Epimorphic regeneration of the distal part of the planarian pharynx

The totipotent stem cells called neoblasts seem to be concerned with the remarkable regeneration ability of planarians. However, the pharynx is able to regenerate after the amputation of its distal part, in spite of a lack of neoblasts in the pharynx. The process of regeneration has been referred to as morphallaxis, based on conventional histochemical observations. We examined it again immuno-histochemically using anti-Dugesia japonica proliferating cell nuclear antigen (DjPCNA) antibody for neoblasts and anti-D. japonica myosin heavy chain-A (DjMHC-A) antibody for pharynx muscle fibers. This immuno-histochemical study, together with observations of the regeneration process of planarians irradiated with X-rays in particular regions, revealed that after the amputation, neoblasts from outside the pharynx entered that organ, moved through the mesenchyme of the pharynx to the wounded area, and differentiated into the cells that had been lost there. We show here that the regeneration after amputation of the distal part of the pharynx is an 'epimorphic' process.     

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     

The process of pharynx regeneration in planarians.

To understand the cellular events during planarian regeneration, we analyzed the process of pharynx regeneration in both head and tail pieces using cell-type-specific markers. Interestingly, cells expressing the pharynx-muscle-specific myosin heavy chain gene (DjMHC-A) appeared within 24 h after amputation (prior to the formation of a pharynx rudiment) in the mesenchymal space of the stump, not in the blastema region. These DjMHC-A-positive cells migrated to the midline and formed the pharynx rudiment. Even after formation of the pharynx rudiment, DjMHC-A-positive cells constantly appeared in the mesenchymal space in the region surrounding the pharynx rudiment and participated in the growth of the pharynx rudiment. These observations clearly indicated that the cells involved in pharynx-muscle formation are committed in the mesenchymal space of the stump, rather than in the blastema region or the pharynx rudiment during planarian regeneration. We also analyzed the process of regeneration of the pharynx epithelia using a monoclonal antibody and investigated the origin of the pharynx epithelia.     

Studies on the Effect of a Weak Electromagnetic Field on Pharynx Regeneration in Dugesia tigrina Planarians

We have studied the effect of a weak electromagnetic field on the morphogenesis of the planarian Dugesia tigrina. The regeneration of the pharynx was examined after its amputation. We have determined the rate of the appearance of the food response of a new pharynx. Experimental conditions were varied, such as dose and duration of irradiation, season, and time of irradiation after surgery. The results of experiments conducted with 2966 planarians have shown that weak electromagnetic field has various effects, which appear either as stimulation of regeneration or as its inhibition. In some experiments, there was no effect at all. These differences depend on numerous factors and may be modulated.     

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.     

Analysis of pharyngeal resistance and genioglossal EMG activity using a model of orifice flow.

A model of orifice flow has been used to analyze the relationships among pressure, flow, and genioglossal electromyographic activity in the human pharynx during inspiration. The orifice flow model permits one to assess the character of airflow (laminar or turbulent) and to estimate the cross-sectional area of the orifice from pressure and flow measurements. On the basis of other data (J. Appl. Physiol. 73: 584-590, 1992), this analysis suggests that pharyngeal airflow is turbulent. Furthermore the area of the pharynx appears to increase as flow increases, but the actual change in pharyngeal diameter necessary to fit the pressure-flow data is quite small (0.11-0.87 cm, depending on the assumptions in the model). The flow-related increase in orifice area can be attributed, in part, to the activation of the genioglossus muscle. However, other flow-related factors may also contribute to pharyngeal dilation as airflow increases. Different airway shapes (circular and elliptical) and orientations (major axis anteroposterior and lateral) were incorporated into the model calculations; these factors modify considerably the apparent efficiency of genioglossal electromyographic activity. Genioglossal muscle shortening increases pharyngeal area and reduces pharyngeal resistance more effectively when the pharynx is elliptical, with the long axis of the ellipse oriented laterally. Hence the genioglossus may operate at a significant mechanical disadvantage in those patients with obstructive sleep apnea with a small sagittally oriented pharyngeal lumen.     

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.     

