Planarian pharynx regeneration revealed by the expression of myosin heavy chain-A

The pharynx is a distinctive organ in the center of the body of planarians. Although the process of pharynx regeneration has been studied previously, the details and mechanism of the process remain controversial. We examined the process of regeneration of the pharynx in the planarian Dugesia japonica in detail by in situ hybridization and immunohistochemistry for myosin heavy chain-A (DjMHC-A), which is mainly expressed in the pharynx muscles and pharynx-anchoring muscles. We also monitored the behavior of the neoblasts in this process. In the regenerating posterior body fragment, the pharyngeal rudiment was formed by accumulation of cells that were probably undifferentiated cells derived from the neoblasts. The pharynx muscles appeared to differentiate in the rudiment in a manner that was coordinated with the differentiation of the pharynx-anchoring muscles in the region surrounding the rudiment. During this process, all cells containing mRNA for DjMHC-A also contained the DjMHC-A protein. These results argue against a previously proposed hypothesis that in the mesenchyme, 'pharynx-forming cells', which are committed to differentiate into the pharyngeal cells but have not yet differentiated, gather in the rudiment to form the pharynx (Agata and Watanabe, 1999). Rather, the present observations suggest that regeneration of the planarian pharynx proceeds by accumulation of cells that are probably undifferentiated cells derived from neoblasts in the rudiment, followed by their differentiation into the pharyngeal cells there.     

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.     

Disto-proximal regional determination and intercalary regeneration in planarians, revealed by retinoic acid induced disruption of regeneration.

The mechanisms that define the body pattern during development and regeneration are the object of major concern in developmental biology. To understand the process and sequence of antero-posterior pattern formation of planarian body regions during regeneration, regenerating organisms were treated with exogenous retinoic acid, which affects development and regeneration in other systems, and the sequence of regional determination has been monitored by a specific molecular marker for the central region, which includes the pharynx. The sequence of gross regional specification have never been analysed in planarians using molecular regional markers or by direct disruption of the regeneration process. Exogenous retinoic acid administration on regenerating planarians disrupts anterior, but not posterior regeneration. The period of maximum sensitivity to exogenous retinoic acid is one day after amputation, during which time the determination of the head has been reported to occur. The data obtained allow us to suggest that gross regional specification during planarian regeneration is disto-proximal, from the regenerative blastema to the old stump, and thus takes place by intercalation of the central region between the anterior and posterior ones.     

Planarian fibroblast growth factor receptor homologs expressed in stem cells and cephalic ganglions

The strong regenerative capacity of planarians is considered to reside in the totipotent somatic stem cell called the 'neoblast'. However, the signal systems regulating the differentiation/growth/migration of stem cells remain unclear. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system is thought to mediate various developmental events in both vertebrates and invertebrates. We examined the molecular structures and expression of DjFGFR1 and DjFGFR2, two planarian genes closely related to other animal FGFR genes. DjFGFR1 and DjFGFR2 proteins contain three and two immunoglobulin-like domains, respectively, in the extracellular region and a split tyrosine kinase domain in the intracellular region. Expression of DjFGFR1 and DjFGFR2 was observed in the cephalic ganglion and mesenchymal space in intact planarians. In regenerating planarians, accumulation of DjFGFR1-expressing cells was observed in the blastema and in fragments regenerating either a pharynx or a brain. In X-ray-irradiated planarians, which had lost regenerative capacity, the number of DjFGFR1-expressing cells in the mesenchymal space decreased markedly. These results suggest that the DjFGFR1 protein may be involved in the signal systems controlling such aspects of planarian regeneration as differentiation/growth/migration of stem cells.     

