Characterization and expression of <Emphasis Type="Italic">DjPreb</Emphasis> gene in the planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

In this study, we report the expression and identification of a PREB-related gene from the planarian Dugesia japonica, DjPreb. The planarian DjPreb cDNA is comprised of 1101 bp and contains a 972 bp open reading frame corresponding to a deduced protein of 323 amino acids with a 69 bp 5’-UTR and a 60 bp 3’-UTR. Phylogenetic analysis shows that DjPreb is PREB/PREB-like members. We examined its spatial and temporal expression and distribution in both intact and regenerating planarians by Relative quantitative real-time PCR and Whole-mount in situ hybridization. The analysis indicates that DjPreb shows a gradient express with peak levels present in the anterior and posterior regions and progressively lower levels in central regions in intact and regenerating planarians. During regeneration the expression of DjPreb is upregulated. Strong expression of DjPreb is observed in the anterior and posterior blastemas. These results suggest that DjPreb may participate in head and tail formation.     

Retinoic acid as a regulator of planarian morphogenesis

The effect of retinoic acid on regeneration of two species of asexual planarian races, Girardia tigrina and Schmidtea mediterranea, was studied. It was established that retinoic acids at physiological concentrations (10⁻⁷–10⁻¹⁰ M) inhibit the regeneration of the head part of planarians but have no effect on tail blastema growth. It is shown that regeneration of the head part is inhibited as a result of arrest of the cell cycle of neoblasts, proliferating stem cells, during the transition from the G ₁/G ₀ to the S phase. Thus, the morphogenetic role of retinoic acids in planarians, primitive bilaterally symmetrical animals, has been demonstrated.     

<Emphasis Type="Italic">Wnt</Emphasis> gene loss in flatworms

Wnt genes encode secreted glycoproteins that act in cell–cell signalling to regulate a wide array of developmental processes, ranging from cellular differentiation to axial patterning. Discovery that canonical Wnt/β-catenin signalling is responsible for regulating head/tail specification in planarian regeneration has recently highlighted their importance in flatworm (phylum Platyhelminthes) development, but examination of their roles in the complex development of the diverse parasitic groups has yet to be conducted. Here, we characterise Wnt genes in the model tapeworm Hymenolepis microstoma and mine genomic resources of free-living and parasitic species for the presence of Wnts and downstream signalling components. We identify orthologs through a combination of BLAST and phylogenetic analyses, showing that flatworms have a highly reduced and dispersed complement that includes orthologs of only five subfamilies (Wnt1, Wnt2, Wnt4, Wnt5 and Wnt11) and fewer paralogs in parasitic flatworms (5–6) than in planarians (9). All major signalling components are identified, including antagonists and receptors, and key binding domains are intact, indicating that the canonical (Wnt/β-catenin) and non-canonical (planar cell polarity and Wnt/Ca²⁺) pathways are functional. RNA-Seq data show expression of all Hymenolepis Wnts and most downstream components in adults and larvae with the notable exceptions of wnt1, expressed only in adults, and wnt2 expressed only in larvae. The distribution of Wnt subfamilies in animals corroborates the idea that the last common ancestor of the Cnidaria and Bilateria possessed all contemporary Wnts and highlights the extent of gene loss in flatworms.     

Simple blood‐feeding method for live imaging of gut tube remodeling in regenerating planarians

Live cell imaging is a powerful technique to study cellular dynamics in vivo during animal development and regeneration. However, few live imaging methods have been reported for studying planarian regeneration. Here, we developed a simple method for steady visualization of gut tube remodeling during regeneration of a living freshwater planarian, Dugesia japonica. When planarians were fed blood several times, gut branches were well‐visualized in living intact animals under normal bright‐field illumination. Interestingly, tail fragments derived from these colored planarians enabled successive observation of the processes of the formation of a single anterior gut branch in the prepharyngeal region from the preexisting two posterior gut branches in the same living animals during head regeneration. Furthermore, we combined this method and RNA interference (RNAi) and thereby showed that a D. japonica raf‐related gene (DjrafA) and mek‐related gene (DjmekA) we identified both play a major role in the activation of extracellular signal‐regulated kinase (ERK) signaling during planarian regeneration, as indicated by their RNAi‐induced defects on gut tube remodeling in a time‐saving initial screening using blood‐feeding without immunohistochemical detection of the gut. Thus, this blood‐feeding method is useful for live imaging of gut tube remodeling, and provides an advance for the field of regeneration study in planarians.     

