EGFR signaling regulates cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis

Similarly to development, the process of regeneration requires that cells accurately sense and respond to their external environment. Thus, intrinsic cues must be integrated with signals from the surrounding environment to ensure appropriate temporal and spatial regulation of tissue regeneration. Identifying the signaling pathways that control these events will not only provide insights into a fascinating biological phenomenon but may also yield new molecular targets for use in regenerative medicine. Among classical models to study regeneration, freshwater planarians represent an attractive system in which to investigate the signals that regulate cell proliferation and differentiation, as well as the proper patterning of the structures being regenerated. Recent studies in planarians have begun to define the role of conserved signaling pathways during regeneration. Here, we extend these analyses to the epidermal growth factor (EGF) receptor pathway. We report the characterization of three epidermal growth factor (EGF) receptors in the planarian Schmidtea mediterranea. Silencing of these genes by RNA interference (RNAi) yielded multiple defects in intact and regenerating planarians. Smed-egfr-1(RNAi) resulted in decreased differentiation of eye pigment cells, abnormal pharynx regeneration and maintenance, and the development of dorsal outgrowths. In contrast, Smed-egfr-3(RNAi) animals produced smaller blastemas associated with abnormal differentiation of certain cell types. Our results suggest important roles for the EGFR signaling in controlling cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis.     

Stem cell-based growth, regeneration, and remodeling of the planarian intestine

Although some animals are capable of regenerating organs, the mechanisms by which this is achieved are poorly understood. In planarians, pluripotent somatic stem cells called neoblasts supply new cells for growth, replenish tissues in response to cellular turnover, and regenerate tissues after injury. For most tissues and organs, however, the spatiotemporal dynamics of stem cell differentiation and the fate of tissue that existed prior to injury have not been characterized systematically. Utilizing in vivo imaging and bromodeoxyuridine pulse-chase experiments, we have analyzed growth and regeneration of the planarian intestine, the organ responsible for digestion and nutrient distribution. During growth, we observe that new gut branches are added along the entire anteroposterior axis. We find that new enterocytes differentiate throughout the intestine rather than in specific growth zones, suggesting that branching morphogenesis is achieved primarily by remodeling of differentiated intestinal tissues. During regeneration, we also demonstrate a previously unappreciated degree of intestinal remodeling, in which pre-existing posterior gut tissue contributes extensively to the newly formed anterior gut, and vice versa. By contrast to growing animals, differentiation of new intestinal cells occurs at preferential locations, including within newly generated tissue (the blastema), and along pre-existing intestinal branches undergoing remodeling. Our results indicate that growth and regeneration of the planarian intestine are achieved by co-ordinated differentiation of stem cells and the remodeling of pre-existing tissues. Elucidation of the mechanisms by which these processes are integrated will be critical for understanding organogenesis in a post-embryonic context.     

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.     

Djeyes absent (Djeya) controls prototypic planarian eye regeneration by cooperating with the transcription factor Djsix-1

A conserved network of nuclear proteins is crucial to eye formation in both vertebrates and invertebrates. The finding that freshwater planarians can regenerate eyes without the contribution of Pax6 suggests that alternative combinations of regulatory elements may control the morphogenesis of the prototypic planarian eye. To further dissect the molecular events controlling eye regeneration in planarians, we investigated the role of eyes absent (Djeya) and six-1 (Djsix-1) genes in Dugesia japonica. These genes are expressed in both regenerating eyes and in differentiated photoreceptors of intact adults. Through RNAi studies, we show that Djsix-1 and Djeya are both critical for the regeneration of normal eyes in planarians and genetically cooperate in vivo to establish correct eye cell differentiation. We further demonstrate that the genetic interaction is mediated by physical interaction between the evolutionarily conserved domains of these two proteins. These data indicate that planarians use cooperatively Djsix-1 and Djeya for the proper specification of photoreceptors, implicating that the mechanism involving their evolutionarily conserved domains can be very ancient. Finally, both Djsix-1 and Djeya double-stranded RNA are substantially more effective at producing no-eye phenotypes in the second round of regeneration. This is probably due to the significant plasticity of the planarian model system, based on the presence of a stable population of totipotent stem cells, which ensure the rapid cell turnover of all differentiated cell types.     