Planaria FoxA (HNF3) homologue is specifically expressed in the pharynx-forming cells

We have isolated a planarian Forkhead box A (FoxA, a new name for a gene group containing HNF3 alpha,beta,gamma)-related gene, DjFoxA, and examined its spatial and temporal distribution in both intact and regenerating planarians by in situ hybridization. In intact worms, DjFoxA is specifically expressed in the cells participating in pharynx development in the region surrounding the pharynx, which is located in the central portion of the body. During regeneration, DjFoxA-positive cells appear in the pharynx-forming region and migrate to the midline to form a pharynx rudiment. These results suggest that DjFoxA is specifically expressed in the cells participating in pharynx formation and has an evolutionarily conserved function in digestive tract formation.     

Regeneration processes in various species of planarians

Blastema growth and functional maturation of the pharynx during regeneration in various planarian species were compared. The intensity of blastema growth was highest in Polycelis tenuis; the lowest, in Schmidtea mediterranea. In the sexual and asexual races of Girardia tigrina blastema growth differed inconsiderably. The function of the pharynx during the regeneration of caudal fragments lacking pharynx was manifested in G. tigrina in the usual amount of time, while in the regeneration of head fragments lacking pharynx, this function occured earlier. In other planarian species of the other two typed, the times of pharynx regeneration had no regular character and took longer compared to the same process in G. tigrina.     

ERK signaling controls blastema cell differentiation during planarian regeneration

The robust regenerative ability of planarians depends on a population of somatic stem cells called neoblasts, which are the only mitotic cells in adults and are responsible for blastema formation after amputation. The molecular mechanism underlying neoblast differentiation associated with blastema formation remains unknown. Here, using the planarian Dugesia japonica we found that DjmkpA, a planarian mitogen-activated protein kinase (MAPK) phosphatase-related gene, was specifically expressed in blastema cells in response to increased extracellular signal-related kinase (ERK) activity. Pharmacological and genetic [RNA interference (RNAi)] approaches provided evidence that ERK activity was required for blastema cells to exit the proliferative state and undergo differentiation. By contrast, DjmkpA RNAi induced an increased level of ERK activity and rescued the differentiation defect of blastema cells caused by pharmacological reduction of ERK activity. These observations suggest that ERK signaling plays an instructive role in the cell fate decisions of blastema cells regarding whether to differentiate or not, by inducing DjmkpA as a negative regulator of ERK signaling during planarian regeneration.     

Mitotic activity in the blastema and stump tissues of regenerating hind limbs of Xenopus laevis larvae after amputation at ankle level. An autoradiographic study

Mitotic activity, as indicated by DNA synthesis, was studied by autoradiographic analysis along the proximodistal axis of regenerating limbs in the early and later larval stages 53 and 57 of Xenopus laevis. Wound-healing, dedifferentiation, blastema formation and growth phases were studied. Most of the various stump tissues, as well as the cell mass of the regeneration blastema, were involved. The study showed an increase in DNA synthesis in the stump tissues during their dedifferentiation as well as during blastema formation. The increase was confined mainly to the distal portion (close to the amputation level), so that a proximodistal gradient was discernible. This could be regarded as valid evidence of contribution of the severed stump tissues to the blastema cells. The mesenchymal blastema cells formed after amputation at stage 53 displayed higher mitotic activity than the fibrocytoid blastema cells formed at stage 57. Although the latter were more differentiated than the former, they still showed DNA replication and mitotic division.     

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.     

Migration of mesenchymal cell fated to blastema is necessary for fish fin regeneration

Urodeles and fish have higher regeneration ability in a variety of tissues and organs than do other vertebrate species including mammals. Though many studies have aimed at identifying the cellular and molecular basis for regeneration, relatively little is known about the detailed cellular behaviors and involved molecular basis. In the present study, a small molecule inhibitor was used to analyzed the role of phosphoinositide 3-kinase (PI3K) signaling during regeneration. We showed that the inhibitor disrupted the formation of blastema including the expression of characteristic genes. The failure of blastema formation was due to the impaired migration of mesenchymal cells to the distal prospective blastema region, although it had a little affect on cell cycle activation in mesenchymal cells. Moreover, we found that the epidermal remodeling including cell proliferation, distal cell migration and Akt phosphorylation was also affected by the inhibitor, implying a possible involvement of epidermis for proper formation of blastema. From these data, we propose a model in which distinct signals that direct the cell cycle activation, mesenchymal cell migration and epidermal remodeling coordinate together to accomplish the correct blastema formation and regeneration.     

