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Studying the Molecular Mechanisms for Generating Progenitor Cells during Tail Regeneration in Ambystoma mexicanum / Studien der molekularen Mechanismen zur Herstellung von Vorläuferzellen während der Schwanzregeneration in Ambystoma mexicanumSchnapp, Esther 10 May 2005 (has links) (PDF)
The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.
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Development of transgenic Ambystoma mexicanum (axolotl) to study cell fate during development and regenerationSobkow, Lidia 18 April 2006 (has links) (PDF)
The establishment of transgenesisi in axolotls is crucial for studying development and regeneration, as it would allow for long-term fate tracing as well as gene expression analysis, therefore we were interested in both obtaining animals expresing the transgene with little mosaicism in F0 generation and transgenesis. We demonstrate here that plasmid injection into one cell stage axolotl embryo generates transgenic animals that display germline transmission of a transgene. However, the efficiency of simple plasmid injection is very low, expression of the transgene is mosaic and seems to be promoter dependant. We have tested several methods of transgenesis developed in other systems. First we used Adeno-Associated Viral Terminal Repeats inserted into the injected construct to enhance the expression level of the transgene and reduce mosaicism. However, in the axolotl system we do not observe the enhancement of expression. Moreover, the expression appeared to be transient and disappeared after two months. Further, we tested the effect of the inclusion of ISceI meganuclease in the injections, succesful transgenesis method in the medaka system. It resulted in a higher percentage of F0 animals displaying strong , stable expression throughout the body. This represents the first demonstration in the axolotl of germline transmission of the transgene. Using this technique we have generated a germline transgenic anima expressing GFP ubiquitously in all tissue examined. We have used this anima to study cell fate in the dirsal fin during development. We have discovered a contribution of somite cells to dorsal fin mesenchyme in the axolotl, which was previously assumed to derive solely from neural crest. We have also studied the role of blood during tail regeneration by transplanting the ventral blood-forming region from GFP+ embryos into unlabeled host. During tail regeneration, we do not observe GFP+ cells contributing to muscle or nerve, suggesting that during tail regeneration blood stem cells do not undergo significant plasticity. We are interested in characterization of pluripotency of blastema cells. Previously, it has been shown that neural progenitor cells form the spinal cord can transdifferentiate to muscle and other tissue types in the regenerating tail. To test if blastema cells have the potency of differentiating into a neural tissue , we transplanted GFP+ 4day blastema into an injured spinal cord. Our result shows that blastema cells don't seem to contribute to the regenerating spinal cord.
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Studying the Molecular Mechanisms for Generating Progenitor Cells during Tail Regeneration in Ambystoma mexicanumSchnapp, Esther 09 June 2005 (has links)
The present thesis is a contribution to unravel the molecular mechanisms that underlie urodele regeneration. Urodele amphibians (newts and salamanders) are among the few vertebrates with the remarkable ability to regenerate lost body appendages, like the limbs and the tail. Urodele tail and limb regeneration occurs via blastemal epimorphic regeneration. A blastema is a mound of progenitor cells that accumulates at the amputation plane and eventually gives rise to the missing structures. It is known today that dedifferentiating muscle fibers at the amputation plane contribute to the blastema cell pool, but how this process occurs on the cellular and molecular level is hardly understood, which is in part due to the lack of molecular methods to test gene function in urodeles. Furthermore, little is known about how coordinated growth and patterning occurs during urodele regeneration, and if the patterning mechanisms in regeneration are related to the ones in development. The goal of this study was to better understand these processes on the molecular level. To address these questions, I first established several methods in our model systems, which are the mexican salamander Ambystoma mexicanum (axolotl) and a cell line derived from the newt Notophthalmus viridescens. In order to monitor gene expression on a cellular level during regeneration, I worked out a good in situ hybridization protocol on axolotl tissue cryosections. To be