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Patterning of stem cells during limb regeneration in Ambystoma mexicanumRönsch, Kathleen 22 January 2018 (has links) (PDF)
Axolotl uniquely generates blastema cells as a pool of progenitor/stem cells to restore an entire limb, a particular property that other organisms, such as humans, do not have. What underlies these differences? Is the main difference that cells residing at the amputation plane (in the stump) undergo reprogramming processes to re-enter the embryonic program, which allows developmental patterning to start, or are there fundamental differences? There is also a significant debate about whether regeneration occurs via stem cell differentiation or by dedifferentiation of mature limb tissue. The aim of my thesis was to address following questions: Are the cells in the blastema reprogrammed or differentiated to regenerate? Are the blastema cells genetically reactivated de novo during regeneration? How does the amputated limb exactly know which part of the limb needs to be regenerate?
Using a novel technique of long-term genetic fate mapping, my team demonstrated that dedifferentiation in regenerated axolotl muscle tissue does not occur. Instead, PAX7+ satellite cells indeed play an important role during muscle regeneration in the axolotl limb. Surprisingly, this is in contrast to the newt, which regenerates muscle cells through a dedifferentiation process. Therefore, there is a fundamental difference that underlies the regenerative mechanism ((Sandoval-Guzman et al., 2014) [KR1]). This demonstrates that there is an unexpected diversity and flexibility of cellular mechanims used during limb regeneration, even among two closely related species. Finally, if one salamander species uses a mammalian regenerative strategy (Cornelison and Wold, 1997; Collins et al., 2005) involving stem cells and another uses a dedifferentiative strategy, this raises the question of whether there are other fundamental aspects of regeneration that could also be anomalous. This hypothesis is promising since there could be more than one possible mechanism to induce mammalian regeneration.
The process of limb regeneration in principle seems to be more similar to those of limb development as historically assumed. We showed molecularly that embryonic players are reused during regeneration by reactivating the position- and tissue-specific developmental gene programs by using the newly isolated Twist sequences as early blastema cell markers ((Kragl et al., 2013) [KR2]). To gain insights into the molecular mechanisms of the P/D limb patterning in general, it was crucial to study the early patterning events of the resident progenitor/stem cells by using the specific blastema cell marker HoxA as a positional marker along the proximo-distal axis. Our HOXA protein analysis using high molecular and cellular resolution as well as transplantation assays demonstrated for the first time that axolotl limb blastema cells acquire their positional identity in a proximal to distal sequence. We found a hierarchy of cellular restrictions in positional identities. Amputation at the level of the upper arm showed that the blastema harbors cells, which convert to lower arm and hand. We observed ((Roensch et al., 2013) [KR3]) for the first time that intercalation- the intermediate element (lower arm) arises later from an interaction between the proximal and distal cells identities- does not occur. Intercalation, which has been an accepted model for a long time, is not the patterning mechanism underlying normal (without any manipulation) limb regeneration that is unique to axolotl. We further demonstrated, using the Hox genes as markers that positional identity is cell-type specific since their effects were confirmed to be present in the lateral plate mesoderm- derived cells of the limb.
As our knowledge about limb blastemas expands concerning cell composition and molecular events controlling patterning, the similarity to development is becoming more and more clear. My work has resolved many ambiguities surrounding the molecularly identification of different types of blastema cells and how P/D limb patterning occurs during regeneration in comparison to development. It has highlighted the importance of combining high-resolution methods, such as in situ hybridizations, single-cell PCR (sc-PCR) of individual dissociated blastema cells and genetic labeling methods with grafting experiments to map cell fates in vivo.
In addition to understanding the processes of regeneration, another long-term goal in the regenerative medicine field is to identify key molecules that trigger the regeneration of tissues. Recently, my colleague Takuji Sugiura (Sugiura et al., 2016) observed that an early event of blastema formation is the secretion of molecules like MLP (MARCKS-like protein), which induces wound-associated cell cycle re-entry. Such findings further increase the enthusiasm of biologists to understand the underlying principles of regeneration. By building our knowledge of the molecules and pathways that are involved in tissue regeneration, we increase the possibility of identifying a way to ‘activate’ regenerative processes in humans and thus reach the final goal of regenerative medicine, which is to use the concepts of cellular reprogramming, stem cell biology and tissue engineering to repair complex body structures.