Two populations of pluripotent stem cells in planarians <Emphasis Type="Italic">Girardia tigrina</Emphasis>

The positional differences in the regenerative capability of individual body parts of the planarian Girardia (Dugesia) tigrina were analyzed. The paper shows the significance of the size and positional differences of individual fragments of planarians for their regenerative capabilities, as well as the fundamental difference in the mechanisms of the head and tail blastema formation. A scheme of regeneration that includes two populations of pluripotent stem cells called neoblasts is suggested. The two populations of neoblasts differ in their role and distribution along the planarian body. Specifically, the population of neoblasts involved in the formation of any blastema migrates to the nearest blastema, and the population participating only in the creation of the head blastema migrates along the planarian body, following the gradient of biomass of the damaged axons arising after the amputation of the head end. The maximal size of the head blastema was found in the fragment obtained after cutting off the head fragment at the eye level, and the maximal portion of all pluripotent stem cells migrating into two blastemas was found in the fragment obtained by cutting the planarian above the mouth, followed by cutting off the head fragment at the eye level.     

Early planarian brain regeneration is independent of blastema polarity mediated by the Wnt/β-catenin pathway

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.     

Wnt signaling is required for antero-posterior patterning of the planarian brain

Wnt signaling functions in axis formation and morphogenesis in various animals and organs. Here we report that Wnt signaling is required for proper brain patterning during planarian brain regeneration. We showed here that one of the Wnt homologues in the planarian Dugesia japonica, DjwntA, was expressed in the posterior region of the brain. When DjwntA-knockdown planarians were produced by RNAi, they could regenerate their heads at the anterior ends of the fragments, but formed ectopic eyes with irregular posterior lateral branches and brain expansion. This suggests that the Wnt signal may be involved in antero-posterior (A-P) patterning of the planarian brain, as in vertebrates. We also investigated the relationship between the DjwntA and nou-darake/FGFR signal systems, as knockdown planarians of these genes showed similar phenotypes. Double-knockdown planarians of these genes did not show any synergistic effects, suggesting that the two signal systems function independently in the process of brain regeneration, which accords with the fact that nou-darake was expressed earlier than DjwntA during brain regeneration. These observations suggest that the nou-darake/FGFR signal may be involved in brain rudiment formation during the early stage of head regeneration, and subsequently the DjwntA signal may function in A-P patterning of the brain rudiment.     

Bromodeoxyuridine specifically labels the regenerative stem cells of planarians

The singular regenerative abilities of planarians require a population of stem cells known as neoblasts. In response to wounding, or during the course of cell turnover, neoblasts are signaled to divide and/or differentiate, thereby replacing lost cell types. The study of these pluripotent stem cells and their role in planarian regeneration has been severely hampered by the reported inability of planarians to incorporate exogenous DNA precursors; thus, very little is known about the mechanisms that control proliferation and differentiation of this stem cell population within the planarian. Here we show that planarians are, in fact, capable of incorporating the thymidine analogue bromodeoxyuridine (BrdU), allowing neoblasts to be labeled specifically during the S phase of the cell cycle. We have used BrdU labeling to study the distribution of neoblasts in the intact animal, as well as to directly demonstrate the migration and differentiation of neoblasts. We have examined the proposal that a subset of neoblasts is arrested in the G2 phase of the cell cycle by double-labeling with BrdU and a mitosis-specific marker; we find that the median length of G2 (approximately 6 h) is sufficient to account for the initial mitotic burst observed after feeding or amputation. Continuous BrdU-labeling experiments also suggest that there is not a large, slow-cycling population of neoblasts in the intact animal. The ability to label specifically the regenerative stem cells, combined with the recently described use of double-stranded RNA to inhibit gene expression in the planarian, should serve to reignite interest in the flatworm as an experimental model for studying the problems of metazoan regeneration and the control of stem cell proliferation.     