Recent identification of an ERK signal gradient governing planarian regeneration

Planarians have strong regenerative abilities derived from their adult pluripotent stem cell (neoblast) system. However, the molecular mechanisms involved in planarian regeneration have long remained a mystery. In particular, no anterior-specifying factor(s) could be found, although Wnt family proteins had been successfully identified as posterior-specifying factors during planarian regeneration (Gurley et al., 2008, Petersen and Reddien, 2008). A recent textbook of developmental biology therefore proposes a Wnt antagonist as a putative anterior factor (Gilbert, 2013). That is, planarian regeneration was supposed to be explained by a single decreasing gradient of the β-catenin signal from tail to head. However, recently we succeeded in demonstrating that in fact the extracellular-signal regulated kinases (ERK) form a decreasing gradient from head to tail to direct the reorganization of planarian body regionality after amputation (Umesono et al., 2013).     

The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration

Planarian flatworms are an exception among bilaterians in that they possess a large pool of adult stem cells that enables them to promptly regenerate any part of their body, including the brain. Although known for two centuries for their remarkable regenerative capabilities, planarians have only recently emerged as an attractive model for studying regeneration and stem cell biology. This revival is due in part to the availability of a sequenced genome and the development of new technologies, such as RNA interference and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular level. Here, we highlight why planarians are an exciting tool in the study of regeneration and its underlying stem cell biology in vivo, and discuss the potential promises and current limitations of this model organism for stem cell research and regenerative medicine.     

Regeneration and the need for simpler model organisms

The problem of regeneration is fundamentally a problem of tissue homeostasis involving the replacement of cells lost to normal 'wear and tear' (cell turnover), and/or injury. This attribute is of particular significance to organisms possessing relatively long lifespans, as maintenance of all body parts and their functional integration is essential for their survival. Because tissue replacement is broadly distributed among multicellular life-forms, and the molecules and mechanisms controlling cellular differentiation are considered ancient evolutionary inventions, it should be possible to gain key molecular insights about regenerative processes through the study of simpler animals. We have chosen to study and develop the freshwater planarian Schmidtea mediterranea as a model system because it is one of the simplest metazoans possessing tissue homeostasis and regeneration, and because it has become relatively easy to molecularly manipulate this organism. The developmental plasticity and longevity of S. mediterranea is in marked contrast to its better-characterized invertebrate cohorts: the fruitfly Drosophila melanogaster and the roundworm Caenorhabditis elegans, both of which have short lifespans and are poor at regenerating tissues. Therefore, planarians present us with new, experimentally accessible contexts in which to study the molecular actions guiding cell fate restriction, differentiation and patterning, each of which is crucial not only for regeneration to occur, but also for the survival and perpetuation of all multicellular organisms.     

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.     

Effects of 17β-estradiol and bisphenol A on the formation of reproductive organs in planarians

Planarians have a remarkable capacity for regeneration after ablation, and they reproduce asexually by fission. However, some planarians can also reproduce and maintain their sexual organs. During the regenerative process, their existing sexual organs degenerate and new ones develop. However, little is known about hormonal regulation during the development of reproductive organs in planarians. In this study, we investigated the effects of 17β-estradiol (a steroid) and bisphenol A (an endocrine disrupter) on the formation of sexual organs in the hermaphroditic planarian Dugesia ryukyuensis. Under control conditions, all worm tissues regenerated into sexual planarians with sexual organs within 4 weeks after ablation. However, in the presence of bisphenol A or 17β-estradiol, although they apparently regenerated into sexual planarians, the yolk glands, which are one of the female sexual organs, failed to regenerate even 7 weeks after ablation. These data suggest that planarians have a steroid hormone system, which plays a key role in the formation and maturation of sexual organs.     

Echinoderm regeneration: an in vitro approach using the crinoid Antedon mediterranea

Among echinoderms, crinoids are well known for their remarkable regenerative potential. Regeneration depends mainly on progenitor cells (undifferentiated or differentiated), which migrate and proliferate in the lesion site. The crucial role of the “progenitor” elements involved in the regenerative processes, in terms of cell recruitment, sources, and fate, is a central problem in view of its topical interest and biological implications. The spectacular regenerative potential of crinoids is used to replace lost internal and external organs. In particular, the process of arm regeneration in the feather star Antedon mediterranea is the regeneration model most extensively explored to date. We have addressed the morphological and functional characterization of the cell phenotypes responsible for the arm regenerative processes by using an in vitro approach. This represents the first successful attempt to culture cells involved in crinoid regeneration. A comparison of these results with others from previous in vivo investigations confirms the diverse cell types contributing to regeneration and underscores their involvement in migration, proliferation, and dedifferentiation processes.     