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.     

Brain regeneration from pluripotent stem cells in planarian

How can planarians regenerate their brain? Recently we have identified many genes critical for this process. Brain regeneration can be divided into five steps: (1) anterior blastema formation, (2) brain rudiment formation, (3) pattern formation, (4) neural network formation, and (5) functional recovery. Here we will describe the structure and process of regeneration of the planarian brain in the first part, and then introduce genes involved in brain regeneration in the second part. Especially, we will speculate about molecular events during the early steps of brain regeneration in this review. The finding providing the greatest insight thus far is the discovery of the nou-darake (ndk; ‘brains everywhere’ in Japanese) gene, since brain neurons are formed throughout the entire body as a result of loss of function of the ndk gene. This finding provides a clue for elucidating the molecular and cellular mechanisms underlying brain regeneration. Here we describe the molecular action of the nou-darake gene and propose a new model to explain brain regeneration and restriction in the head region of the planarians.     

Two msh/msx-related genes, Djmsh1 and Djmsh2, contribute to the early blastema growth during planarian head regeneration

Regeneration in planarians is an intriguing phenomenon, based on the presence of pluripotent stem cells, known as neoblasts. Following amputation, these cells activate mitotic divisions, migrate distally and undergo differentiation, giving rise to the regeneration blastema. We have identified two msh/msx-related genes, Djmsh1 and Djmsh2, which are expressed in distinct cell populations of the planarian Dugesia japonica and activated, with different patterns, during head regeneration. We demonstrate that RNA interference of Djmsh1 or Djmsh2 generates a delay in the growth of cephalic blastema, interfering with the dynamics of mitoses during its initial formation. Our data also reveal that the activity of the two planarian msh genes is required to regulate Djbmp expression during head regeneration. This study identifies, for the first time, a functional association between muscle segment homeobox (MSH) homeoproteins and BMP signaling during stem cell-based regeneration of the planarian head and provides a functional analysis of how msh genes may regulate in vivo the regenerative response of planarian stem cells.     

Retinoic acid signaling controls the formation, proliferation and survival of the blastema during adult zebrafish fin regeneration

Adult teleosts rebuild amputated fins through a proliferation-dependent process called epimorphic regeneration, in which a blastema of cycling progenitor cells replaces the lost fin tissue. The genetic networks that control formation of blastema cells from formerly quiescent stump tissue and subsequent blastema function are still poorly understood. Here, we investigated the cellular and molecular consequences of genetically interfering with retinoic acid (RA) signaling for the formation of the zebrafish blastema. We show that RA signaling is upregulated within the first few hours after fin amputation in the stump mesenchyme, where it controls Fgf, Wnt/β-catenin and Igf signaling. Genetic inhibition of the RA pathway at this stage blocks blastema formation by inhibiting cell cycle entry of stump cells and impairs the formation of the basal epidermal layer, a signaling center in the wound epidermis. In the established blastema, RA signaling remains active to ensure the survival of the highly proliferative blastemal population by controlling expression of the anti-apoptotic factor bcl2. In addition, RA signaling maintains blastema proliferation through the activation of growth-stimulatory signals mediated by Fgf and Wnt/β-catenin signaling, as well as by reducing signaling through the growth-inhibitory non-canonical Wnt pathway. The endogenous roles of RA in adult vertebrate appendage regeneration are uncovered here for the first time. They provide a mechanistic framework to understand previous observations in salamanders that link endogenous sources of RA to the regeneration process itself and support the hypothesis that the RA signaling pathway is an essential component of vertebrate tissue regeneration.     