able to test gene function, I established electroporation conditions to both overexpress genes in the cultured newt cells and to deliver morpholinos into axolotl cells in vivo and newt cells in culture. I demonstrate here that morpholinos are an effective tool to downregulate protein expression in urodele cells in vivo and in culture. Testing the role of two candidate genes in muscle fiber dedifferentiation, the homeobox containing transcription factor Msx1 and Rad, a GTP-binding protein of a new Ras-related protein family, revealed that neither seems to play a major role in muscle dedifferentiation, both in culture and in vivo. In addition to testing gene function I have examined the muscle dedifferentiation process in more detail. I show here that dedifferentiating muscle fiber nuclei undergo morphological changes that are likely due to chromatin remodeling events. I also demonstrate that the axolotl spinal cord expresses embryonic dorsoventral (d/v) patterning markers of the neural tube. The transcription factors Msx1, Pax7 and Pax6 are expressed in their respective d/v domains in both the differentiated and the regenerating axolotl spinal cord. Furthermore, the secreted signaling molecule sonic hedgehog (Shh) is expressed in the floor plate in both the differentiated and the regenerating cord. Using a chemical inhibitor (cyclopamine) and an activator of the hedgehog pathway, I discovered that hedgehog signaling is required for overall tail regeneration. Blocking hedgehog signaling does not only result in d/v patterning defects of the regenerating spinal cord, but it also strongly reduces blastema cell proliferation. In addition, I identified cartilage and putative muscle progenitor cells in the blastema, marked by the expression of the transcription factors Sox9 and Pax7, respectively. Both progenitor populations are reduced in the blastema in the absence of hedgehog signaling. The continuous expression of marker genes for embryonic progenitor cell domains in the mature axolotl may be related to their ability to regenerate.
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Development of transgenic Ambystoma mexicanum (axolotl) to study cell fate during development and regenerationSobkow, Lidia 03 May 2006 (has links)
The establishment of transgenesisi in axolotls is crucial for studying development and regeneration, as it would allow for long-term fate tracing as well as gene expression analysis, therefore we were interested in both obtaining animals expresing the transgene with little mosaicism in F0 generation and transgenesis. We demonstrate here that plasmid injection into one cell stage axolotl embryo generates transgenic animals that display germline transmission of a transgene. However, the efficiency of simple plasmid injection is very low, expression of the transgene is mosaic and seems to be promoter dependant. We have tested several methods of transgenesis developed in other systems. First we used Adeno-Associated Viral Terminal Repeats inserted into the injected construct to enhance the expression level of the transgene and reduce mosaicism. However, in the axolotl system we do not observe the enhancement of expression. Moreover, the expression appeared to be transient and disappeared after two months. Further, we tested the effect of the inclusion of ISceI meganuclease in the injections, succesful transgenesis method in the medaka system. It resulted in a higher percentage of F0 animals displaying strong , stable expression throughout the body. This represents the first demonstration in the axolotl of germline transmission of the transgene. Using this technique we have generated a germline transgenic anima expressing GFP ubiquitously in all tissue examined. We have used this anima to study cell fate in the dirsal fin during development. We have discovered a contribution of somite cells to dorsal fin mesenchyme in the axolotl, which was previously assumed to derive solely from neural crest. We have also studied the role of blood during tail regeneration by transplanting the ventral blood-forming region from GFP+ embryos into unlabeled host. During tail regeneration, we do not observe GFP+ cells contributing to muscle or nerve, suggesting that during tail regeneration blood stem cells do not undergo significant plasticity. We are interested in characterization of pluripotency of blastema cells. Previously, it has been shown that neural progenitor cells form the spinal cord can transdifferentiate to muscle and other tissue types in the regenerating tail. To test if blastema cells have the potency of differentiating into a neural tissue , we transplanted GFP+ 4day blastema into an injured spinal cord. Our result shows that blastema cells don't seem to contribute to the regenerating spinal cord.
