DDX24 is required for muscle fiber organization and the suppression of wound-induced Wnt activity necessary for pole re-establishment during planarian regeneration.

Planarians have a remarkable ability to undergo whole-body regeneration. Successful regeneration outcome is determined by processes like polarity establishment at the wound site, which is followed by pole (organizer) specification. Interestingly, these determinants are almost exclusively expressed by muscles in these animals. However, the molecular toolkit that enables the functional versatility of planarian muscles remains poorly understood. Here we report that SMED_DDX24, a D-E-A-D Box RNA helicase, is necessary for planarian survival and regeneration. We found that DDX24 is enriched in muscles and its knockdown disrupts muscle fiber organization. This leads to defective pole specification, which in turn results in misregulation of many positional control genes specifically during regeneration. ddx24 RNAi also upregulates wound-induced Wnt signalling. Suppressing this ectopic Wnt activity rescues the knockdown phenotype by enabling better anterior pole regeneration. To summarize, our work highlights the role of an RNA helicase in muscle fiber organization, and modulating amputation-induced wnt levels, both of which seem critical for pole re-organization, thereby regulating whole-body regeneration.

Effects of large gradient high magnetic field (LG-HMF) on the long-term culture of aquatic organisms: Planarians example.

Large gradient high magnetic field (LG-HMF) is a powerful tool to study the effects of altered gravity on organisms. In our study, a platform for the long-term culture of aquatic organisms was designed based on a special superconducting magnet with an LG-HMF, which can provide three apparent gravity levels (µ g, 1 g, and 2 g), along with a control condition on the ground. Planarians, Dugesia japonica, were head-amputated and cultured for 5 days in a platform for head reconstruction. After planarian head regeneration, all samples were taken out from the superconducting magnet for a behavioral test under geomagnetic field and normal gravity conditions. To analyze differences among the four groups, four aspects of the planarians were considered, including head regeneration rate, phototaxis response, locomotor velocity, and righting behavior. Data showed that there was no significant difference in the planarian head regeneration rate under simulated altered gravity. According to statistical analysis of the behavioral test, all of the groups had normal functioning of the phototaxis response, while the planarians that underwent head reconstruction under the microgravity environment had significantly slower locomotor velocity and spent more time in righting behavior. Furthermore, histological staining and immunohistochemistry results helped us reveal that the locomotor system of planarians was affected by the simulated microgravity environment. We further demonstrated that the circular muscle of the planarians was weakened (hematoxylin and eosin staining), and the epithelial cilia of the planarians were reduced (anti-acetylated tubulin staining) under the simulated microgravity environment. Bioelectromagnetics. 2018;39:428-440. © 2018 Wiley Periodicals, Inc.

Morphogenic Effect of Exogenous Glucocorticoid Hormones in the Girardia tigrina Planarian (Turbellaria, Tricladida).

We have studied the effect of two glucocorticoid hormones: hydrocortisone and its synthetic analogue methylprednisolone on the regeneration activity of head and tail blastema of the Girardia tigrina planarian. The regeneration activity was studied in head and tail blastema formed after resection by means of lifetime computer morphometry and immunohistochemical labeling of neoblasts. The search for orthologous proteins-glucocorticoid receptors (hydrocortisone) was performed using the SmedGD database of the Schmidtea mediterranea planarian. The results indicate that both hormones influence the recovery rate of the regenerating head and tail blastema. The worms with regenerating tail blastema have less sensitivity to the hormones' treatment compared to the ones with regenerating head blastema. Hydrocortisone at a high concentration (10-3 M) suppressed the regeneration rate, while stimulating it at lower concentrations (10-4-10-6 M). The same concentrations of methylprednisolone inhibited the regeneration of head blastema, but did not affect the tail blastema regeneration. The two hormones acted differently: while hydrocortisone stimulated the proliferation of neoblasts in the periwound region, methylprednisolone reduced the mitotic activity, mainly on the tail zone furthest from the wound surface. We suggest that exogenous glucocorticoids can influence endogenous mechanisms of hormone-dependent regeneration.

Schmidtea happens: Re-establishing the planarian as a model for studying the mechanisms of regeneration.