显示三角涡虫神经系统的三种方法

涡虫在动物系统演化史中占有十分重要的地位,同时又具有很强的再生能力,如何显示其正常组织结构,对研究涡虫的再生具有重要意义。本文用3种染色方法(H.E染色、Masson染色、Van Gieson染色)显示了日本三角涡虫(Dugesia japonica)的神经系统,结果表明,尽管3种方法都能很清晰地显示涡虫的神经系统,但在显示咽部神经时,只有Masson染色能够很清晰地显示。     

Formation of malformed pharynx and neoplasia in the planarian Bdellocephala brunnea following treatment with a carcinogen

The effect of benzo(a)pyrene on regeneration of the pharynx in Bdellocephala brunnea Ijima et Kaburaki was studied by inserting a 5-µg crystal of this carcinogen into the pharyngeal cavity of worms from which the pharynx had been extirpated 2 d previously. Multiple pharynges formed in the regenerates, and neoplasia was induced in a few cases. The malformed pharynges appeared as protrusions overlapping the anterior part of the main pharynx. Electron microscopy of the pharynx in normal, intact worms shows eight layers comprising inner and outer ciliated epithelia, muscle, and glands.     

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.     

Effect of weak electromagnetic radiation on regeneration of the pharynx in Dugesia tigrina planaria]

We have studied the effect of weak electromagnetic field on the morphogenesis of the planarian Dugesia tigrina. Regeneration of the pharynx was examined after its amputation. We have determined the rate of the appearance of the food response of a new pharynx. Experimental conditions were varied, such as dose and duration of irradiation, season, and time of irradiation after surgery. The results of experiments conducted with 2966 planarians have shown that weak electromagnetic field has various effects, which appear either as stimulation of regeneration or as its inhibition. In some experiments, there was no effect at all. These differences depend on numerous factors and may be modulated.     

Cell proliferation is necessary for the regeneration of oral structures in the anthozoan cnidarian Nematostella vectensis

The contribution of cell proliferation to regeneration varies greatly between different metazoan models. Planarians rely on pluripotent neoblasts and amphibian limb regeneration depends upon formation of a proliferative blastema, while regeneration in Hydra can occur in the absence of cell proliferation. Recently, the cnidarian Nematostella vectensis has shown potential as a model for studies of regeneration because of the ability to conduct comparative studies of patterning during embryonic development, asexual reproduction, and regeneration. The present study investigates the pattern of cell proliferation during the regeneration of oral structures and the role of cell proliferation in this process. In intact polyps, cell proliferation is observed in both ectodermal and endodermal tissues throughout the entire oral-aboral axis, including in the tentacles and physa. Following bisection, there is initially little change in proliferation at the wound site of the aboral fragment, however, beginning 18 to 24?hours after amputation there is a dramatic increase in cell proliferation at the wound site in the aboral fragment. This elevated level of proliferation is maintained throughout the course or regeneration of oral structures, including the tentacles, the mouth, and the pharynx. Treatments with the cell proliferation inhibitors hydroxyurea and nocodazole demonstrate that cell proliferation is indispensable for the regeneration of oral structures. Although inhibition of regeneration by nocodazole was generally irreversible, secondary amputation reinitiates cell proliferation and regeneration. The study has found that high levels of cell proliferation characterize the regeneration of oral structures in Nematostella, and that this cell proliferation is necessary for the proper progression of regeneration. Thus, while cell proliferation contributes to regeneration of oral structures in both Nematostella and Hydra, Nematostella lacks the ability to undergo the compensatory morphallactic mode of regeneration that characterizes Hydra. Our results are consistent with amputation activating a quiescent population of mitotically competent stem cells in spatial proximity to the wound site, which form the regenerated structures.     

