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 βcatenin-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 (βcatenin 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

Analysis of Fox genes in Schmidtea mediterranea reveals new families and a conserved role of Smed‑foxO in controlling cell death

The forkhead box (Fox) genes encode transcription factors that control several key aspects of development. Present in the ancestor of all eukaryotes, Fox genes underwent several duplications followed by loss and diversification events that gave rise to the current 25 families. However, few Fox members have been identified from the Lophotrochozoa clade, and specifically from planarians, which are a unique model for understanding development, due to the striking plasticity of the adult. The aim of this study was to identify and perform evolutionary and functional studies of the Fox genes of lophotrochozoan species and, specifically, of the planarian Schmidtea mediterranea. Generating a pipeline for identifying Forkhead domains and using phylogenetics allowed us the phylogenetic reconstruction of Fox genes. We corrected the annotation for misannotated genes and uncovered a new family, the QD, present in all metazoans. According to the new phylogeny, the 27 Fox genes found in Schmidtea mediterranea were classified into 12 families. In Platyhelminthes, family losses were accompanied by extensive gene diversification and the appearance of specific families, the A(P) and N(P). Among the newly identified planarian Fox genes, we found a single copy of foxO, which shows an evolutionary conserved role in controlling cell death

WNT-FRIZZLED-LRP5/6 Signaling Mediates Posterior Fate and Prolifer-ation 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/ -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/ -catenin signal in posterior-facing wounds. We found that Wnt1-Fzd1-LRP5/6 signaling is evolutionarily conserved in executing a WNT/ -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

FoxK1 is required for ectodermal cell differentiation during planarian regeneration

Forkhead box (Fox) genes belong to the 'winged helix' transcription factor superfamily. The function of some Fox genes is well known, such as the role of foxO in controlling metabolism and longevity and foxA in controlling differentiation of endodermal tissues. However, the role of some Fox factors is not yet well characterized. Such is the case of FoxK genes, which are mainly studied in mammals and have been implicated in diverse processes including cell proliferation, tissue differentiation and carcinogenesis. Planarians are free-living flatworms, whose importance in biomedical research lies in their regeneration capacity. Planarians possess a wide population of pluripotent adult stem cells, called neoblasts, which allow them to regenerate any body part after injury. In a recent study, we identified three foxK paralogs in the genome of Schmidtea mediterranea. In this study, we demonstrate that foxK1 inhibition prevents regeneration of the ectodermal tissues, including the nervous system and the epidermis. These results correlate with foxK1 expression in neoblasts and in neural progenitors. Although the triggering of wound genes expression, polarity reestablishment and proliferation was not affected after foxK1 silencing, the apoptotic response was decreased. Altogether, these results suggest that foxK1 would be required for differentiation and maintenance of ectodermal tissues

Wnt/β-catenin signalling is required for pole-specific chromatin remodeling during planarian regeneration.

For successful regeneration, the identity of the missing tissue must be specified according to the pre-existing tissue. Planarians are ideal for the study of the mechanisms underlying this process; the same field of cells can regrow a head or a tail according to the missing body part. After amputation, the differential activation of the Wnt/β-catenin signal specifies anterior versus posterior identity. Initially, both wnt1 and notum (Wnt inhibitor) are expressed in all wounds, but 48 hours later they are restricted to posterior or anterior facing wounds, respectively, by an unknown mechanism. Here we show that 12 hours after amputation, the chromatin accessibility of cells in the wound region changes according to the polarity of the pre-existing tissue in a Wnt/β-catenin-dependent manner. Genomic analyses suggest that homeobox transcription factors and chromatin-remodeling proteins are direct Wnt/β-catenin targets, which trigger the expression of posterior effectors. Finally, we identify FoxG as a wnt1 up-stream regulator, probably via binding to its first intron enhancer region

Autonomous and non-autonomous regulation on planarian growth and regeneration: Smed-bls, canonical Wnt signalling and Fox family

