Regeneration in <Emphasis Type="Italic">Macrostomum lignano</Emphasis> (Platyhelminthes): cellular dynamics in the neoblast stem cell system

Neoblasts are potentially totipotent stem cells and the only proliferating cells in adult Platyhelminthes. We have examined the cellular dynamics of neoblasts during the posterior regeneration of Macrostomum lignano. Double-labeling of neoblasts with bromodeoxyuridine and the anti-phospho histone H3 mitosis marker has revealed a complex cellular response in the first 48 h after amputation; this response is different from that known to occur during regeneration in triclad platyhelminths and in starvation/feeding experiments in M. lignano. Mitotic activity is reduced during the first 8 h of regeneration but, at 48 h after amputation, reaches almost twice the value of control animals. The total number of S-phase cells significantly increases after 1 day of regeneration. A subpopulation of fast-cycling neoblasts surprisingly shows the same dynamics during regeneration as those in control animals. Wound healing and regeneration are accompanied by the formation of a distinct blastema. These results present new insights, at the cellular level, into the early regeneration of rhabditophoran Platyhelminthes. This work was supported by FWF Grant (P16618; P.I. Rieger, Innsbruck).     

Evaluation of endogenous reference genes for analysis of gene expression with real-time RT-PCR during planarian regeneration

It is important that endogenous reference genes for real-time RT-PCR be empirically evaluated for stability in different cell types, developmental stages, and/or sample treatment. To select the most stable endogenous reference genes during planarian regeneration, three housekeeping genes, 18S rRNA, ACTB and DjEF2, were identified and established expression levels by real-time RT-PCR. The data were analyzed by GeNorm and NormFinder software. Expression levels of the Djsix-1 gene were studied in parallel with ACTB and DjEF2 both or each and 18S rRNA as reference during regeneration. The results showed that ACTB was the most stable expressed reference gene in the planarian regeneration.     

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.     

Regeneration in two freshwater planarian species exposed to methylmercury compounds

The regeneration rate was studied by the morphometric method in planarians Polycelis tenuis and Dugesia lugubris after different periods of exposure on food substrates with high (0.30–0.50 mg/kg wet weight) and low (0.02–0.07 mg/kg) concentration of methylmercury compounds. The planarian growth and the ratio of regenerating tissue area to the total fragment area after transverse cutting were evaluated. The rate of size increment was lower in animals with high level than with low level of methylmercury than with low level of methylmercury. The highest relative and absolute body area increment after amputation was observed in D. lugubris and P. tenius, respectively. Thus, natural methylmercury compounds were shown to inhibit tissue regeneration in planarians in a dose-dependent manner.     

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.     

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.     

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.     

Regeneration of freshwater planarians <Emphasis Type="Italic">Dugesia tigrina</Emphasis> and <Emphasis Type="Italic">Polycelis tenuis</Emphasis> under the influence of methyl mercury compounds of natural origin

We studied the effects of methyl mercury compounds of natural origin on regeneration of the planarians Dugesia tigrina and Polycelis tenuis. Accumulation of methyl mercury in the planarian body leads to a delayed formation of photoreceptor organs in planarians of both species. After a significant traumatic load, the regeneration is suppressed and the death of some control and most experimental animals was observed. The intensity of joining additional cuts depends on the localization of body fragment with a cut and localization of a cut itself.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 35–40.Original Russian Text Copyright © 2005 by Medvedev, Komov.     

Proliferative response of the stem cell system during regeneration of the rostrum in <Emphasis Type="Italic">Macrostomum lignano</Emphasis> (Platyhelminthes)

Macrostomum lignano (Platyhelminthes) possesses pluripotent stem cells, also called neoblasts, which power its extraordinary regeneration capacity. We have examined the cellular dynamics of neoblasts during regeneration of the rostrum in M. lignano. First, using live squeeze observations, the growth curve of the rostrum was determined. Second, neoblasts were labelled with 5-bromo-2'-deoxyuridine (BrdU) and an anti-phospho-histone H3 mitosis marker (anti-phos-H3) to analyze their proliferative response to amputation. During the regeneration process, both S- and M-phase cells were present anterior to the eyes, a region that is devoid of proliferating cells during homeostasis. Furthermore, BrdU pulse experiments revealed a biphasic S-phase pattern, different from the pattern known to occur during regeneration of the tail plate in M. lignano. During a first systemic phase, S-phase numbers significantly increased, both in the region adjacent to the wound (the anterior segment) and the region far from the wound (the posterior segment). During the second, spatially restricted phase, S-phase numbers in the anterior segment rose to a peak at 3 to 5 days post-amputation (p-a), while in the posterior segment, S-phase activity approached control values again. A blastema, characterized as a build-up of S- and M-phase cells, was formed 1 day p-a.     