Ectopic pharynxes arise by regional reorganization after anterior/posterior chimera in planarians

To elucidate the mechanisms underlying pharynx regeneration in planarians, we transplanted pieces excised from various regions of the body into the prepharyngeal or postpharyngeal region, since it has been shown that such transplantation experiments can induce ectopic pharynx formation. We confirmed the ectopic formation of pharynxes by expression of the myosin heavy chain gene specific to pharynx muscles (DjMHC-A). To investigate the cellular events after grafting, we also stained such transplanted worms by in situ hybridization using neuronal cell- and mucous producing cell-type-specific marker genes which can detect formation of brain and prepharyngeal region, respectively. When the head piece was transplanted into the tail region, ectopic formation of the head, prepharyngeal and pharynx region was observed in the postpharyngeal region anterior to the graft, while these organs were formed in the reversed polarity along the anterior-posterior (A-P) axis. Furthermore, in the tail region posterior to the graft, ectopic formation of the prepharyngeal and pharynx region was observed. In the reverse combination, when a tail piece was transplanted into the prepharyngeal region, ectopic formation of prepharyngeal and pharynx region was observed in the region between the head and the graft, and an additional ectopic pharynx was also formed in reverse polarity in the region between the graft and host pharynx. These results clearly indicated that ectopic pharynxes were formed as a consequence of the regional reorganization induced by interaction between the host and graft. Furthermore, chimeric analyses demonstrated that the cells participating in ectopic pharynx formation were not exclusively derived from the host or donor cells in the worm, suggesting that the stem cells of the host and donor may change their differentiation pattern due to altered regionality. To further investigate if regional reorganization is induced after grafting, expression of a Hox gene was analyzed in the transplanted worms by whole-mount in situ hybridization. The expression of the Hox gene along the A-P axis was apparently rearranged after grafting of the head piece into the tail region. These results suggest that grafting of the head piece may rearrange the regionality of the host tail, and that stem cells in the region newly defined as pharynx-forming may start to regenerate a pharynx.     

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.     

The regeneration capacity of the flatworm Macrostomum lignano—on repeated regeneration, rejuvenation, and the minimal size needed for regeneration

The lion’s share of studies on regeneration in Plathelminthes (flatworms) has been so far carried out on a derived taxon of rhabditophorans, the freshwater planarians (Tricladida), and has shown this group’s outstanding regeneration capabilities in detail. Sharing a likely totipotent stem cell system, many other flatworm taxa are capable of regeneration as well. In this paper, we present the regeneration capacity of Macrostomum lignano, a representative of the Macrostomorpha, the basal-most taxon of rhabditophoran flatworms and one of the most basal extant bilaterian protostomes. Amputated or incised transversally, obliquely, and longitudinally at various cutting levels, M. lignano is able to regenerate the anterior-most body part (the rostrum) and any part posterior of the pharynx, but cannot regenerate a head. Repeated regeneration was observed for 29 successive amputations over a period of almost 12 months. Besides adults, also first-day hatchlings and older juveniles were shown to regenerate after transversal cutting. The minimum number of cells required for regeneration in adults (with a total of 25,000 cells) is 4,000, including 160 neoblasts. In hatchlings only 1,500 cells, including 50 neoblasts, are needed for regeneration. The life span of untreated M. lignano was determined to be about 10 months.An erratum to this article can be found at     

Ein überzähliger genitalapparat bei einem rhabdocölen Turbellar

A specimen of Ethmorhynchus anophthalmus Meixner 1938 contains a complex of supernumerary genital organs in front of the pharynx. The phenomenon elucidates some traits in the morphogenesis of the atrial organs.

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Abkürzungen in den Abbildungen At Atrium genitale - Bu Bursa - Ci Cirrus des Kopulationsorganes - Dc Ductus communis - Ho Hoden - Ke Keimstock - Kop Kopulationsorgan - Ph Pharynx - R Rüssel - Sa Samenblase     

Inflamed adult pharynx tissues and swimming larva of Ciona intestinalis share CiTNFα-producing cells

In situ hybridisation and immunohistochemistry analyses have shown that the Ciona intestinalis tumour necrosis factor alpha gene (CiTNFα), which has been previously cloned and sequenced, is expressed either during the inflammatory pharynx response to lipopolysaccharide (LPS) or during the swimming larval phase of development. Granulocytes with large granules and compartment/morula cells are CiTNFα-producing cells in both inflamed pharynx and larvae. Pharynx vessel endothelium also takes part in the inflammatory response. Haemocyte nodules in the vessel lumen or associated with the endothelium suggest the involvement of CiTNFα in recruiting lymphocyte-like cells and promoting the differentiation of inflammatory haemocytes. Specific antibodies against a CiTNFα peptide have identified a 43-kDa cell-bound form of the protein. Observations of pharynx histological sections (at 4 and 8 h post-LPS inoculation) from naive and medium-inoculated ascidians have confirmed the CiTNFα-positive tissue response. Larval histological sections and whole-mount preparations have revealed that CiTNFα is expressed by trunk mesenchyme, preoral lobe and tunic cells, indicating CiTNFα-expressing cell immigration events and an ontogenetic role.     

