WNT-FRIZZLED-LRP5/6 Signaling Mediates Posterior Fate and Proliferation during Planarian Regeneration.

An organizer is defined as a group of cells that secrete extracellular proteins that specify the fate of surrounding cells according to their concentration. Their function during embryogenesis is key in patterning new growing tissues. Although organizers should also participate in adult development when new structures are regenerated, their presence in adults has only been identified in a few species with striking regenerative abilities, such as planarians. Planarians provide a unique model to understand the function of adult organizers, since the presence of adult pluripotent stem cells provides them with the ability to regenerate any body part. Previous studies have shown that the differential activation of the WNT/ß-catenin signal in each wound is fundamental to establish an anterior or a posterior organizer in the corresponding wound. Here, we identify the receptors that mediate the WNT/ß-catenin signal in posterior-facing wounds. We found that Wnt1-Fzd1-LRP5/6 signaling is evolutionarily conserved in executing a WNT/ß-catenin signal to specify cell fate and to trigger a proliferative response. Our data allow a better understanding of the mechanism through which organizers signal to a "competent" field of cells and integrate the patterning and growth required during de novo formation of organs and tissues.

Planarian organizers.

An organizer is defined as a group of cells that secrete specific factors and can change the fate of adjacent cells and instruct a specific pattern. Spemann and Mangold were the first to use the term, when in 1938 they discovered that the dorsal blastopore lip of a salamander embryo induced a secondary axis after transplantation. Since then, several such regions have been identified in the embryos of many animal species. However, little is known about the presence of organizers at the adult stage, although some organizing activity must be required during regenerative processes to pattern the new tissue. In this study we review the current knowledge on planarians, flatworms that can regenerate any lost body parts, including their heads, within a few days. We will summarize the current data that made it possible to identify planarian anterior and posterior tips as regenerative organizers. We will present the current knowledge about the molecular networks that define each organizer, and we will discuss the presence of organizers in planarians during normal homeostasis. We will propose some unanswered questions concerning both planarian regeneration and regenerative medicine, and examine future research prospects in this field.

Immunohistochemistry on Paraffin-Embedded Planarian Tissue Sections.

Planarians are flatworms with almost unlimited regenerative abilities, which make them an excellent model for stem cell-based regeneration. To study the process of regeneration at the cellular level, immunohistochemical staining methods are an important tool, and the availability of such protocols is one of the prerequisites for mechanistic experiments in any animal model. Here, we detail protocols for paraffin embedding and immunostaining of paraffin sections of the model species Schmidtea mediterranea. This protocol yields robust results with a variety of commercially available antibodies. Further, the procedures provide a useful starting point for customizing staining procedures for new antibodies and/or different planarian species.

A C-terminally truncated form of ß-catenin acts as a novel regulator of Wnt/ß-catenin signaling in planarians.

ß-Catenin, the core element of the Wnt/ß-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/ß-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a 'whole animal' developmental framework to approach this question. Here we identify a C-terminally truncated ß-catenin (ß-catenin4), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of ß-catenin4 is to modulate the activity of ß-catenin1, the planarian ß-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of ß-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear ß-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of ß-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/ß-catenin signaling in specific cellular contexts.

Localization of planarian ß-CATENIN-1 reveals multiple roles during anterior-posterior regeneration and organogenesis.

The ß-catenin-dependent Wnt pathway exerts multiple context-dependent roles in embryonic and adult tissues. In planarians, ß-catenin-1 is thought to specify posterior identities through the generation of an anteroposterior gradient. However, the existence of such a gradient has not been directly demonstrated. Here, we use a specific polyclonal antibody to demonstrate that nuclear ß-CATENIN-1 exists as an anteroposterior gradient from the pre-pharyngeal region to the tail of the planarian Schmidtea polychroa High levels in the posterior region steadily decrease towards the pre-pharyngeal region but then increase again in the head region. During regeneration, ß-CATENIN-1 is nuclearized in both anterior and posterior blastemas, but the canonical WNT1 ligand only influences posterior nuclearization. Additionally, ß-catenin-1 is required for proper anterior morphogenesis, consistent with the high levels of nuclear ß-CATENIN-1 observed in this region. We further demonstrate that ß-CATENIN-1 is abundant in developing and differentiated organs, and is particularly required for the specification of the germline. Altogether, our findings provide the first direct evidence of an anteroposterior nuclear ß-CATENIN-1 gradient in adult planarians and uncover novel, context-dependent roles for ß-catenin-1 during anterior regeneration and organogenesis.

Posterior Wnts Have Distinct Roles in Specification and Patterning of the Planarian Posterior Region.

The wnt signaling pathway is an intercellular communication mechanism essential in cell-fate specification, tissue patterning and regional-identity specification. A ßcatenin-dependent signal specifies the AP (Anteroposterior) axis of planarians, both during regeneration of new tissues and during normal homeostasis. Accordingly, four wnts (posterior wnts) are expressed in a nested manner in central and posterior regions of planarians. We have analyzed the specific role of each posterior wnt and the possible cooperation between them in specifying and patterning planarian central and posterior regions. We show that each posterior wnt exerts a distinct role during re-specification and maintenance of the central and posterior planarian regions, and that the integration of the different wnt signals (ßcatenin dependent and independent) underlies the patterning of the AP axis from the central region to the tip of the tail. Based on these findings and data from the literature, we propose a model for patterning the planarian AP axis.

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 (ß-catenin-dependent) and non-canonical (ß-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.