A powerful and versatile new fixation protocol for immunohistology and in situ hybridization that preserves delicate tissues in planaria

Whole-mount in situ hybridization (WISH) is a powerful and widely used technique to visualize the expression pattern of genes in different biological systems. Here we describe a new protocol for ISH and immunostaining in the planarian Schmidtea mediterranea. The new Nitric Acid/Formic Acid (NAFA) protocol is compatible with both assays and prevents degradation of the epidermis or blastema. Instead of proteinase K digestion, formic acid treatment is used to permeabilize tissues and preserve antigen epitopes. We show that the NAFA protocol successfully permits development of chromogenic and fluorescent signals in situ, while preserving the anatomy of the animal. Further, the immunostaining of different proteins was compatible with the NAFA protocol following fluorescent in situ hybridization. Finally, we demonstrate with high resolution confocal imaging that the regeneration blastema is preserved when using the new method. This new NAFA protocol will be a valuable technique to study the process of wounding response and regeneration.

Intestine-enriched apolipoprotein b orthologs regulate stem cell differentiation and regeneration in planarians

Little is known about how lipid mobilization and utilization are modulated during stem-cell-driven tissue growth during regeneration. Planarian flatworms can regenerate all missing tissues in 10 days due to the proliferation and differentiation of pluripotent somatic stem cells called neoblasts. In planarians, diet-derived neutral lipids are stored in the intestine. Here, we identify two intestine-enriched paralogs of apolipoprotein b, apob-1 and apob-2, that are required for regeneration. Consistent with apolipoproteins' known roles regulating neutral lipid (NL) transport in lipoprotein particles (LPs), NLs increased in the intestine upon simultaneous dsRNA-mediated knockdown of apob-1 and apob-2, but were depleted in neoblasts and their progeny. apob knockdown reduced regeneration blastema morphogenesis, and delayed re-establishment of axial polarity and regeneration of multiple organs. Using flow cytometry, we found that neoblast progeny accumulated in apob(RNAi) animals, with minimal effects on neoblast maintenance or proliferation. In addition, ApoB reduction primarily dysregulated expression of transcripts enriched in neoblast progeny and mature cell types, compared to cycling neoblasts. Together, our results provide evidence that intestine-derived lipids serve as a source of metabolites required for neoblast differentiation. In addition, these findings demonstrate that planarians are a tractable model for elucidating specialized mechanisms by which lipid metabolism must be regulated during animal regeneration.

Calcineurin controls proximodistal blastema polarity in zebrafish fin regeneration

Planarian flatworms regenerate their heads and tails from anterior or posterior wounds and this regenerative blastema polarity is controlled by Wnt/β-catenin signaling. It is well known that a regeneration blastema of appendages of vertebrates such as fish and amphibians grows distally. However, it remains unclear whether a regeneration blastema in vertebrate appendages can grow proximally. Here, we show that a regeneration blastema in zebrafish fins can grow proximally along the proximodistal axis by calcineurin inhibition. We used fin excavation in adult zebrafish to observe unidirectional regeneration from the anterior cut edge (ACE) to the posterior cut edge (PCE) of the cavity and this unidirectional regeneration polarity occurs as the PCE fails to build blastemas. Furthermore, we found that calcineurin activities in the ACE were greater than in the PCE. Calcineurin inhibition induced PCE blastemas, and calcineurin hyperactivation suppressed fin regeneration. Collectively, these findings identify calcineurin as a molecular switch to specify the PCE blastema of the proximodistal axis and regeneration polarity in zebrafish fin.

Regeneration of the flatworm Prosthiostomum siphunculus (Polycladida, Platyhelminthes)

Fueled by the discovery of head regeneration in triclads (planarians) two and a half centuries ago, flatworms have been the focus of regeneration research. But not all flatworms can regenerate equally well and to obtain a better picture of the characteristics and evolution of regeneration in flatworms other than planarians, the regeneration capacity and stem cell dynamics during regeneration in the flatworm order Polycladida are studied. Here, we show that as long as the brain remained at least partially intact, the polyclad Prosthiostomum siphunculus was able to regenerate submarginal eyes, cerebral eyes, pharynx, intestine and sucker. In the complete absence of the brain only wound closure was observed but no regeneration of missing organs. Amputated parts of the brain could not be regenerated. The overall regeneration capacity of P. siphunculus is a good fit for category III after a recently established system, in which most polyclads are currently classified. Intact animals showed proliferating cells in front of the brain which is an exception compared with most of the other free-living flatworms that have been observed so far. Proliferating cells could be found within the regeneration blastema, similar to all other flatworm taxa except triclads. No proliferation was observed in epidermis and pharynx. In pulse-chase experiments, the chased cells were found in all regenerated tissues and thereby shown to differentiate and migrate to replace the structures lost upon amputation.

No head regeneration here: regeneration capacity and stem cell dynamics of Theama mediterranea (Polycladida, Platyhelminthes)

Research on the regeneration potential of flatworms (Platyhelminthes) has been mainly undertaken with planarians (Tricladida), where most species can regenerate a head and no proliferation takes place in the blastema, i.e. the early undifferentiated regenerative tissue. Only few studies are available for an early-branching group within the Platyhelminthes, the Polycladida. Head regeneration in polyclads is not possible, with a single exception from a study performed more than 100 years ago: Cestoplana was reported to be able to regenerate a head if cut a short distance behind the brain. Here, we show that ‘Cestoplana’ was misdetermined and most likely was the small interstitial polyclad Theama mediterranea. We revisited regeneration capacity and dynamics of T. mediterranea with live observations and stainings of musculature, nervous system, and proliferating and differentiating stem cells. In our experiments, after transversal amputation, only animals retaining more than half of the brain could fully restore the head including the brain. If completely removed, the brain was never found to regenerate to any extent. Different from planarians, but comparable to other free-living flatworms we detected cell proliferation within the posterior regeneration blastema in T. mediterranea. Similar to other free-living flatworms, proliferation did not occur within, but only outside, the differentiating organ primordia. Our results strongly imply that brain regeneration in the absence of the latter is not possible in any polyclad studied so far. Also, it appears that proliferation of stem cells within the regeneration blastema is a plesiomorphy in flatworms and that planarians are derived in this character.