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.     

Seasonal variability of wing length and eyespots in populations of <Emphasis Type="Italic">Erebia ligea</Emphasis> (L.) (Lepidoptera,Satyridae) in the Middle Urals

The flight period of Erebia ligea (Linnaeus, 1758) in the Sysert District of Sverdlovsk Province lasts from early July until early August only in the odd-numbered years. The species is characterized by protandry, its males emerging 5–7 days before females. Erebia ligea manifests sexual and seasonal variation of the wing length and diameter of the eyespots in the Middle Urals. According to the literature and newly obtained data, the relative male/female wing length varies within the species range. The vectors of the seasonal variation of the body size are opposite in the males and females: in early summer the largest males and the smallest females occur, then mediumsized individuals appear, while the smallest males and the largest females fly at the end of the warm period. Although the males of Erebia ligea are larger than females, the latter have larger eyespots on the wings. Seasonal variation of the diameters of the forewing eyespots is largely similar in the two sexes: individuals with larger spots appear earlier in the season than those with small spots. The eyespots treated as phenes in this study develop on the wings only if the remaining spots of the wing pattern attain a threshold size. A threshold nature was shown for P4 spot in M ₃-Cu ₁ cell of the fore wing and G2 spot in cell M ₁–M ₂ of the hind wing. Individuals with and without phene G2 have opposite vectors of seasonal variation of the sizes of the rest spots of the hind wing pattern. It is apparently the threshold character of variation of these elements of the wing pattern that defines the variation of the proportion of phenetic combinations of the eyespots in the individuals emerging in different periods of the season. Describing phenetic variation at the population level, one should remember that the percentage of the “morphs” or phenetic combinations may vary during a season, especially in the protandric species.     

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.     

Acute phase response in amputated tail stumps and neural tissue‐preferential expression in tail bud embryos of the Xenopus neuronal pentraxin I gene

Regeneration of lost organs involves complex processes, including host defense from infection and rebuilding of lost tissues. We previously reported that Xenopus neuronal pentraxin I (xNP1) is expressed preferentially in regenerating Xenopus laevis tadpole tails. To evaluate xNP1 function in tail regeneration, and also in tail development, we analyzed xNP1 expression in tailbud embryos and regenerating/healing tails following tail amputation in the ‘regeneration’ period, as well as in the ‘refractory’ period, when tadpoles lose their tail regenerative ability. Within 10 h after tail amputation, xNP1 was induced at the amputation site regardless of the tail regenerative ability, suggesting that xNP1 functions in acute phase responses. xNP1 was widely expressed in regenerating tails, but not in the tail buds of tailbud embryos, suggesting its possible role in the immune response/healing after an injury. xNP1 expression was also observed in neural tissues/primordia in tailbud embryos and in the spinal cord in regenerating/healing tails in both periods, implying its possible roles in neural development or function. Moreover, during the first 48 h after amputation, xNP1 expression was sustained at the spinal cord of tails in the ‘regeneration’ period tadpoles, but not in the ‘refractory’ period tadpoles, suggesting that xNP1 expression at the spinal cord correlates with regeneration. Our findings suggest that xNP1 is involved in both acute phase responses and neural development/functions, which is unique compared to mammalian pentraxins whose family members are specialized in either acute phase responses or neural functions.     

Overexpression of <Emphasis Type="Italic">WUSCHEL</Emphasis> in <Emphasis Type="Italic">C. chinense</Emphasis> causes ectopic morphogenesis

Capsicum chinense is a recalcitrant species for in vitro morphogenesis, and up to date there is no efficient system for genetic transformation and regeneration of this species via somatic embryogenesis. Here, we carried out an in vitro transformation of C. chinense via Agrobacterium tumefaciens co-cultivation with a system that expresses the heterologous gene WUSCHEL from Arabidopsis thaliana. WUSCHEL has been shown to promote the transition from vegetative to embryogenic state when overexpressed. We tested if the expression of WUSCHEL in C. chinense would promote an embryogenic response in this species. After 15 days of induction, the segments of transformed stems begun to form globular structures, suggesting that heterologus WUSCHEL was active and involved in the process of morphogenesis.