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Patterning of stem cells during limb regeneration in Ambystoma mexicanumRönsch, Kathleen 30 November 2017 (has links)
Axolotl uniquely generates blastema cells as a pool of progenitor/stem cells to restore an entire limb, a particular property that other organisms, such as humans, do not have. What underlies these differences? Is the main difference that cells residing at the amputation plane (in the stump) undergo reprogramming processes to re-enter the embryonic program, which allows developmental patterning to start, or are there fundamental differences? There is also a significant debate about whether regeneration occurs via stem cell differentiation or by dedifferentiation of mature limb tissue. The aim of my thesis was to address following questions: Are the cells in the blastema reprogrammed or differentiated to regenerate? Are the blastema cells genetically reactivated de novo during regeneration? How does the amputated limb exactly know which part of the limb needs to be regenerate?
Using a novel technique of long-term genetic fate mapping, my team demonstrated that dedifferentiation in regenerated axolotl muscle tissue does not occur. Instead, PAX7+ satellite cells indeed play an important role during muscle regeneration in the axolotl limb. Surprisingly, this is in contrast to the newt, which regenerates muscle cells through a dedifferentiation process. Therefore, there is a fundamental difference that underlies the regenerative mechanism ((Sandoval-Guzman et al., 2014) [KR1]). This demonstrates that there is an unexpected diversity and flexibility of cellular mechanims used during limb regeneration, even among two closely related species. Finally, if one salamander species uses a mammalian regenerative strategy (Cornelison and Wold, 1997; Collins et al., 2005) involving stem cells and another uses a dedifferentiative strategy, this raises the question of whether there are other fundamental aspects of regeneration that could also be anomalous. This hypothesis is promising since there could be more than one possible mechanism to induce mammalian regeneration.
The process of limb regeneration in principle seems to be more similar to those of limb development as historically assumed. We showed molecularly that embryonic players are reused during regeneration by reactivating the position- and tissue-specific developmental gene programs by using the newly isolated Twist sequences as early blastema cell markers ((Kragl et al., 2013) [KR2]). To gain insights into the molecular mechanisms of the P/D limb patterning in general, it was crucial to study the early patterning events of the resident progenitor/stem cells by using the specific blastema cell marker HoxA as a positional marker along the proximo-distal axis. Our HOXA protein analysis using high molecular and cellular resolution as well as transplantation assays demonstrated for the first time that axolotl limb blastema cells acquire their positional identity in a proximal to distal sequence. We found a hierarchy of cellular restrictions in positional identities. Amputation at the level of the upper arm showed that the blastema harbors cells, which convert to lower arm and hand. We observed ((Roensch et al., 2013) [KR3]) for the first time that intercalation- the intermediate element (lower arm) arises later from an interaction between the proximal and distal cells identities- does not occur. Intercalation, which has been an accepted model for a long time, is not the patterning mechanism underlying normal (without any manipulation) limb regeneration that is unique to axolotl. We further demonstrated, using the Hox genes as markers that positional identity is cell-type specific since their effects were confirmed to be present in the lateral plate mesoderm- derived cells of the limb.
As our knowledge about limb blastemas expands concerning cell composition and molecular events controlling patterning, the similarity to development is becoming more and more clear. My work has resolved many ambiguities surrounding the molecularly identification of different types of blastema cells and how P/D limb patterning occurs during regeneration in comparison to development. It has highlighted the importance of combining high-resolution methods, such as in situ hybridizations, single-cell PCR (sc-PCR) of individual dissociated blastema cells and genetic labeling methods with grafting experiments to map cell fates in vivo.