Cell-, tissue-, and position-specific monoclonal antibodies against the planarian Dugesia (Girardia) tigrina

 To obtain specific immunological probes for studying molecular mechanisms involved in cell renewal, cell differentiation, and pattern formation in intact and regenerating planarians, we have produced a hybridoma library specific for the asexual race of the freshwater planarian Dugesia (Girardia) tigrina. Among the 276 monoclonal antibodies showing tissue-, cell-, cell subtype-, subcellular- and position-specific staining, we have found monoclonal antibodies against all tissues and cell types with the exception of neoblasts, the undifferentiated totipotent stem-cells in planarians. We have also detected position-specific antigens that label anterior, central, and posterior regions. Patterns of expression uncovered an unexpected heterogeneity among previously thought single cell types, as well as interesting cross-reactivities that deserve further study. Characterization of some of these monoclonal antibodies suggests they may be extremely useful as molecular markers for studying cell renewal and cell differentiation in the intact and regenerating organism, tracing the origin, lineage, and differentiation of blastema cells, and characterizing the stages and mechanisms of early pattern formation. Moreover, two position-specific monoclonals, the first ones isolated in planarians, will be instrumental in describing in molecular terms how the new pattern unfolds during regeneration and in devising the pattern formation model that best fits classical data on regeneration in planarians. Accepted: 16 September 1996     

Djrho2 is involved in regeneration of visual nerves in Dugesia japonica

The freshwater planarian is a powerful animal model for studying regeneration and stem cell activity in vivo.During regeneration,stem ceils (neoblasts in planarian) migrated to the wounding edge to re-build missing parts of the body.However, proteins involved in regulating cell migration during planarian regeneration have not been studied extensively.Here we report two small GTPase genes (Djrho2 and Djrho3) of Dugesia japonica (strain Pek-1).In situ hybridization results indicated that Djrho2 was expressed throughout the body with the exception of the pharynx region while Djrho3 was specifically expressed along the gastro-vaseular system.Djrho2 was largely expressed in neoblasts since its expression was sensitive to X-ray irradiation.In Djrho2-RNAi planarians, smaller anterior blaste-mas were observed in tail fragments during regeneration.Consistently, defective regeneration of visual nerve was detected by immu-nostainning with VC-1 antibody.These results suggested that Djrho2 is required for proper anterior regeneration in planairan.In contrast,no abnormality was observed after RNAi of Djrho3.We compared protein compositions of control and Djrho2-RNAi planarians using an optimized proteomic approach.Twenty-two up-regulated and 26 de-regulated protein spots were observed in the two-dimensional elec-trophoresis gels, and 17 proteins were successfully identified by Mass Spectrometry (MS) analysis.Among them, 6 actin-binding or cy-toskeleton-related proteins were found de-expressed in Djrho2-RNAi animals, suggesting that abnormal cytoskeleton assembling and cell migration were likely reasons of defected regeneration.     

Expression of secreted Wnt pathway components reveals unexpected complexity of the planarian amputation response

Regeneration is widespread throughout the animal kingdom, but our molecular understanding of this process in adult animals remains poorly understood. Wnt/β-catenin signaling plays crucial roles throughout animal life from early development to adulthood. In intact and regenerating planarians, the regulation of Wnt/β-catenin signaling functions to maintain and specify anterior/posterior (A/P) identity. Here, we explore the expression kinetics and RNAi phenotypes for secreted members of the Wnt signaling pathway in the planarian Schmidtea mediterranea. Smed-wnt and sFRP expression during regeneration is surprisingly dynamic and reveals fundamental aspects of planarian biology that have been previously unappreciated. We show that after amputation, a wounding response precedes rapid re-organization of the A/P axis. Furthermore, cells throughout the body plan can mount this response and reassess their new A/P location in the complete absence of stem cells. While initial stages of the amputation response are stem cell independent, tissue remodeling and the integration of a new A/P address with anatomy are stem cell dependent. We also show that WNT5 functions in a reciprocal manner with SLIT to pattern the planarian mediolateral axis, while WNT11-2 patterns the posterior midline. Moreover, we perform an extensive phylogenetic analysis on the Smed-wnt genes using a method that combines and integrates both sequence and structural alignments, enabling us to place all nine genes into Wnt subfamilies for the first time.     