Understanding the Role of the Clitellum in the Regeneration Events of the Earthworm Eudrilus eugeniae

Regeneration is a complex mechanism to restore lost or damaged body parts. In earthworms, regeneration capability varies among different species, and it is important to explore the mechanism behind the regeneration process. Interestingly, regeneration in earthworms is either dependent or independent of clitellum segments. In the present study, juvenile earthworms (Eudrilus eugeniae) were amputated at 3 different sites, namely the head, clitellum, and tail segments (at segments 10, 15, and 30, respectively), and their regeneration ability was documented using a foldscope. The amputated segments having the intact clitellum were able to heal the wounds and form the regenerative blastema. The smaller portions of the amputated segments (segments 1–10 and 1–15) without intact clitellum were unable to heal the wound, and death occurs within 12–24 h. The larger portions of the amputated segments (segments 15 and 30 to anus) without intact clitellum were able to heal the wound but lacked the regeneration capability. In control worms, alkaline phosphatase (ALP) signals were observed at the anterior tip, clitellum, and gut epithelium tissues, whereas, upon amputation, the enriched signals from the clitellum diminished, but profound signals were observed at the amputation site and regenerative blastema. Interestingly, on days 3 and 4, blastemal tips lacked ALP signals due to initiation of the differentiation process in the regeneration blastema. In summary, using a foldscope microscope, the role of the clitellum in the regeneration mechanism was indicated by ALP activity.

Morphological, cellular and molecular characterization of posterior regeneration in the marine annelid Platynereis dumerilii

ABSTRACTRegeneration, the ability to restore body parts after an injury or an amputation, is a widespread but highly variable and complex phenomenon in animals. While having fascinating scientists for centuries, fundamental questions about the cellular basis of animal regeneration as well as its evolutionary history remain largely unanswered. We study regeneration of the marine annelid Platynereis dumerilii, an emerging comparative developmental biology model, which, like many other annelids, displays important regenerative abilities. If the posterior part of the body is amputated, P. dumerilii worms are able to regenerate the posteriormost differentiated part of the body and stem cell-rich growth zone that allows to make new segments which replace the amputated ones. We show that posterior regeneration is a rapid process that follows a well reproducible paths and timeline, going through specific stages that we thoroughly defined. Wound healing is achieved by one day post-amputation and a regeneration blastema forms one day later. At this time point, some tissue specification already occurs, and a functional posterior growth zone is re-established as early as three days after amputation. Regeneration is only influenced in a minor manner by worm size and position of the amputation site along the antero-posterior axis of the worm and regenerative abilities persist upon repeated amputations without important alterations of the process. We also show that intense cell proliferation occurs during regeneration and that cell divisions are strictly required for regeneration to normally proceed. Finally, through several 5-ethynyl-2’-deoxyuridine (EdU) pulse and chase experiments, we provide evidence in favor of a local origin of the blastema, whose constituting cells mostly derive from the segment immediately abutting the amputation plane. The detailed characterization of P. dumerilii posterior body regeneration presented in this article provides the foundation for future mechanistic and comparative studies of regeneration in this species.

Macrophages are necessary for epimorphic regeneration in African spiny mice

How the immune system affects tissue regeneration is not well understood. In this study, we used an emerging mammalian model of epimorphic regeneration, the African spiny mouse, to examine cell-based inflammation and tested the hypothesis that macrophages are necessary for regeneration. By directly comparing inflammatory cell activation in a 4 mm ear injury during regeneration (Acomys cahirinus) and scarring (Mus musculus), we found that both species exhibited an acute inflammatory response, with scarring characterized by stronger myeloperoxidase activity. In contrast, ROS production was stronger and more persistent during regeneration. By depleting macrophages during injury, we demonstrate a functional requirement for these cells to stimulate regeneration. Importantly, the spatial distribution of activated macrophage subtypes was unique during regeneration with pro-inflammatory macrophages failing to infiltrate the regeneration blastema. Together, our results demonstrate an essential role for inflammatory cells to regulate a regenerative response.

Epigenetics and Shared Molecular Processes in the Regeneration of Complex Structures

The ability to regenerate complex structures is broadly represented in both plant and animal kingdoms. Although regenerative abilities vary significantly amongst metazoans, cumulative studies have identified cellular events that are broadly observed during regenerative events. For example, structural damage is recognized and wound healing initiated upon injury, which is followed by programmed cell death in the vicinity of damaged tissue and a burst in proliferation of progenitor cells. Sustained proliferation and localization of progenitor cells to site of injury give rise to an assembly of differentiating cells known as the regeneration blastema, which fosters the development of new tissue. Finally, preexisting tissue rearranges and integrates with newly differentiated cells to restore proportionality and function. While heterogeneity exists in the basic processes displayed during regenerative events in different species—most notably the cellular source contributing to formation of new tissue—activation of conserved molecular pathways is imperative for proper regulation of cells during regeneration. Perhaps the most fundamental of such molecular processes entails chromatin rearrangements, which prime large changes in gene expression required for differentiation and/or dedifferentiation of progenitor cells. This review provides an overview of known contributions to regenerative processes by noncoding RNAs and chromatin-modifying enzymes involved in epigenetic regulation.