MicroRNAs from the Planarian Schmidtea mediterranea: a model system for stem cell biology

MicroRNAs (miRNAs) are approximately 22-nt RNA molecules that typically bind to the 3' untranslated regions of target mRNAs and function to either induce mRNA degradation or repress translation. miRNAs have been shown to play important roles in the function of stem cells and cell lineage decisions in a variety of organisms, including humans. Planarians are bilaterally symmetric metazoans that have the unique ability to completely regenerate lost tissues or organs. This regenerative capacity is facilitated by a population of stem cells known as neoblasts. Planarians are therefore an excellent model system for studying many aspects of stem cell biology. Here we report the cloning and initial characterization of 71 miRNAs from the planarian Schmidtea mediterranea. While several of the S. mediterranea miRNAs are members of miRNA families identified in other species, we also identified a number of planarian-specific miRNAs. This work lays the foundation for functional studies aimed at addressing the role of these miRNAs in regeneration, cell lineage decisions, and basic stem cell biology.     

Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting

The remarkable capability of planarian regeneration is mediated by a group of adult stem cells referred to as neoblasts. Although these cells possess many unique cytological characteristics (e.g. they are X-ray sensitive and contain chromatoid bodies), it has been difficult to isolate them after cell dissociation. This is one of the major reasons why planarian regenerative mechanisms have remained elusive for a long time. Here, we describe a new method to isolate the planarian adult stem cells as X-ray-sensitive cell populations by fluorescence-activated cell sorting (FACS). Dissociated cells from whole planarians were labeled with fluorescent dyes prior to fractionation by FACS. We compared the FACS profiles from X-ray-irradiated and non-irradiated planarians, and thereby found two cell fractions which contained X-ray-sensitive cells. These fractions, designated X1 and X2, were subjected to electron microscopic morphological analysis. We concluded that X-ray-sensitive cells in both fractions possessed typical stem cell morphology: an ovoid shape with a large nucleus and scant cytoplasm, and chromatoid bodies in the cytoplasm. This method of isolating X-ray-sensitive cells using FACS may provide a key tool for advancing our understanding of the stem cell system in planarians.     

皮毛之道：以表皮器官为研究模型探讨再生生物学与再生医学的基础概念

再生医学是一个具有巨大潜力的新兴医学领域。该文以此为方向讨论了再生医学研究中的三个关键问题,并以非神经外胚层器官的干细胞行为为例做进一步的探讨。第一,如何获取干细胞,介绍了包括胚胎干细胞、组织干细胞和诱导性多能干细胞的获得途径,以及若干组织细胞重编程的成功范例;第二,如何将干细胞转化为组织和器官,这需要了解干细胞分化以及形态发生的机制,并以羽毛的形态发生为模型,引入了千细胞拓扑生物学的概念以及干细胞微环境调控塑造器官形态的机制;第三,如何将干细胞及其转化产物置于患者体内,并以鼠毛生长周期波为例,阐明了宏观环境因素如何调控干细胞的活性：最后,还分析了在器官发生中干细胞的自组织对于新生毛发组织工程的重要意义。该文的许多原则不仅限于皮肤,同时也适用于其它体内器官。通过对生物再生的过程的基础研究,我们可以受到生物再生之道的启发,逐渐理解组织修复及再生的机制,并提高分子和细胞水平上的干细胞操作技术,希望在不久的将来将干细胞研究成果应用于临床医学。     

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.     

Ultrastructure of the protonephridial system in Neodasys chaetonotoideus (Gastrotricha: Chaetonotida) and in the ground pattern of Gastrotricha

The taxon Neodasys has a basal position within Gastrotricha. This makes it very interesting for phylogenetic considerations in this group. To complete the reconstruction of the nephridial system in the stem species of Gastrotricha started earlier, we have studied the whole protonephridial system of Neodasys chaetonotoideus by means of complete sets of ultrathin sections and TEM. In many characters, protonephridia of N. chaetonotoideus resemble those of macrodasyidan gastrotrich species. For example, each of the six protonephridia, arranged in three pairs, consists of three distinct cells that constitute the continuous protonephridial lumen. Especially, the terminal cell of the protonephridia of N. chaetonotoideus shows a striking pattern: The perforation of the filter region is a meandering cleft that is continuous with the seam of the enfolded lumen of that cell. With the results presented here and that of former TEM studies, we give a comprehensive idea of the excretory organs in the ground pattern of Gastrotricha. Moreover, we can elaborate on the hypothesized protonephridial system in the stem species of Bilateria. We suggest that a meandering filtration cleft is a feature of the ground pattern of the Bilateria.