Role of c-Jun N-terminal kinase activation in blastema formation during planarian regeneration

The robust regenerative abilities of planarians absolutely depend on a unique population of pluripotent stem cells called neoblasts, which are the only mitotic somatic cells in adult planarians and are responsible for blastema formation after amputation. Little is known about the molecular mechanisms that drive blastema formation during planarian regeneration. Here we found that treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 blocked the entry of neoblasts into the M-phase of the cell cycle, while allowing neoblasts to successfully enter S-phase in the planarian Dugesia japonica. The rapid and efficient blockage of neoblast mitosis by treatment with the JNK inhibitor provided a method to assess whether temporally regulated cell cycle activation drives blastema formation during planarian regeneration. In the early phase of blastema formation, activated JNK was detected prominently in a mitotic region (the "postblastema") proximal to the blastema region. Furthermore, we demonstrated that undifferentiated mitotic neoblasts in the postblastema showed highly activated JNK at the single cell level. JNK inhibition by treatment with SP600125 during this period caused a severe defect of blastema formation, which accorded with a drastic decrease of mitotic neoblasts in regenerating animals. By contrast, these animals still retained many undifferentiated neoblasts near the amputation stump. These findings suggest that JNK signaling plays a crucial role in feeding into the blastema neoblasts for differentiation by regulating the G2/M transition in the cell cycle during planarian regeneration.     

The planarian <Emphasis Type="Italic">nanos</Emphasis>-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration

Planarians are highly regenerative organisms with the ability to remake all their cell types, including the germ cells. The germ cells have been suggested to arise from totipotent neoblasts through epigenetic mechanisms. Nanos is a zinc-finger protein with a widely conserved role in the maintenance of germ cell identity. In this work, we describe the expression of a planarian nanos-like gene Smednos in two kinds of precursor cells namely, primordial germ cells and eye precursor cells, during both development and regeneration of the planarian Schmidtea mediterranea. In sexual planarians, Smednos is expressed in presumptive male primordial germ cells of embryos from stage 8 of embryogenesis and throughout development of the male gonads and in the female primordial germ cells of the ovary. Thus, upon hatching, juvenile planarians do possess primordial germ cells. In the asexual strain, Smednos is expressed in presumptive male and female primordial germ cells. During regeneration, Smednos expression is maintained in the primordial germ cells, and new clusters of Smednos-positive cells appear in the regenerated tissue. Remarkably, during the final stages of development (stage 8 of embryogenesis) and during regeneration of the planarian eye, Smednos is expressed in cells surrounding the differentiating eye cells, possibly corresponding to eye precursor cells. Our results suggest that similar genetic mechanisms might be used to control the differentiation of precursor cells during development and regeneration in planarians. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inhibition of planarian regeneration by melatonin

Melatonin, which is a substance produced by the pineal body in vertebrates, inhibited regeneration in the planarian Dugesia japonica japonica Ichikawa et Kawakatsu. When decapitated planarians were maintained in a 1 mmol dm^–3 solution of melatonin, formation of the head was retarded; formation of the eyes, however, was not disturbed. Similarly in animals from which the tail was cut, regeneration of the tail was retarded if the animals were kept in melatonin solution of 1 mmol dm^–3. The effect was reversible once the melatonin was removed. Retardation of regeneration did not occur with similar application of three melatonin derivatives, serotonin hydrochloride, N-acetylserotonin, and 6-hydroxymelatonin. Melatonin endogenous to the planarian could be demonstrated by means of radio-immunoassay and was more abundant in the head region than other regions of the body. Melatonin, thus, appears to play a role in regulating regeneration in planarians and conceivably provides positional information in that process.     

