βcatenin1 is involved in Wnt signalling.

Smed-Evi/Wntless is required for β-catenin-dependent and -independent processes during planarian regeneration

Planarians can regenerate a whole animal from only a small piece of their body, and have become an important model for stem cell biology. To identify regenerative processes dependent on Wnt growth factors in the planarian Schmidtea mediterranea (Smed), we analyzed RNAi phenotypes of Evi, a transmembrane protein specifically required for the secretion of Wnt ligands. We show that, during regeneration, Smed-evi loss-of-function prevents posterior identity, leading to two-headed planarians that resemble Smed-β-catenin1 RNAi animals. In addition, we observe regeneration defects of the nervous system that are not found after Smed-β-catenin1 RNAi. By systematic knockdown of all putative Smed Wnts in regenerating planarians, we identify Smed-WntP-1 and Smed-Wnt11-2 as the putative posterior organizers, and demonstrate that Smed-Wnt5 is a regulator of neuronal organization and growth. Thus, our study provides evidence that planarian Wnts are major regulators of regeneration, and that they signal through β-catenin-dependent and -independent pathways.

Planarian regeneration: achievements and future directions after 20 years of research

Planarians can undergo dramatic changes in body size and regenerate their entire body plan from small pieces after cutting. This remarkable morphological plasticity has made them an excellent model in which to analyze phenomena such as morphogenesis, restoration of pattern and polarity, control of tissue proportions and tissue homeostasis. They have a unique population of pluripotent stem cells in the adult that can give rise to all differentiated cell types, including the germ cells. These cellular characteristics provide an excellent opportunity to study the mechanisms involved in the maintenance and differentiation of cell populations in intact and regenerating animals. Until recently, the planarian model system lacked opportunities for genetic analysis; however, this handicap was overcome in the last decade through the development of new molecular methods which have been successfully applied to planarians. These techniques have allowed analysis of the temporal and spatial expression of genes, as well as interference with gene function, generating the first phenotypes by loss or gain of function. Finally, the sequencing of the planarian genome has provided the essential tools for an in-depth analysis of the genomic regulation of this model system. In this review, we provide an overview of planarians as a model system for research into development and regeneration and describe new lines of investigation in this area.

Gradients in Planarian Regeneration and Homeostasis

Planarian regeneration was one of the first models in which the gradient concept was developed. Morphological studies based on the analysis of the regeneration rates of planarian fragments from different body regions, the generation of heteromorphoses, and experiments of tissue transplantation led T.H. Morgan (1901) and C.M Child (1911) to postulate different kinds of gradients responsible for the regenerative process in these highly plastic animals. However, after a century of research, the role of morphogens in planarian regeneration has yet to be demonstrated. This may change soon, as the sequencing of the planarian genome and the possibility of performing gene functional analysis by RNA interference (RNAi) have led to the isolation of elements of the bone morphogenetic protein (BMP), Wnt, and fibroblast growth factor (FGF) pathways that control patterning and axial polarity during planarian regeneration and homeostasis. Here, we discuss whether the actions of these molecules could be based on morphogenetic gradients.

Dishevelled is essential for neural connectivity and planar cell polarity in planarians

The Wingless/Integrated (Wnt) signaling pathway controls multiple events during development and homeostasis. It comprises multiple branches, mainly classified according to their dependence on β-catenin activation. The Wnt/β-catenin branch is essential for the establishment of the embryonic anteroposterior (AP) body axis throughout the phylogenetic tree. It is also required for AP axis establishment during planarian regeneration. Wnt/β-catenin–independent signaling encompasses several different pathways, of which the most extensively studied is the planar cell polarity (PCP) pathway, which is responsible for planar polarization of cell structures within an epithelial sheet. Dishevelled (Dvl) is the hub of Wnt signaling because it regulates and channels the Wnt signal into every branch. Here, we analyze the role of Schmidtea mediterranea Dvl homologs (Smed-dvl-1 and Smed-dvl-2) using gene silencing. We demonstrate that in addition to a role in AP axis specification, planarian Dvls are involved in at least two different β-catenin–independent processes. First, they are essential for neural connectivity through Smed-wnt5 signaling. Second, Smed-dvl-2, together with the S. mediterranea homologs of Van-Gogh (Vang) and Diversin (Div), is required for apical positioning of the basal bodies of epithelial cells. These data represent evidence not only of the function of the PCP network in lophotrocozoans but of the involvement of the PCP core elements Vang and Div in apical positioning of the cilia.