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TRANSCRIPTIONAL AND MORPHOLOGICAL CHANGES DURING THYROXINE-INDUCED METAMORPHOSIS OF THE MEXICAN AXOLOTL AND AXOLOTL-TIGER SALAMANDER HYBRIDSPage, Robert Bryce 01 January 2009 (has links)
For nearly a century, amphibian metamorphosis has served as an important model of how thyroid hormones regulate vertebrate development. Consequently metamorphosis has been studied in a number of ways including: morphologically, developmentally, ecologically, and from an endocrine perspective. Over the last two decades, much has been learned about the molecular basis of anuran (frog) metamorphosis. However, very little is known about the molecular underpinnings of urodele (salamander) metamorphosis. Using the axolotl and axolotl hybrids as models, I present some of the first studies on the gene expression changes that occur during urodele metamorphosis. In Chapter 1, the motivation for the research described in the subsequent chapters is presented and the literature is briefly reviewed. In Chapter 2, the first microarray analysis of urodele metamorphosis is presented. This analysis shows that hundreds of genes are differentially expressed during thyroid hormone-induced metamorphic skin remodeling. Chapter 3 extends the analysis presented in Chapter 2 by showing that the transcriptional patterns associated with metamorphic skin remodeling are robust even when the concentration of thyroid hormone used to induce metamorphosis is varied by an order of magnitude. Chapter 4 makes use of the differentially expressed genes identified in Chapters 2 and 3 to articulate the first model of urodele metamorphosis to integrate changes in morphology, gene expression, and histology. In addition, Chapter 4 outlines a novel application for piecewise linear regression. In turn, Chapter 5 makes use of the model presented in Chapter 4 to demonstrate that full siblings segregating profound variation in metamorphic timing begin to diverge in phenotype early during larval development. In Chapter 6 the conclusions drawn from the research are summarized and future directions are suggested.
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Clonage et analyse de la protéine suppresseur de tumeur P53 chez l'axolotlVilliard, Éric January 2006 (has links)
Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.
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A Comparative Study of Head Development in Mexican Axolotl and Australian Lungfish: Cell Migration, Cell Fate and MorphogenesisEricsson, Rolf January 2003 (has links)
<p>The development of the vertebrate head is a complex process involving interactions between a multitude of cell types and tissues. This thesis describes the development of the cranial neural crest and of the visceral arch muscles in the head of two species. One, the Mexican axolotl (<i>Ambystoma mexicanum</i>), is a basal tetrapod, whereas the other, the Australian lungfish (<i>Neoceratodus forsteri</i>), belongs to the Dipnoi, the extant sister group of the Tetrapoda. </p><p>The migration of neural crest cells, which form most of the bones and connective tissues in the head, and the morphogenesis of the jaw, was determined in the Mexican axolotl. It was shown that both the upper and lower jaws form from ventral condensations of neural crest cells in the mandibular arch. The dorsal condensation, earlier considered to give rise to the upper jaw, was shown to form the trabecula cranii.</p><p>The normal spatio-temporal development of visceral arch muscles was investigated in both the Mexican axolotl and the Australian lungfish. In axolotl, the muscles tended to start forming almost simultaneously in all visceral arches at their future origins and extend towards their future insertions at the onset of muscle fibre formation. In lungfish, fibres formed simultaneously throughout most of each muscle anlage in the first and second visceral arch, but were delayed in the branchial arches. The anlagen were first observed at their future insertion, from which they developed towards future origins. </p><p>To test the ability of neural crest cells to pattern the visceral arch muscles, migrating crest cells were extirpated from axolotl embryos, which resulted in a wide range of muscle malformations. In most cases, the muscles appeared in the right position but were small and extended in abnormal directions. This shows that neural crest cells are responsible not for the position of the muscles but for their correct anatomical pattern. Fate mapping showed that connective tissue surrounding myofibers is, at least partly, neural crest derived.</p><p>In conclusion, the work presented in this thesis shows that although early development may map out the patterns of later development, the differences between axolotl and lungfish head development are not seen until during morphogenesis. Further investigation of morphogenesis is needed to explain the great variation of head morphology seen in vertebrates today.</p>
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A Comparative Study of Head Development in Mexican Axolotl and Australian Lungfish: Cell Migration, Cell Fate and MorphogenesisEricsson, Rolf January 2003 (has links)