The Planarian HOM/HOX Homeobox Genes (Plox) Expressed along the Anteroposterior Axis

In the freshwater planarian Dugesia japonica, five cDNAs for HOM/HOX homeobox genes were cloned and sequenced. Together with sequence data on HOM/HOX homeobox genes of platyhelminthes deposited in databases, comparison of the deduced amino acid sequences revealed that planarians have at least seven HOM/HOX homeobox genes, Plox1 to Plox7 ( anarian HOM/H homeobox genes). Whole-mount in situ hybridization and RT-PCR revealed that Plox4 and Plox5 were increasingly expressed along a spatial gradient in the posterior region of intact animals. During regeneration, Plox5 was expressed only in the posterior region of regenerating body pieces, suggesting that the gene is involved in the anteroposterior patterning in planarians. Plox5 was not found to be expressed in a blastema-specific manner, which contradicts a previous report (J. R. Bayascas, E. Castillo, A. M. Muños-Mármol, and E. Saló. Development 124, 141–148, 1997). X-ray irradiation experiments showed that Plox5 was expressed at least in some cells other than neoblasts, but that the induction of Plox5 expression during regeneration might require neoblasts.     

Distribution of the stem cells (neoblasts) in the planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

It has been postulated that the high regeneration ability of planarians is supported by totipotent stem cells, called neoblasts. There have been a few reports showing the distribution of neoblasts in planarians. However, the findings were not completely consistent. To determine the distribution of neoblasts, we focused on proliferating cell nuclear antigen (PCNA), which is present in proliferative cells. We cloned and sequenced the cDNA of PCNA from the planarian Dugesia japonica and produced an antiserum recognizing the gene product. X-ray irradiation caused rapid loss of all PCNA-positive cells and loss of the neoblasts (which were morphologically defined by the presence of the chromatoid body), strongly suggesting that all PCNA-positive cells were true neoblasts. Using the antiserum, we were successful in identifying the neoblasts more clearly than any previous work. In addition to their dispersed distribution in the dorsal and ventral mesenchyme, the neoblasts were distributed as clusters along the midline and bilateral lines in the dorsal mesenchyme. We also examined the behavior of the neoblasts after decapitation. Decapitation did not seem to affect the migration of neoblasts far from the wound. We demonstrated here that DjPCNA is a powerful tool for identifying planarian neoblasts.Edited by D.A. Weisblat     

Constitutive gene expression and the specification of tissue identity in adult planarian biology

Planarians are flatworms that constitutively maintain adult tissues through cell turnover and can regenerate entire organisms from tiny body fragments. In addition to requiring new cells (from neoblasts), these feats require mechanisms that specify tissue identity in the adult. Crucial roles for Wnt and BMP signaling in the regeneration and maintenance of the body axes have been uncovered, among other regulatory factors. Available data indicate that genes involved in positional identity regulation at key embryonic stages in other animals display persisting regionalized expression in adult planarians. These expression patterns suggest that a constitutively active gene expression map exists for the maintenance of the planarian body. Planarians thus present a fertile ground for the identification of factors regulating the regionalization of the metazoan body plan and for the study of the attributes of these factors that can lead to the maintenance and regeneration of adult tissues.     

TINP1 homolog is required for planarian regeneration

The planarian flatworm is an ideal system for the study of regeneration in vivo. In this study, we focus on TINP1, which is one of the most conserved proteins in eukaryotic organisms. We found that TINP1 was expressed in parenchymal region through whole body as well as central nervous system (CNS) during the course of regeneration. RNA interference targeting DjTINP1 caused lysis defects in regenerating tissues and a decreased in cell division and expression levels of DjpiwiA and Djpcna. Furthermore, the expression levels of DjTINP1 were decreased when we inhibited the TGF-β signal by knockdown of smad4, which is the sole co-smad and has been proved to control the blastema patterning and central nervous system (CNS) regeneration in planarians. These findings suggest that DjTINP1 participate in the maintenance of neoblasts and be required for proper cell proliferation in planarians as a downstream gene of the TGF-β signal pathway.