In addition to understanding the processes of regeneration, another long-term goal in the regenerative medicine field is to identify key molecules that trigger the regeneration of tissues. Recently, my colleague Takuji Sugiura (Sugiura et al., 2016) observed that an early event of blastema formation is the secretion of molecules like MLP (MARCKS-like protein), which induces wound-associated cell cycle re-entry. Such findings further increase the enthusiasm of biologists to understand the underlying principles of regeneration. By building our knowledge of the molecules and pathways that are involved in tissue regeneration, we increase the possibility of identifying a way to ‘activate’ regenerative processes in humans and thus reach the final goal of regenerative medicine, which is to use the concepts of cellular reprogramming, stem cell biology and tissue engineering to repair complex body structures.
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Los aspectos fantásticos en "Noche boca arriba", "Continuidad de los parques" y "Axolotl" de Julio Cortázar / Fantastic aspects in "Noche boca arriba", "Continuidad de los parques" and "Axolotl" by Julio CortázarKlintman, Ina January 2021 (has links)
This essay deals with the fantasy aspects that exist in work of Julio Cortázar. It is knownthat Cortázar frequently uses certain narrative tools in order to make a story to one of thefantastic kind. The purpose of the study is therefore to plunge into some of the fantasyaspects that are included in his work. The questions that the thesis answers are: which narrative techniques does the author use in the chosen stories to make these storiesfantastic? How does he utilize these techniques in the three stories? The corpus consistsof a short synopsis of the plots of the stories “Axolotl”, “Noche boca arriba” and“Continuidad de los parques” from the compilation Final del juego (1956). The methodof analysis used for the three stories consists of a reading that focuses on narrative aspects,including the characteristics of the fantasy genre. The results show role of the aspects of fantasy such as animalization, anthropomorphism, and identity change in “Axolotl”. Inaddition, the narrating voice is revealed to be an important literary aspect in this story. When it comes to "Noche boca arriba" and "Continuidad de los parques" the presence ofparallel realitiesis noticeable. Both of those story worlds are equally possible andimportant, according tothe theory of possible worlds (PW-theory). In "Continuidad de los parques", we can clearly see two worlds that experience a fusion in the moment they collide. Again, according to the theory of possible worlds, both are equally important. As a theoretical tool, Tzvetan Todorov's theory of the fantastic is used. The application of Todorov's theory is useful for the study as it serves as the basis in order to define the threestories aspart of the fantasy genre. / Este ensayo aborda los aspectos fantásticos que existen en la obra de Julio Cortázar. Essabido que Cortázar utiliza con frecuencia ciertas herramientas narrativas que hacen elcuento fantástico. El propósito del estudio es, por lo tanto, profundizar en algunos de losaspectos fantásticos incluidos en su obra. Las preguntas que la tesina responde son: ¿Quétécnicas narrativas utiliza el autor en los cuentos elegidos para hacerlos fantásticos?;¿Cómo utiliza estas técnicas en los tres cuentos? El corpus consiste en una breve sinopsisde las tramas de los cuentos “Axolotl”, “Noche boca arriba” y “Continuidad de losparques” de la compilación Final del juego (1956). El método de análisis utilizado paralos tres relatos consiste en una lectura que se enfoca en aspectos narrativos, inclusive enlas características de lo fantástico. Los resultados muestran el papel de los aspectosfantásticos como la animalización, el antropomorfismo y el cambio de identidad en “Axolotl”. Además, se revela que la voz narradora es un aspecto literario importante en este cuento. En cuanto a “Noche boca arriba” y “Continuidad de los parques” se nota la presencia de realidades paralelas. Ambos de estos universos narrativos son igualmenteposibles e importantes, deacuerdo con la teoría de mundos posibles. En "Continuidad delos parques", podemos verclaramente dos mundos que experimentan una fusión en elmomento en el que colisionan.Otra vez, de acuerdo con la teoría de mundos posibles,ambos son igualmente importantes. Como herramienta teórica se utiliza la teoría sobre lo fantástico de TzvetanTodorov. La aplicación de la teoría de Todorov es útil para el estudioya que sirve de basepara definir a los tres relatos como parte del género fantástico.