[eng] Development requires an increment of cell growth and cell number, concomitant to a tightly control of cell differentiation. Thanks to cell communication, cells can be spatiotemporal patterned to acquire the required fate. Planarians are a unique model to study developmental processes due to their ability to regenerate and modulate their body size according to the nutrient availability. This body plasticity is based on the presence of pluripotent adult stem cells (neoblasts) and the continuous activation of the intercellular communication mechanisms. This active regulation of stem cells fate make them perfect models to study processes as growth, patterning, differentiation cell proliferation or cell death. In this thesis we have studied different molecular mechanisms that control planarian growth and pattern. We have described a novel gene family, blitzschnell (bls), formed by de novo and taxonomically restricted genes, which control cell number trough the regulation of cell proliferation and cell death. Nutrient intake controls its expression suggesting that bls family have evolved in planarians as a mechanism by which to restrict cell number in nutrient-fluctuating environments. During growth and regeneration, planarians are not only able regulate their body and organ size accordingly but they also maintain a proper pattern. This regulation is mediated by different signalling centres that specify different regions along the 3 body axes (AP, DV and ML). Particularly, after an amputation, the anterior and the posterior planarian tips behave as organizers (signalling centre), specifying the fate of each planarian pole. The anterior organizer is defined by notum (a Wnt inhibitor) and the posterior by wnt1 expression. The inhibition of any of those elements leads to a shift in polarity. During the first hours of regeneration both notum and wnt1 are expressed in both poles, and it’s around 36 hours that their expression becomes restricted to their respective tip. To decipher the molecular interactions that restrict the expression of wnt1 to the posterior tip and confer the organizing activity we used genome wide approaches. ATAC-seq and RNA-seq analysis of regenerating wild-type and wnt1 (RNAi) planarians allowed the identification of specific Cis-Regulatory Elements (CREs) of posterior regeneration. We found that already at 12 hours of regeneration the accessible CREs in posterior and anterior blastemas have essentially changed, indicating that specific posterior chromatin changes induced by amputation occur much earlier than the formation of the organizers. Furthermore, we have identified specific transcription factors (TF) of the Otx and Fox families, which are enriched in posterior CREs. Particularly, pitx and foxG regulates wnt1+ cells and are essential for the specification of the posterior cells. TFs regulate patterning events and developmental specification, particularly the Fox Family exerts crucial roles defining cell types of all germ cell layers or regulating cell cycle. Before this Thesis, poorly was known about the Fox family in Schmidtea mediterranea (Smed) neither in the Lophocotrozoan clade. In this study we have identified 27 Fox genes in Smed, classified in 13 families: A, At, C, D, E, G, L1t, QD, J1, N2/3, Nt, O and P. We have performed an extensive phylogenetic study of the family to understand the evolution of the Fox family in this clade. Furthermore, we have studied the sequence, expression and function of several planarian Fox genes. Overall, we studied different molecular mechanisms that regulate planarian growth and regeneration, and that provide novel data concerning development and evolution

Autonomous and non-autonomous regulation on planarian growth and regeneration: Smed-bls, canonical Wnt signalling and Fox family

Development requires an increment of cell growth and cell number, concomitant to a tightly control of cell differentiation. Thanks to cell communication, cells can be spatiotemporal patterned to acquire the required fate. Planarians are a unique model to study developmental processes due to their ability to regenerate and modulate their body size according to the nutrient availability. This body plasticity is based on the presence of pluripotent adult stem cells (neoblasts) and the continuous activation of the intercellular communication mechanisms. This active regulation of stem cells fate make them perfect models to study processes as growth, patterning, differentiation cell proliferation or cell death. In this thesis we have studied different molecular mechanisms that control planarian growth and pattern. We have described a novel gene family, blitzschnell (bls), formed by de novo and taxonomically restricted genes, which control cell number trough the regulation of cell proliferation and cell death. Nutrient intake controls its expression suggesting that bls family have evolved in planarians as a mechanism by which to restrict cell number in nutrient-fluctuating environments. During growth and regeneration, planarians are not only able regulate their body and organ size accordingly but they also maintain a proper pattern. This regulation is mediated by different signalling centres that specify different regions along the 3 body axes (AP, DV and ML). Particularly, after an amputation, the anterior and the posterior planarian tips behave as organizers (signalling centre), specifying the fate of each planarian pole. The anterior organizer is defined by notum (a Wnt inhibitor) and the posterior by wnt1 expression. The inhibition of any of those elements leads to a shift in polarity. During the first hours of regeneration both notum and wnt1 are expressed in both poles, and it’s around 36 hours that their expression becomes restricted to their respective tip. To decipher the molecular interactions that restrict the expression of wnt1 to the posterior tip and confer the organizing activity we used genome wide approaches. ATAC-seq and RNA-seq analysis of regenerating wild-type and wnt1 (RNAi) planarians allowed the identification of specific Cis-Regulatory Elements (CREs) of posterior regeneration. We found that already at 12 hours of regeneration the accessible CREs in posterior and anterior blastemas have essentially changed, indicating that specific posterior chromatin changes induced by amputation occur much earlier than the formation of the organizers. Furthermore, we have identified specific transcription factors (TF) of the Otx and Fox families, which are enriched in posterior CREs. Particularly, pitx and foxG regulates wnt1+ cells and are essential for the specification of the posterior cells. TFs regulate patterning events and developmental specification, particularly the Fox Family exerts crucial roles defining cell types of all germ cell layers or regulating cell cycle. Before this Thesis, poorly was known about the Fox family in Schmidtea mediterranea (Smed) neither in the Lophocotrozoan clade. In this study we have identified 27 Fox genes in Smed, classified in 13 families: A, At, C, D, E, G, L1t, QD, J1, N2/3, Nt, O and P. We have performed an extensive phylogenetic study of the family to understand the evolution of the Fox family in this clade. Furthermore, we have studied the sequence, expression and function of several planarian Fox genes. Overall, we studied different molecular mechanisms that regulate planarian growth and regeneration, and that provide novel data concerning development and evolution