JNK Controls the Onset of Mitosis in Planarian Stem Cells and Triggers Apoptotic Cell Death Required for Regeneration and Remodeling

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun–NH2–kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal.

β-Catenin-Dependent Control of Positional Information along the AP Body Axis in Planarians Involves a Teashirt Family Member

Wnt/β-catenin signaling regulates tissue homeostasis and regeneration in metazoans. In planarians-flatworms with high regenerative potential-Wnt ligands are thought to control tissue polarity by shaping a β-catenin activity gradient along the anterior-posterior axis, yet the downstream mechanisms are poorly understood. We performed an RNA sequencing (RNA-seq)-based screen and identified hundreds of β-catenin-dependent transcripts, of which several were expressed in muscle tissue and stem cells in a graded fashion. In particular, a teashirt (tsh) ortholog was induced in a β-catenin-dependent manner during regeneration in planarians and zebrafish, and RNAi resulted in two-headed planarians. Strikingly, intact planarians depleted of tsh induced anterior markers and slowly transformed their tail into a head, reminiscent of β-catenin RNAi phenotypes. Given that β-catenin RNAi enhanced the formation of muscle cells expressing anterior determinants in tail regions, our study suggests that this pathway controls tissue polarity through regulating the identity of differentiating cells during homeostasis and regeneration.

Wnt signaling in planarians: new answers to old questions.

Wnts are secreted glycoproteins involved in a broad range of essential cell functions, including proliferation, migration and cell-fate determination. Recent years have seen substantial research effort invested in elucidating the role of the Wnt signaling pathway in planarians, flatworms with incredible regenerative capacities. In this review, we summarize current knowledge on the role of canonical (β-catenin-dependent) and non-canonical (β-catenin-independent) Wnt signaling in planarians, not only during regeneration, but also during normal homeostasis. We also describe some of the preliminary data that has been obtained regarding the role of these pathways during embryogenesis. Models are proposed to integrate the different results which have been obtained to date and highlight those questions that still remain to be answered.

Complete Functional Segregation of Planarian β-Catenin-1 and -2 in Mediating Wnt Signaling and Cell Adhesion

β-Catenin is a bifunctional protein participating in both cell adhesion and canonical Wnt signaling. In cell adhesion, it bridges the transmembrane cadherin and the actin-binding protein α-catenin and is essential for adherens junction formation, whereas in canonical Wnt signaling, it shuttles between the cytosol and nucleus and functions as an essential transcriptional activator. Schmidtea mediterranea β-catenin-1 was identified as a determinant of antero-posterior polarity during body regeneration by mediating Wnt signaling. Here we show that S. mediterranea β-catenin-2 is specifically expressed in epithelial cells in the gut and pharynx, where it has a putative role in mediating cell adhesion. We show evidence that planarian β-catenin-1 and -2 have distinct biochemical properties. β-Catenin-1 can interact with the components of the canonical Wnt signaling pathway but not with α-catenin, whereas β-catenin-2 interacts with cell adhesion molecules, including E-cadherin and α-catenin, but not with Wnt signaling components. Consistent with their specific function, β-catenin-1 is a potent transcriptional activator, whereas β-catenin-2 has no transcriptional activity. Protein sequence alignment also indicates that the planarian β-catenin-1 and -2 retain distinct critical residues and motifs, which are in agreement with the differences in their biochemical properties. At last, phylogenetic analysis reveals a probable Platyhelminthes- specific structural and functional segregation from which the monofunctional β-catenins evolved. Our results thus identify the first two monofunctional β-catenins in metazoans.

Planarian GSK3s are involved in neural regeneration

Glycogen synthase kinase-3 (GSK3) is a key element in several signaling cascades that is known to be involved in both patterning and neuronal organization. It is, therefore, a good candidate to play a role in neural regeneration in planarians. We report the characterization of three GSK3 genes in Schmidtea mediterranea. Phylogenetic analysis shows that Smed-GSK3.1 is highly conserved compared to GSK3 sequences from other species, whereas Smed-GSK3.2 and Smed-GSK3.3 are more divergent. Treatment of regenerating planarians with 1-azakenpaullone, a synthetic GSK3 inhibitor, suggests that planarian GSK3s are essential for normal differentiation and morphogenesis of the nervous system. Cephalic ganglia appear smaller and disconnected in 1-azakenpaullone-treated animals, whereas visual axons are ectopically projected, and the pharynx does not regenerate properly. This phenotype is consistent with a role for Smed-GSK3s in neuronal polarization and axonal growth.