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Meningeal Fibrosis in the Axolotl Spinal Cord: Extracellular Matrix and Cellular ResponsesDeborah Anne Sarria (18405282) 03 June 2024 (has links)
<p dir="ltr">Though mammalian spinal cord injury (SCI) has long been a topic of study, effective therapies that promote functional recovery are not yet available. The axolotl, <i>Ambystoma mexicanum</i>, is a valuable animal model in the investigation of spinal cord regeneration, as this urodele is able to achieve functional recovery even after complete spinal cord transection. Understanding the similarities and differences between the mammalian SCI response and that of the axolotl provides insight into the process of successful regeneration, and bolsters the fundamental knowledge used in the development of future mammalian SCI treatments. This thesis provides a detailed analysis of the ultrastructure of the axolotl meninges, as this has not yet been presented in existing literature, and reveals that the axolotl meninges consist of 3 distinct layers as does mammalian meninges; the dura mater, arachnoid mater, and pia mater. The role of reactive meningeal and ependymal cells is also investigated in regard to the deposition and remodeling of the fibrotic ECM, which is found to be similar in composition to hydrogel scaffolds being studied in mammalian SCI. It is shown that meningeal fibroblasts are the primary source of the extensive fibrillar collagen deposition that fills the entire spinal canal, peaking at approximately 3 weeks post transection and remaining until approximately 5 weeks post transection, and that there is no deposition of type IV collagen within the lesion site. Mesenchymal ependymal cells are shown to contribute to the ECM deposition through the production of glycosaminoglycans that are used in sidechains of both unsulfated and sulfated proteoglycans, while simultaneously remodeling the ECM through the production of MMPs and phagocytosis of cellular debris. Further, this study shows that mesenchymal ependymal cells and a population of foamy macrophages contribute to the degradation of the fibrin clot that forms in the acute phase of injury, and that this fibrin clot provides a necessary and permissive substrate for early mesenchymal outgrowth.</p>
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Osteoclast-mediated resorption primes the skeleton for successful integration during axolotl limb regenerationRiquelme-Guzmán, Camilo, Tsai, Stephanie L, Karen Carreon, Nguyen, Congtin, Oriola, David, Schuez, Maritta, Brugués, Jan, Currie, Joshua D, Sandoval-Guzmán, Tatiana 21 May 2024 (has links)
Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven, and highly regulated event. This process, carried out by osteoclasts, is essential for a successful integration of the newly formed skeleton. Indeed, the extent of resorption is directly correlated with the integration efficiency, and treatment with zoledronic acid resulted in osteoclast function inhibition and failed tissue integration. Moreover, we identified the wound epithelium as a regulator of skeletal resorption, likely releasing signals involved in recruitment/differentiation of osteoclasts. Finally, we reported a correlation between resorption and blastema formation, particularly, a coordination of resorption with cartilage condensation. In sum, our results identify resorption as a major event upon amputation, playing a critical role in the overall process of skeletal regeneration.
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Étude du rôle de la signalisation canonique des Bmp lors de la régénération de la patte d'axolotlVincent, Etienne 05 1900 (has links)
No description available.