The development of the vertebrate head is a complex process involving interactions between a multitude of cell types and tissues. This thesis describes the development of the cranial neural crest and of the visceral arch muscles in the head of two species. One, the Mexican axolotl (Ambystoma mexicanum), is a basal tetrapod, whereas the other, the Australian lungfish (Neoceratodus forsteri), belongs to the Dipnoi, the extant sister group of the Tetrapoda. The migration of neural crest cells, which form most of the bones and connective tissues in the head, and the morphogenesis of the jaw, was determined in the Mexican axolotl. It was shown that both the upper and lower jaws form from ventral condensations of neural crest cells in the mandibular arch. The dorsal condensation, earlier considered to give rise to the upper jaw, was shown to form the trabecula cranii. The normal spatio-temporal development of visceral arch muscles was investigated in both the Mexican axolotl and the Australian lungfish. In axolotl, the muscles tended to start forming almost simultaneously in all visceral arches at their future origins and extend towards their future insertions at the onset of muscle fibre formation. In lungfish, fibres formed simultaneously throughout most of each muscle anlage in the first and second visceral arch, but were delayed in the branchial arches. The anlagen were first observed at their future insertion, from which they developed towards future origins. To test the ability of neural crest cells to pattern the visceral arch muscles, migrating crest cells were extirpated from axolotl embryos, which resulted in a wide range of muscle malformations. In most cases, the muscles appeared in the right position but were small and extended in abnormal directions. This shows that neural crest cells are responsible not for the position of the muscles but for their correct anatomical pattern. Fate mapping showed that connective tissue surrounding myofibers is, at least partly, neural crest derived. In conclusion, the work presented in this thesis shows that although early development may map out the patterns of later development, the differences between axolotl and lungfish head development are not seen until during morphogenesis. Further investigation of morphogenesis is needed to explain the great variation of head morphology seen in vertebrates today.
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ROLE OF THE NEURONAL SPECIFIC TRANSCRIPTION COREGULATOR NPDC-1 IN RETINOID AND THYROID RECEPTOR SIGNALING IN HUMAN AND THE AXOLOTL AMBYSTOMA MEXICANUMTheodosiou, Maria 01 January 2006 (has links)
Section I: This section is an introduction to the field of nuclear receptors. A general overview of nuclear receptor-mediated transcriptional regulation is followed by a review of literature on retinoid and thyroid receptor-mediated signaling. Section II: An introduction to NPDC-1 (neural proliferation, differentiation, and control), its discovery and characterization with regards to developmental expression and cellular localization. In addition NPDC-1 has been found to associate with a number of cell cycle regulatory proteins. NPDC-1 is characterized as a regulator of nuclear receptor-mediated transcriptional regulation. NPDC-1 was also demonstrated to be regulated post-transcriptionally through the ubiquitin/proteosome degradation pathway. Section III: Axolotl NPDC-1 (aNPDC-1) was cloned from axolotl brain and analyzed for homology to NPDC-1 from higher vertebrates. The tissue distribution and developmental expression of axolotl NPDC-1 were also examined. Section IV: The axolotl homolog for RAR (aRAR) was isolated from axolotl brain. Axolotl NPDC-1 and aRAR were then examined in a series of assays for interactions. Axolotl NPDC-1 was found to repress transcription mediated by aRAR to a smaller extent than the repression observed in higher vertebrates. The DNA binding of aRAR-RXR was increased in the presence of aNPDC-1 and complex mobility was also observed. The domain of interaction between aNPDC-1, aRAR and hRXR was localized in the amino terminus of aNPDC-1. Axolotl NPDC-1 was also demonstrated to repress proliferation as measured by reduced [3H] thymidine incorporation. Section V: The axolotl homologs of TR and TR (aTR) genes were isolated and utilized in a series of experiments to demonstrate an interaction between aTRs and aNPDC-1. As observed for RE, aNPDC-1 increases the binding of aTR-RXR heterodimer to xDR4, but no change in the mobility of the complex was observed. Interaction between aNPDC-1, aTR and aTR was localized to the amino terminus of aNPDC-1. In contrast to previous observations for other nuclear receptors, aNPDC-1 was found to stimulate transcription mediated by axolotl TRs, suggesting a role for aNPDC-1 in axolotl metamorphosis. Section VI: A summary of data presented in the previous sections as well as a presentation of future directions and a proposed model for NPDC-1 actions in retinoid and thyroid-receptor mediated signaling in axolotl.
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Revealing the Dynamics of the Limb-Brain Axis During Axolotl Limb RegenerationTornes, Jason Andrew 15 May 2023 (has links)
No description available.
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