Autonomous and non-autonomous regulation on planarian growth and regeneration: Smed-bls, canonical Wnt signalling and Fox family

Development requires an increment of cell growth and cell number, concomitant to a tightly control of cell differentiation. Thanks to cell communication, cells can be spatiotemporal patterned to acquire the required fate. Planarians are a unique model to study developmental processes due to their ability to regenerate and modulate their body size according to the nutrient availability. This body plasticity is based on the presence of pluripotent adult stem cells (neoblasts) and the continuous activation of the intercellular communication mechanisms. This active regulation of stem cells fate make them perfect models to study processes as growth, patterning, differentiation cell proliferation or cell death. In this thesis we have studied different molecular mechanisms that control planarian growth and pattern. We have described a novel gene family, blitzschnell (bls), formed by de novo and taxonomically restricted genes, which control cell number trough the regulation of cell proliferation and cell death. Nutrient intake controls its expression suggesting that bls family have evolved in planarians as a mechanism by which to restrict cell number in nutrient-fluctuating environments. During growth and regeneration, planarians are not only able regulate their body and organ size accordingly but they also maintain a proper pattern. This regulation is mediated by different signalling centres that specify different regions along the 3 body axes (AP, DV and ML). Particularly, after an amputation, the anterior and the posterior planarian tips behave as organizers (signalling centre), specifying the fate of each planarian pole. The anterior organizer is defined by notum (a Wnt inhibitor) and the posterior by wnt1 expression. The inhibition of any of those elements leads to a shift in polarity. During the first hours of regeneration both notum and wnt1 are expressed in both poles, and it’s around 36 hours that their expression becomes restricted to their respective tip. To decipher the molecular interactions that restrict the expression of wnt1 to the posterior tip and confer the organizing activity we used genome wide approaches. ATAC-seq and RNA-seq analysis of regenerating wild-type and wnt1 (RNAi) planarians allowed the identification of specific Cis-Regulatory Elements (CREs) of posterior regeneration. We found that already at 12 hours of regeneration the accessible CREs in posterior and anterior blastemas have essentially changed, indicating that specific posterior chromatin changes induced by amputation occur much earlier than the formation of the organizers. Furthermore, we have identified specific transcription factors (TF) of the Otx and Fox families, which are enriched in posterior CREs. Particularly, pitx and foxG regulates wnt1+ cells and are essential for the specification of the posterior cells. TFs regulate patterning events and developmental specification, particularly the Fox Family exerts crucial roles defining cell types of all germ cell layers or regulating cell cycle. Before this Thesis, poorly was known about the Fox family in Schmidtea mediterranea (Smed) neither in the Lophocotrozoan clade. In this study we have identified 27 Fox genes in Smed, classified in 13 families: A, At, C, D, E, G, L1t, QD, J1, N2/3, Nt, O and P. We have performed an extensive phylogenetic study of the family to understand the evolution of the Fox family in this clade. Furthermore, we have studied the sequence, expression and function of several planarian Fox genes. Overall, we studied different molecular mechanisms that regulate planarian growth and regeneration, and that provide novel data concerning development and evolution

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 βcatenin-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 (βcatenin 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