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Purification of A Serum Factor That Triggers Cell Cycle Re-entry In Differentiated Newt MyotubesStraube, Werner 26 June 2006 (has links)
In contrast to mammals, some fish and amphibians have retained the ability to regenerate complex body structures or organs, such as the limb, the tail, the eye lens or even parts of the heart. One major difference in the response to injury is the appearance of a mesenchymal growth zone or blastema in these regenerative species instead of the scarring seen in mammals. This blastema is thought to largely derive from the dedifferentiation of various functional cell types, such as skeletal muscle, skin and cartilage. In the case of multinucleated skeletal muscle fibres, cell cycle re-entry into S-phase as well as fragmentation into mononucleated progenitors is observed both in vitro and in vivo. In order to identify molecules that initiate dedifferentiation of cells at the wound site in amphibians we have established a cellular assay with a cultured newt myogenic cell line. Using this assay we have found a serum activity that stimulates cell cycle re-entry in differentiated multinucleated newt myotubes. The activity is present in serum of all mammalian species tested so far and, interestingly, thrombin proteolysis amplifies the activity from both serum and plasma. We think this serum factor provides a link between wounding and regeneration and its identification will be a key step in understanding the remarkable differences in wound healing between mammals and amphibians. In the course of this PhD thesis we have characterized the serum factor as a thermo-labile, pH- and proteinase K-sensitive, high molecular weight protein that is resistant to denaturing conditions such as SDS, urea or organic solvents. Surprisingly, under denaturing conditions the activity behaves as a low molecular weight protein that displays charge heterogeneity on isoelectric focusing. Using these characteristics of the serum factor we have performed a systematic investigation of commonly used protein chromatography modes and separation techniques to develop a successful purification procedure. After four column chromatography steps -- cation exchange, hydrophobic interaction, heparin affinity and size exclusion chromatography under denaturing conditions -- we have achieved a 2,000-fold purification starting from a commercially available Crude Bovine Thrombin preparation. This represents about 40,000-fold purification over bovine serum. Silver stained gels of the most purified fractions revealed ten major protein bands. In order to finally identify the cell cycle re-entry factor, we are currently analyzing the purification by quantitative mass spectrometry by correlating the abundance of tryptic peptides with activity in sequential fractions across a chromatography run.
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Étude du rôle des gènes TGF-β1 et HSP-70 lors du processus de régénération du membre chez l’axolotlLévesque, Mathieu 08 1900 (has links)
Les urodèles amphibiens, dont fait partie l’axolotl (Ambystoma mexicanum), ont la capacité de régénérer leurs organes et membres suite à une amputation, tout au long de leur vie. La patte est l’organe dont le processus de régénération est le mieux caractérisé et ce dernier est divisé en deux phases principales. La première est la phase de préparation et commence immédiatement suite à l’amputation. Elle renferme des étapes essentielles au processus de régénération comme la guérison de la plaie et la formation d’une coiffe apicale ectodermique. Par la suite, les fibroblastes du derme et certaines cellules musculaires vont revenir à un état pluripotent via un processus appelé dédifférenciation cellulaire. Une fois dédifférenciées, ces cellules migrent et s’accumulent sous la coiffe apicale pour former le blastème. Lors de la phase de redéveloppement, les cellules du blastème se divisent puis se redifférencient pour régénérer la partie amputée. Fait intéressant, la régénération d’un membre ou la guérison d’une plaie chez l’axolotl ne mène jamais à la formation d’une cicatrice. Afin d’en apprendre plus sur le contrôle moléculaire de la régénération, les gènes Heat-shock protein-70 (Hsp-70) et Transforming growth factor-β1 (Tgf-β1) ont été sélectionnés. Ces gènes jouent un rôle important dans la réponse au stress et lors de la guérison des plaies chez les mammifères. HSP-70 est une chaperonne moléculaire qui est produite pour maintenir l’intégrité des protéines cellulaires lorsqu’un stress se présente. TGF-β1 est une cytokine produite suite à une blessure qui active la réponse inflammatoire et qui stimule la fermeture de la plaie chez les amniotes. Les résultats présentés dans cette thèse démontrent que Hsp-70 est exprimé et régulé lors du développement et de la régénération du membre chez l’axolotl. D’autre part, nos expériences ont mené à l’isolation de la séquence codante pour Tgf-β1 chez l’axolotl. Nos résultats