Understanding the remarkable regenerative abilities of freshwater planarians was a classic problem of developmental biology. These animals were widely studied until the late 1960s, when their use as experimental subjects declined precipitously after some infamous experiments on memory transfer. By the mid-1990s, only a handful of laboratories worldwide were investigating the mechanisms of planarian regeneration. Here, we provide the personal stories behind our work to reinvigorate studies of these fascinating animals. We recount many of the challenges that had to be overcome and reflect on some of the fortuitous events that helped launch the planarian Schmidtea mediterranea as a model organism for studying the molecular basis of regeneration.

Gamma-Secretase Inhibitor (DAPT), a potential therapeutic target drug, caused neurotoxicity in planarian regeneration by inhibiting Notch signaling pathway.

DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester) is a γ-secretase inhibitor that indirectly blocks the activity of Notch pathway. It is a potential therapeutic target drug for many diseases, such as cancer, neurological, cardiovascular, and cerebrovascular diseases. However, the pharmacological action and specific mechanisms of DAPT are not clear. Planarians have strong regenerative capacity and can regenerate a new individual with a complete nervous system in one week. Thus, they are used as an ideal indicator of environmental toxicants and a novel model for studying neurodevelopmental toxicology. In this study, different concentrations and treatment times of DAPT are used to analyze the gene expression levels of major components in Notch pathway. The results show that the optimal concentration and exposure time of DAPT is 100 nM for 10 days in planarians and indicate that the inhibitory of DAPT treatment on Notch pathway is time- and concentration-dependent. The potential impact of DAPT is effectively analyzed by qPCR, WISH, and Immunofluorescence. The results indicate that DAPT exposure causes intact planarian wavy or swollen, and regenerative planarians asymmetric growth or muti-eye. Moreover, DAPT exposure increases cell proliferation and apoptosis, results in neurodevelopmental defects and dynamic changes of some marker genes. These results suggest that the balance of proliferation and apoptosis is disturbed, and then, affecting tissue homeostasis and differentiation. These findings demonstrate that DAPT has serious side effects in organisms and relies on Notch pathway to determine cell fate, it is cautious in the use of DAPT as a potential therapeutic approach for the disease in clinical trials.

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 ß-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).

Tissue absence initiates regeneration through follistatin-mediated inhibition of activin signaling.

Regeneration is widespread, but mechanisms that activate regeneration remain mysterious. Planarians are capable of whole-body regeneration and mount distinct molecular responses to wounds that result in tissue absence and those that do not. A major question is how these distinct responses are activated. We describe a follistatin homolog (Smed-follistatin) required for planarian regeneration. Smed-follistatin inhibition blocks responses to tissue absence but does not prevent normal tissue turnover. Two activin homologs (Smed-activin-1 and Smed-activin-2) are required for the Smed-follistatin phenotype. Finally, Smed-follistatin is wound-induced and expressed at higher levels following injuries that cause tissue absence. These data suggest that Smed-follistatin inhibits Smed-Activin proteins to trigger regeneration specifically following injuries involving tissue absence and identify a mechanism critical for regeneration initiation, a process important across the animal kingdom. DOI:http://dx.doi.org/10.7554/eLife.00247.001.

Cytophotometric evidence for cell 'transdifferentiation' in planarian regeneration.

The role and fate of male germ cells in planarian regeneration was studied in a population ofDugesia lugubris s.1. which provided a suitable karyological marker to distinguish diploid male germ cells from triploid embryonic and somatic cells. The nuclear Feulgen-DNA content in non-replicating triploid muscle cells of the pharynx and in non-replicating male gonia of testes from intact animals were measured by the cytophotometric technique. The pharynx was then removed by transection and each anterior regenerant was allowed to completely regenerate this organ. Measurements of the Feulgen-DNA content in muscle cells of the regenerated pharynx showed that most of these cells (∼95%) have a DNA content typical of triploid cells; however, some muscle cells (∼5%) with a nuclear DNA content typical of male gonia alone were observed.These results were interpreted in the following way. After transection, young male germ cells move from the testes to the wound where they participate in blastema formation along with reserve and/or somatic dedifferentiated cells. During regeneration some of these cells of male origin differentiate into pharyngeal muscle cells. Our findings are discussed in relation to the occurrence of mataplasia in planarians.