Manuel Marí-Beffa

Old questions, new tools, and some answers to the mystery of fin regeneration†

Pluridisciplinary approaches led to the notion that fin regeneration is an intricate phenomenon involving epithelial–mesenchymal and reciprocal exchanges throughout the process as well as interactions between ray and interray tissue. The establishment of a blastema after fin amputation is the first event leading to the reconstruction of the missing part of the fin. Here, we review our knowledge on the origin of the blastema, its formation and growth, and of the mechanisms that control differentiation and patterning of the regenerate. Our current understanding results from studies of fin regeneration performed in various teleost fish over the past century. We also report the recent breakthroughs that have been made in the past decade with the arrival of a new model, the zebrafish, Danio rerio, which now offers the possibility to combine cytologic, molecular, and genetic analyses and open new perspectives in this field. Developmental Dynamics 226:190–201, 2003.

The Stem Cell Niche Should be a Key Issue for Cell Therapy in Regenerative Medicine

Recent advances in stem cell research have highlighted the role played by such cells and their environment (the stem cell niche) in tissue renewal and homeostasis. The control and regulation of stem cells and their niche are remaining challenges for cell therapy and regenerative medicine on several tissues and organs. These advances are important for both, the basic knowledge of stem cell regulation, and their practical translational applications into clinical medicine. This article is primarily concerned with the mesenchymal stem cells (MSCs) and it reviews the current aspects of their own niche. We discuss on the need for a deeper understanding of the identity of this cell type and its microenvironment in order to improve the effectiveness of any cell therapy for regenerative medicine. Ex vivo reproduction of the conditions of the natural stem cell niche, when necessary, would provide success to tissue engineering. The first challenge of regenerative medicine is to find cells able to replace and/or repair the lost function of tissues and organs by disease or aging and the trophic and immunomodulatory effects recently found for MSCs open up for new opportunities. If MSCs are pericytes, as it has been proposed, perhaps it may explain the ubiquity of these cells and their possible role in miscellaneous repairs throughout the body opening for new chances for extensive tissue repair.

Actinotrichia collagens and their role in fin formation.

The skeleton of zebrafish fins consists of lepidotrichia and actinotrichia. Actinotrichia are fibrils located at the tip of each lepidotrichia and play a morphogenetic role in fin formation. Actinotrichia are formed by collagens associated with non-collagen components. The non-collagen components of actinotrichia (actinodins) have been shown to play a critical role in fin to limb transition. The present study has focused on the collagens that form actinotrichia and their role in fin formation. We have found actinotrichia are formed by Collagen I plus a novel form of Collagen II, encoded by the col2a1b gene. This second copy of the collagen II gene is only found in fishes and is the only Collagen type II expressed in fins. Both col1a1a and col2a1b were found in actinotrichia forming cells. Significantly, they also expressed the lysyl hydroxylase 1 (lh1) gene, which encodes an enzyme involved in the post-translational processing of collagens. Morpholino knockdown in zebrafish embryos demonstrated that the two collagens and lh1 are essential for actinotrichia and fin fold morphogenesis. The col1a1 dominant mutant chihuahua showed aberrant phenotypes in both actinotrichia and lepidotrichia during fin development and regeneration. These pieces of evidences support that actinotrichia are composed of Collagens I and II, which are post-translationally processed by Lh1, and that the correct expression and assembling of these collagens is essential for fin formation. The unique collagen composition of actinotrichia may play a role in fin skeleton morphogenesis.

Differential regulation of msx genes in the development of the gonopodium, an intromittent organ, and of the ''sword,'' a sexually selected trait of swordtail fishes (Xiphophorus)