montrent que Tgf-β1 est exprimé spécifiquement lors de la phase de préparation dans le membre en régénération. De plus, le blocage de la voie des Tgf-β avec l’inhibiteur pharmacologique SB-431542, lors de la régénération, mène à l’inhibition du processus. Ceci démontre que la signalisation via la voie des Tgf-β est essentielle à la régénération du membre chez l’axolotl. / Urodele amphibians, such as the axolotl (Ambystoma mexicanum), have the unique ability, among vertebrates, to perfectly regenerate many parts of their body throughout their life. Among the complex structures that can be regenerated, the limb is the most widely studied. Limb regeneration is divided in two main phases. The preparation phase, which begins right after amputation, includes wound healing and the formation of an apical ectodermal cap. During this phase, dermal fibroblasts and muscle cells will lose their characteristics and become pluripotent through a process called cellular dedifferentiation. The dedifferentiated cells migrate and accumulate under the apical ectodermal cap to form the blastema. During the redevelopment phase, the cells in the blastema proliferate and redifferentiate to regenerate the lost structures. It is interesting to highlight the fact that regeneration never leads to scar formation in the axolotl. In order to learn more about the molecular control of limb regeneration, the genes Heat-shock protein-70 (Hsp-70) and Transforming growth factor-β1 (Tgf- β1) were selected for their important roles in stress response and wound healing in mammals. HSP-70 is a molecular chaperone which is produced to protect cellular proteins when the cell faces a stress. TGF-β1 is a cytokine produced after wounding that activates the inflammatory response and stimulates wound closure in amniotes. Results presented in this thesis show that Hsp-70 is expressed and regulated during limb development and regeneration in the axolotl. We were also able to isolate the cDNA coding for axolotl Tgf-β1 and our results show that this gene is expressed specifically during the preparation phase of limb regeneration. Treatment of regenerating axolotls with a specific inhibitor of Tgf-β signalling, SB-431542, led to complete inhibition of regeneration. This directly implies that Tgf-β signalling is essential for limb regeneration in axolotl.
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Étude du rôle des gènes TGF-β1 et HSP-70 lors du processus de régénération du membre chez l’axolotlLévesque, Mathieu 08 1900 (has links)
Les urodèles amphibiens, dont fait partie l’axolotl (Ambystoma mexicanum), ont la capacité de régénérer leurs organes et membres suite à une amputation, tout au long de leur vie. La patte est l’organe dont le processus de régénération est le mieux caractérisé et ce dernier est divisé en deux phases principales. La première est la phase de préparation et commence immédiatement suite à l’amputation. Elle renferme des étapes essentielles au processus de régénération comme la guérison de la plaie et la formation d’une coiffe apicale ectodermique. Par la suite, les fibroblastes du derme et certaines cellules musculaires vont revenir à un état pluripotent via un processus appelé dédifférenciation cellulaire. Une fois dédifférenciées, ces cellules migrent et s’accumulent sous la coiffe apicale pour former le blastème. Lors de la phase de redéveloppement, les cellules du blastème se divisent puis se redifférencient pour régénérer la partie amputée. Fait intéressant, la régénération d’un membre ou la guérison d’une plaie chez l’axolotl ne mène jamais à la formation d’une cicatrice. Afin d’en apprendre plus sur le contrôle moléculaire de la régénération, les gènes Heat-shock protein-70 (Hsp-70) et Transforming growth factor-β1 (Tgf-β1) ont été sélectionnés. Ces gènes jouent un rôle important dans la réponse au stress et lors de la guérison des plaies chez les mammifères. HSP-70 est une chaperonne moléculaire qui est produite pour maintenir l’intégrité des protéines cellulaires lorsqu’un stress se présente. TGF-β1 est une cytokine produite suite à une blessure qui active la réponse inflammatoire et qui stimule la fermeture de la plaie chez les amniotes. Les résultats présentés dans cette thèse démontrent que Hsp-70 est exprimé et régulé lors du développement et de la régénération du membre chez l’axolotl. D’autre part, nos expériences ont mené à l’isolation de la séquence codante pour Tgf-β1 chez l’axolotl. Nos résultats montrent que Tgf-β1 est exprimé spécifiquement lors de la phase de préparation dans le membre en régénération. De plus, le blocage de la voie des Tgf-β avec l’inhibiteur pharmacologique SB-431542, lors de la régénération, mène à l’inhibition du processus. Ceci démontre que la signalisation via la voie des Tgf-β est essentielle à la régénération du membre chez l’axolotl. / Urodele amphibians, such as the axolotl (Ambystoma mexicanum), have the unique ability, among vertebrates, to perfectly regenerate many parts of their body