Research into dedifferentiation during epimorphic regeneration in Xenopus and Zebrafish
Year: 2013
Type: dissertation
Abstract: Humans, as every mammalian animal, have a limited capacity to repair or regenerate damaged body parts. In contrast, certain amphibian species possess extraordinary regenerative capacities that, for example, allow them to reform severed appendages such as limbs and tails. The phenomenon of appendage regeneration in vertebrate animals, formally called epimorphic regeneration, has attracted the attention of scientists for centuries. Understanding which factors are behind the capacity to restore a lost or severely injured body part in animal models has obvious implications to human health. Cellular, genetic or chemical therapies are some of the procedures that already are or may soon be applied to human patients to ameliorate conditions related to diseased, injured or lost body structures. An immense research effort has been made into uncovering the molecular and cellular differences between lower, regenerating vertebrates, and the non-regenerating, higher vertebrates. One of the main differences resides in the formation of the blastema, a specialized structure at the tip of the stump that will grow in size and reform the lost appendage. This structure contains highly proliferative stem-like cells that migrated from the tissues in the distal part of the stump. Mammals generally fail to form a blastema, while in most amphibians and fish, a blastema starts to form right after wound healing. The process of blastema formation relies on the activation of adult stem cells and, in most cases, in the dedifferentiation of mature cells. Cellular dedifferentiation is characterized by the loss of differentiation or specialization characteristics and by the acquisition of stem cell features such as proliferation and migration. In short, differentiated cells return to a stem-like state, what gives them the capacity to contribute to the blastema. The presence of stem cells in a tissue and/or the ability to dedifferentiate mature cells is generally associated with the capacity to regenerate. Skin, bone and skeletal muscle tissues can regenerate independently (to some extent) in mammalian animals because these tissues possess a population of adult stem cells that proliferates and substitutes the damaged cells in each tissue. On the other hand, a severed limb does not regenerate, although it is mainly formed by these same tissues. Several scientists believe that mammals and many other animals do not undergo epimorphic regeneration because their cells do not have the capacity to dedifferentiate in vivo, failing thus to form a blastema. Accordingly, various studies have shown that mammalian cells in culture are much harder to dedifferentiate than amphibian cells, but still do, what indicates that it could be possible to induce dedifferentiation in mammalians and, maybe, stimulate limb regeneration in the process. In this thesis I studied the phenomenon of epimorphic regeneration in two species of aquatic animals, the frog Xenopus laevis and the “zebrafish” Danio rerio. I focused on the detection of dedifferentiation in Xenopus and zebrafish, and on the possibility to enhance Xenopus limb regeneration through the induction of dedifferentiation. The choice to work with Xenopus was based on the fact that Xenopus larvae (tadpoles) gradually lose the capacity to regenerate their limbs during larval development and metamorphosis, in a somewhat analogous way to what happens in mammalian embryos during gestation. Moreover, Xenopus and mammals apparently share the incapacity to induce dedifferentiation during regeneration. This puts Xenopus laevis closer to mammalian animals in respect to the regenerative capacity, making it a very interesting choice to study and test ways to improve the regenerative capacity in mammals. On the other hand, zebrafish was studied because this animal model allows the quick creation of transgenic lines and, at the same time, it has a great regenerative ability. Since one of the main tissues contributing to limb or tail blastemas is the skeletal muscle, I centred most effort on the study of this tissue. Skeletal muscle is mainly composed of elongated cells called muscle fibres, or myofibres, that form during development and regeneration by the fusion of multiple myogenic progenitor cells known as myoblasts. For this reason, myofibres are multinucleated, or syncytial, cells. Muscle fibres are also characterized by a highly organized internal structure, necessary for proper contraction of the muscle. Microscopically, this complex internal structure is built up by aligned proteins that create the characteristic striated pattern of the muscle. Early studies in urodele amphibians (salamanders, the “champions” of regeneration) showed that the striated pattern of limb muscle is lost during early stages of regeneration, an indicative feature of muscle dedifferentiation. Further studies strongly suggested that limb and tail muscle fibres were able to fragment and that the resulting mononuclear cells proliferated and contributed to the blastema. Along with the described muscle dedifferentiation, also muscle stem cells (satellite cells) were recently shown to contribute to the new muscle during regeneration. In contrast to what is described in urodeles, recent studies on Xenopus tail regeneration revealed that muscle fibres do not contribute to the blastema, strongly suggesting the absence of muscle dedifferentiation. Instead, muscle satellite cells were shown to be the main contributor to the new tail muscle. The absence of dedifferentiation during Xenopus regeneration could explain why these animals gradually lose their limb regenerative capacity while limb cells differentiate. In my thesis I examined the relation between dedifferentiation and regenerative capacity of Xenopus, dividing this study in two main projects. The first project was intended to confirm if dedifferentiation was in fact absent from limb regeneration, through the use of diverse assessment techniques. In the second project I tested several methods expected to induce dedifferentiation and, possibly also, to enhance limb regeneration. To confirm the absence of (muscle) dedifferentiation during limb regeneration I tested different cell labelling techniques to trace muscle fibres during regeneration (the best technique would be also used for the second project), analysed the ultrastructure of regenerating limbs, and measured the expression levels of some muscle specific genes. Although no cell labelling technique worked well enough to confirm the absence of dedifferentiation, histological and gene expression analysis showed no clear evidences of limb muscle dedifferentiation. On the other hand, tail muscle regeneration, which was initially meant to serve only as a negative control for dedifferentiation, surprisingly revealed dedifferentiation phenotypes both histologically and by gene expression analysis. Nevertheless, I concluded that this dedifferentiation was incomplete since it neither resulted in the cell cycle re-entry of myonuclei nor in the fragmentation of labelled muscle fibres. Consequently, no labelled myofibres contributed with nuclei to the regenerated tail muscle. Similar observations were made in the tail of zebrafish larvae. Labelled muscle fibres did not contribute to the regenerated muscle and, on contrary to tadpole tail, no signs of histological dedifferentiation were found. The complete absence of dedifferentiation on zebrafish tail muscle revealed that skeletal muscle dedifferentiation is less common than previously expected in lower vertebrates. The in vivo tracing of labelled muscle fibres in the tadpole tail also evidenced a relation between the histological dedifferentiation phenotype and the retraction of muscle fibres upon amputation. Both phenotypes were mostly present in a specific type of muscle fibre and after amputation at a certain level, shared similar myofibre shapes or morphologies, and gradually disappeared with time. Importantly, the absence of both phenotypes in a more proximal level of amputation indicates that the observed “incomplete” dedifferentiation is not necessary for muscle regeneration. In addition, the apparent cause-effect relation between retraction and dedifferentiation suggests that dedifferentiation is a stress- or damage-related response and, not necessarily, regeneration-related. In the second project I investigated the possibility to stimulate Xenopus limb regeneration through the induction of dedifferentiation. This idea was not new and had previously given few positive results, although no clear proof of dedifferentiation induction was presented. I tested various strategies, from chemical treatments to transgene overexpression, but they were mostly unsuccessful. A few treatments showed a tendency to improve limb regeneration but I was not able to verify if dedifferentiation was indeed induced, since the cell labelling techniques from the first project were ineffective in the limbs. In a complementary study I analysed the expression pattern of bmp2, 4 and 7 genes during limb development and regeneration and found a clear relation between bmp expression and successful regeneration.
Language: Portuguese
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W15635017