Summary The possession of a conspicuous extension of colored ventral rays of the caudal fin in male fish of swordtails (genus Xiphophorus) is a prominent example for a trait that evolved by sexual selection. To understand the evolutionary history of this so‐called sword molecularly, it is of interest to unravel the developmental pathways responsible for extended growth of sword rays during development of swordtail males. We isolated two msx genes and showed that they are differentially regulated during sword outgrowth. During sword growth in juvenile males, as well as during testosterone‐induced sword development and fin ray regeneration in the sword after amputation, expression of msxC is markedly up‐regulated in the sword forming fin rays. In contrast, msxE/1 is not differentially expressed in ventral and dorsal male fin rays, suggesting a link between the development of male secondary sexual characters in fins and up‐regulation of msxC expression. In addition, we showed that msx gene expression patterns differ significantly between Xiphophorus and zebrafish. We also included in our study the gonopodium, a testosterone‐dependent anal fin modification that serves as a fertilization organ in males of live‐bearing fishes. Our finding that increased levels of msxC expression are associated with the testosterone‐induced outgrowth of the gonopodium might suggest either that at least parts of the signaling pathways that pattern the evolutionary older gonopodium have been coopted to evolve a sexually selected innovation such as the sword or that increased msxC expression may be inherent to the growth process of long fin rays in general.

Dimethylfumarate Inhibits Angiogenesis In Vitro and In Vivo: A Possible Role for Its Antipsoriatic Effect?

The fumaric acid esters (FAEs) have been used for the oral treatment of psoriasis for some 50 years. Given that a persistent and maintained angiogenesis is associated with several cutaneous diseases, including psoriasis, we sought in our study to gain further insight into their mechanism of action by investigating whether FAEs are able to interfere with angiogenesis mechanisms. Our results demonstrate that dimethylfumarate (DMF) inhibits certain functions of endothelial cells, namely, differentiation, proliferation, and migration. This activity was not exhibited by similar concentrations of monomethylfumarate or fumaric acid. Our data indicate that DMF inhibits the growth of transformed and nontransformed cells in a dose-dependent manner. The growth-inhibitory effect exerted by this compound on proliferating endothelial cells could be due, at least in part, to an induction of apoptosis. Inhibition by DMF of the mentioned essential steps of in vitro angiogenesis is consistent with the observed inhibition of in vivo angiogenesis, substantiated using chick chorioallantoic membrane and live fluorescent zebrafish embryo neovascularization assays. The antiangiogenic activity of DMF may contribute to the antipsoriatic, antitumoral, and antimetastatic activities of this compound and suggests its potential in the treatment of angiogenesis-related malignancies.

Zebrafish fins as a model system for skeletal human studies.

Recent studies on the morphogenesis of the fins of Danio rerio (zebrafish) during development and regeneration suggest that a number of inductive signals involved in the process are similar to some of those that affect bone and cartilage differentiation in mammals and humans. Akimenko et al. (2002) has shown that bone morphogenetic protein-2b (BMP2b) is involved in the induction of dermal bone differentiation during fin regeneration. Many other groups have also shown that molecules from the transforming growth factor-beta superfamily (TGFβ), including BMP2, are effective in promoting chondrogenesis and osteogenesis in vivo in higher vertebrates, including humans. In the present study, we review the state of the art of this topic by a comparative analysis of skeletal tissue development, regeneration and renewal processes in tetrapods, and fin regeneration in fishes. A general conclusion of this study states that lepidotrichia is a special skeletal tissue different to cartilage, bone, enamel, or dentine in fishes, according to its extracellular matrix (ECM) composition. However, the empirical analysis of inducing signals of skeletal tissues in fishes and tetrapods suggests that lepidotrichia is different to any responding features with main skeletal tissues. A number of new inductive molecules are arising from fin development and regeneration studies that might establish an empirical basis for further molecular approaches to mammal skeletal tissues differentiation. Despite the tissue dissimilarity, this empirical evidence might finally lead to clinical applications to skeletal disorders in humans.

Dermoskeleton morphogenesis in zebrafish fins

Zebrafish fins have a proximal skeleton of endochondral bones and a distal skeleton of dermal bones. Recent experimental and genetic studies are discovering mechanisms to control fin skeleton morphogenesis. Whereas the endochondral skeleton has been extensively studied, the formation of the dermal skeleton requires further revision. The shape of the dermal skeleton of the fin is generated in its distal growing margin and along a proximal growing domain. In these positions, dermoskeletal fin morphogenesis can be explained by intertissue interactions and the function of several genetic pathways. These pathways regulate patterning, size, and cell differentiation along three axes. Finally, a common genetic control of late development, regeneration, and tissue homeostasis of the fin dermoskeleton is currently being analyzed. These pathways may be responsible for the similar shape obtained after each morphogenetic process. This provides an interesting conceptual framework for future studies on this topic. Developmental Dynamics 239:2779–2794, 2010.