throughout their life. Among the complex structures that can be regenerated, the limb is the most widely studied. Limb regeneration is divided in two main phases. The preparation phase, which begins right after amputation, includes wound healing and the formation of an apical ectodermal cap. During this phase, dermal fibroblasts and muscle cells will lose their characteristics and become pluripotent through a process called cellular dedifferentiation. The dedifferentiated cells migrate and accumulate under the apical ectodermal cap to form the blastema. During the redevelopment phase, the cells in the blastema proliferate and redifferentiate to regenerate the lost structures. It is interesting to highlight the fact that regeneration never leads to scar formation in the axolotl. In order to learn more about the molecular control of limb regeneration, the genes Heat-shock protein-70 (Hsp-70) and Transforming growth factor-β1 (Tgf- β1) were selected for their important roles in stress response and wound healing in mammals. HSP-70 is a molecular chaperone which is produced to protect cellular proteins when the cell faces a stress. TGF-β1 is a cytokine produced after wounding that activates the inflammatory response and stimulates wound closure in amniotes. Results presented in this thesis show that Hsp-70 is expressed and regulated during limb development and regeneration in the axolotl. We were also able to isolate the cDNA coding for axolotl Tgf-β1 and our results show that this gene is expressed specifically during the preparation phase of limb regeneration. Treatment of regenerating axolotls with a specific inhibitor of Tgf-β signalling, SB-431542, led to complete inhibition of regeneration. This directly implies that Tgf-β signalling is essential for limb regeneration in axolotl.
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The role of retinoids in the regeneration of the axolotl spinal cordKirk, Maia P. 17 July 2015 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Retinoids play an important role in tissue patterning during development as well as in epithelial formation and health. In the mammalian central nervous system, the meninges are a source of retinoids for brain tissue. Retinoid production has been described in juvenile Axolotl ependymal cells. Retinoid effects may possess a significant role in the regeneration-permissive interaction of the meninges and ependyma of the Axolotl spinal cord after penetrating injury. During spinal cord regeneration in urodele amphibians, the pattern of retinoid production changes as the meninges interact with the injury-reactive ependymal cells reconstructing the injured spinal cord. In order to determine which components of the retinoid metabolism and intracellular signaling pathway act in Urodele spinal cord regeneration, we employed antibody/horseradish peroxidase staining of both intact and regenerating Axolotl spinal cord tissues obtained from adult animals as well as cell culture techniques to determine expression of three retinoid pathway components: Cellular Retinoic Acid Binding Protein II (CRABP 2), Cellular Retinol Binding Protein I (CRBP 1), and Retinaldehyde Dehydrogenase II (RALDH 2). Current results demonstrate the following in the intact cord: 1) CRBP 1 is expressed in the pia and dura mater meningeal layers, in gray matter neurons (including their axonal processes), and the ependymal cell radial processes that produce the glia limitans, 2) CRABP 2 is expressed in the arachnoid and/or dura mater meningeal layers surrounding the spinal cord, and 3) RALDH 2 is expressed in the meninges as well as
cytoplasm of grey matter neurons and some ependymal/sub-ependymal cells. In the regenerating cord, CRBP 1 is expressed in ependymal cells that are undergoing epithelial-to-mesenchymal transition (EMT), as is CRABP 2. RALDH 2 staining is very strong in the reactive meninges; in addition, expression is also upregulated in the cytoplasmic and perinuclear regions of reactive grey matter neurons, including motor neurons and in the apical region of ependymal. Preliminary studies culturing reactive meninges and ependymal cells together suggested that the meninges could drive re-epithelialization of the reactive ependymal cells. Experiments to characterize this interaction show an unusual proliferation pattern: Proliferating Cell Nuclear Antigen (PCNA) labeling is present in intact and regenerating cord ependymal cells. However, in culture, the presence of meninges results in no proliferation proximal to the explant, but extensive proliferation in leading cell outgrowth; also, the cultured meninges is positive for RALDH2. In summary, the intact adult cord shows meningeal production of RA, which is upregulated following injury; in addition, during this time, RA production is upregulated in the adult ependymal cells as well. In culture, the reactive meninges appears to modulate the behavior of reactive ependymal cells.
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