Intercalary muscle cell renewal in planarian pharynx

 Planarian cell renewal is achieved as a result of proliferation and differentiation of totipotent undifferentiated cells called neoblasts. The absence of mitosis within the planarian pharynx raises the question as to how cell renewal and growth occur within this organ. Two explanations have been advanced: one proposes that new cells remain close to the base of the pharynx, which then grows by distal displacement of older cells, and the other suggests that the new cells are intercalated between older cells throughout the pharynx. The second alternative, however, does not explain how new cells enter the pharynx or how they reach their final destination. In this study of myosin heavy-chain gene expression within planarian pharynx, a row of differentiating myocytes was detected all along the pharynx parenchyma. According to the hybridization pattern, all these myocytes appeared to be at early stages of differentiation. These data favour an intercalary model for muscle cell renewal within the pharynx. According to this model, neoblasts at the base of the pharynx would enter the pharynx, where they would start differentiation to myocytes, move to the subepithelial musculature and intercalate between the old muscle cells. The possible application of this intercalary model to other pharynx cell types is also discussed. Received: 30 July 1998 / Accepted: 20 November 1998     

中国东北森林生长演替模拟模型及其在全球变化研究中的应用

 NEWCOP模型是一个新的适于模拟东北森林的种类组成动态的林窗类计算机模拟模型,它通过模拟在每一个林分斑块上的每株树木的更新、生长和死亡的全过程来反映森林群落的中长期生长和演替动态。由于 NEWCOP模型是一个由气候变量驱动的生态系统模型,故可用于评价气候变化对东北森林生长和演替的影响。在东北大兴安岭、小兴安岭和长白山地区对NEWCOP模型进行了验证和校准。沿环境梯度对NEWCOP模型的数字模拟实验表明：它能准确地再现顶极森林中树种组成及其在东北地区的垂直分布规律和水平分布规律;能准确地再现大兴安岭、小兴安岭和长白山的主要类型森林的生长和演替规律;在一定的场合NEWCOP还可反映林分的径级结构;NEWCOP模型还具有对现有森林的跟踪模拟能力。应用NEWCOP模型评估了东北森林生态系统对可能气候变化的敏感性。在GFDL 2×CO2和GISS 2×CO2气候变化情景下,东北森林的种类组成将发生很大变化,落叶阔叶树将取代目前长白山、小兴安岭的红松(Pinus koraiensis)和大兴安岭的兴安落叶松(Larix gmelinii)成为东北森林主要树种,而针叶树将在地带性森林中占很小的比重,阔叶树中蒙古栎(Quercus mongolica)将是最重要的树种,它将成为小兴安岭和大兴安岭最主要树种;东北地区适于森林生长的区域将大幅度减少,这些变化主要发生在气候变化过渡期。东北森林对不同的气候变化情景有不同响应。但是,总的趋势是未来东北森林中落叶阔叶树的比重将大幅度增加。这些结论对在全球气候变化背景下,我国东北合理地选择造林树种和制定现有森林的保护经营策略具有一定参考价值。     

Modeling of airflow in the pharynx with application to sleep apnea.

A three-dimensional numerical modeling of airflow in the human pharynx using an anatomically accurate model was conducted. The pharynx walls were assumed to be passive and rigid. The results showed that the pressure drop in the pharynx lies in the range 200-500 Pa. The onset of turbulence was found to increase the pressure drop by 40 percent. A wide range of pharynx geometries covering three sleep apnea treatment therapies (CPAP, mandibular repositioning devices, and surgery) were modeled and the resulting flow characteristics were investigated and compared. The results confirmed that the airflow in the pharynx lies in the laminar-to-turbulence transitional flow regime and thus, a subtle change in the morphology caused by these treatment therapies can significantly affect the airflow characteristics.