Morphometric study of the regeneration of individual rays in teleost tail fins

The results obtained using morphometric variables which describe fin ray regeneration patterns are reported for individual fin ray amputations in the goldfish (Carassius auratus) and zebrafish (Brachydanio rerio). Classical and updated experiments are compared to verify previous morphogenetic models of cell tractions (Oster et al. 1983) or epidermis‐mesenchyme induction (Saunders et al. 1959) applied to the limb of other vertebrates. Position‐dependent patterns within the fin of Carassius auratus are analysed under a comparative protocol using morphometric methods. Conditions in which the apical epidermis is separated from blastema may differentiate small fin rays, thus suggesting this epidermis is involved in blastemal formation. Blastemal cells differentiating as lepidotrichia forming cells (LFCs) may also be related to morphological changes in covering epidermis. Long‐range interactions from neighbouring fin ray blastemas or short‐range interactions within the blastema, may be postulated through the analysis of segmentation.

Toluquinol, a marine fungus metabolite, is a new angiosuppresor that interferes the Akt pathway

Toluquinol, a methylhydroquinone produced by a marine fungus, was selected in the course of a blind screening for new potential inhibitors of angiogenesis. In the present study we provide the first evidence that toluquinol is a new anti-angiogenic-compound. In a variety of experimental systems, representing the sequential events of the angiogenic process, toluquinol treatment of activated endothelial cells resulted in strong inhibitory effect. Toluquinol inhibited the growth of endothelial and tumor cells in culture in the micromolar range. Our results indicate that the observed growth inhibitory effect could be due, at least in part, to an induction of apoptosis. Toluquinol induced endothelial cell death is mediated via apoptosis after a cell cycle block and caspase activation. Capillary tube formation on Matrigel and migratory, invasive and proteolytic capabilities of endothelial cells were inhibited by addition of toluquinol at subtoxic concentrations. Inhibition of the mentioned essential steps of in vitro angiogenesis agrees with the observed inhibition of the in vivo angiogenesis, substantiated by using the chick chorioallatoic membrane assay and confirmed by the murine Matrigel plug, the zebrafish embryo neovascularization and the zebrafish caudal fin regeneration assays. Data here shown altogether indicate that toluquinol has antiangiogenic effects both in vitro and in vivo that are exerted partly by suppression of the VEGF and FGF-induced Akt activation of endothelial cells. These effects are carried out at lower concentrations to those required for other inhibitors of angiogenesis, what makes toluquinol a promising drug candidate for further evaluation in the treatment of cancer and other angiogenesis-related pathologies.

Freeze substitution followed by low melting point wax embedding preserves histomorphology and allows protein and mRNA localization techniques.

Fixation and embedding are major steps in tissue preservation for histological analysis. However, conventional fixatives like aldehyde‐based solutions usually mask tissular epitopes preventing their immunolocalization. Alternative fixation methods used to avoid this drawback, such as cryopreservation, alcohol‐ or zinc salts‐based fixatives do not efficiently preserve tissue and cell morphology. Likewise, paraffin and resin embedding, commonly used for thin sectioning, frequently damage epitopes due to the clearing agents and high temperatures needed along the embedding procedure. Alternatives like cryosectioning avoid the embedding steps but yield sections of poorer quality and are not suitable for all kinds of samples. To overcome these handicaps, we have developed a method that preserves histoarchitecture as well as tissue antigenic properties. This method, which we have named CryoWax, involves freeze substitution of the samples in isopentane and methanol, followed by embedding in low melting point polyester wax. CryoWax has proven efficient in obtaining thin sections of embryos and adult tissues from different species, including amphioxus, zebrafish, and mouse. CryoWax sections displayed optimal preservation of tissue morphology and were successfully immunostained for fixation‐ and temperature‐sensitive antigens. Furthermore, CryoWax has been tested for in situ hybridization application, obtaining positive results. Microsc. Res. Tech., 2011.