Planarian pharynx regeneration in regenerating tail fragments monitored with cell-specific monoclonal antibodies

 The special morphological features of freshwater planarians make them an attractive and informative model for studying the processes of regeneration and pattern formation. In this work, we investigate pattern formation and maturation of the planarian pharynx during regeneration in tail fragments. Using three monoclonal antibodies (TCAV-1, TF-26 and TMUS-13) specific for epithelial, secretory and muscle cells, respectively, we followed the sequence and timing of differentiation and maturation of these three cell types within the regenerating pharynx. Two of these monoclonal antibodies, TCAV-1 and TMUS-13, also labelled morphologically immature cells that appear to be committed to the differentiation pathway leading to their respective adult cell types. Our results show that the cells forming the new pharynx come from undifferentiated cells through proliferation and differentiation processes rather than from differentiated cells of the old stump. We describe three stages of pharynx regeneration according to the immunoreactivity shown: (1) no immunoreactivity, corresponding to the accumulation of undifferentiated cells that form the pharynx primordium; (2) immunoreactivity to TCAV-1 and TMUS-13, corresponding to the re-building of the pharynx; and (3) immunoreactivity to TF-26, corresponding to a fully mature and functional pharynx. The sequence of differentiation of these three cell types suggests that the pharynx grows by intercalation of new undifferentiated cells coming from the parenchyma between the older pharyngeal cells, in agreement with existing models of pharynx regeneration. Finally, our results suggest an intercalary model for pharynx epithelial cell renewal. Received: 30 September 1996 / Accepted: 6 December 1996     

Circular form of regeneration in an unidentified species of land planarians, Bipalium sp

The communication describes an interesting and possibly novel finding regarding a species of land planarians (Bipalium sp.) from a high altitude of Himalayan range. The regeneration in Bipalium sp. is highly interesting and the process takes about a fortnight for the completion. Accumulation of numerous basophilic cells in parenchyma at the blastema region occurs within 5 days after excision and in 7 to 9 days the head and tail regions become reorganized with the formation of functional pharynx in the tail piece. The neoblast cells in the mesenchyme and the gastrodermis cells proliferate continuously replenishing the population of stem cell for growth, reproduction, and regeneration. Many undifferentiated cells are present even in completely regenerated specimens. Sometimes middle piece regenerates apparently fuse at the blastema surface forming a peculiar circular form of Bipalium sp. where both the head and tail cut ends join completely. These regenerated individuals excepting the middle piece regenerate behave in a normal fashion within 12-14 days.     

Wound healing and blastema formation in regenerating digit tips of adult mice

Amputation of the distal region of the terminal phalanx of mice causes an initial wound healing response followed by blastema formation and the regeneration of the digit tip. Thus far, most regeneration studies have focused in embryonic or neonatal models and few studies have examined adult digit regeneration. Here we report on studies that include morphological, immunohistological, and volumetric analyses of adult digit regeneration stages. The regenerated digit is grossly similar to the original, but is not a perfect replacement. Re-differentiation of the digit tip occurs by intramembranous ossification forming a trabecular bone network that replaces the amputated cortical bone. The digit blastema is comprised of proliferating cells that express vimentin, a general mesenchymal marker, and by comparison to mature tissues, contains fewer endothelial cells indicative of reduced vascularity. The majority of blastemal cells expressing the stem cell marker SCA-1, also co-express the endothelial marker CD31, suggesting the presence of endothelial progenitor cells. Epidermal closure during wound healing is very slow and is characterized by a failure of the wound epidermis to close across amputated bone. Instead, the wound healing phase is associated with an osteoclast response that degrades the stump bone allowing the wound epidermis to undercut the distal bone resulting in a novel re-amputation response. Thus, the regeneration process initiates from a level that is proximal to the original plane of amputation.     

Analysis of the expression and function of Wnt-5a and Wnt-5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs

Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non-canonical Wnt genes, Wnt-5a and Wnt-5b . We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl ( Ambystoma mexicanum ), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt-5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration-competent cells of the blastema.