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Tgf-β, Wnt, and Fgf signaling pathways are required for many developmental processes. Here, I investigated the requirement of these signaling pathways during tail regeneration and limb development in the Mexican axolotl (Ambystoma mexicanum). Using small chemical inhibitors during tail regeneration, I found that the Tgf-β signaling pathway was required from 0-24 and 48-72 hours post tail amputation (hpa), the Wnt signaling pathway was required from 0-120 hpa, and the Fgf signaling pathway was required from 0-12hpa. Tgf-β1 was upregulated after amputation and thus may mediate Tgf-β signaling pathway during tail regeneration. Both Smad-mediated and non-Smad mediated Tgf-β signaling were activated as early as 1hpa. Smad-mediated Tgf-β signaling via activated pSmad2 and pSmad3, and via phosphorylated Erk and Akt. Two different Tgf-β signaling pathway inhibitors, SB505124 and Naringenin, differentially regulated pSmad2, pSmad3, p-Erk, and p-Akt, while SB505124 and Naringenin both inhibited tail regeneration; only SB505124 reduced cell proliferation. Wnt/β-Catenin signaling was increased and was enhanced by Wnt-C59. Disruption of the Wnt signaling pathway directly or indirectly activated Erk and Akt signaling. Disruption of the Fgf signaling pathway decreased p-Erk and increased p-Akt. All three signaling pathways affected cell proliferation and mitosis during tail regeneration. The Wnt pathway inhibitor Wnt-C59 prevented forelimb bud outgrowth. The critical window for Wnt signaling regulating forelimb bud outgrowth was approximately developmental stage 40-42. Wnt signaling ligand Wnt3a and tight junction protein Zo-1 were expressed in the epidermis of the forelimb bud and both were down-regulated by Wnt-C59. Moreover, both Wnt and Fgf signaling pathways affected cell proliferation and mitosis of mesodermal cells during forelimb bud outgrowth. Overall, my results show that Tgf-β, Wnt, and Fgf signaling pathways are required for axolotl tail regeneration. All three pathways affect Erk and Akt signaling and guide cell proliferation and mitosis. The Wnt signaling pathway is required for forelimb bud outgrowth, and it appears to regulate expression of Wnt3a and Zo1, and control cell proliferation and mitosis of mesodermal cells underlying the forelimb epidermis. These data enrich understanding of signaling network dynamics that underlie tissue regeneration and vertebrate limb development.

A robotic microscope was designed using a microcontroller to take time-lapse digital photographs of developing salamander embryos. The microcontroller operated three stepper motors to control three-axis movement accurately, and two six mega-pixel digital cameras to capture through-focus time-lapse digital pictures of six views of Ambystoma mexicanum embryos (axolotl, a salamander). The device is designed to take images every five minutes for 80 hours of early development, from fertilization to stage 20, when the neural tube closes to form the brain and spinal column. Techniques to enhance the embryo images were investigated including image fusion to get in-focus views from a stack of images. In the early embryo surface epithelial cells differentiate to form neural tissue and external skin tissue. Observing the whole embryo surface at cellular level will give a better idea of the stress and strain each cell undergoes and what physical forces are involved in cell differentiation. / May 2007

Tissue regeneration in salamanders is a robust process that is not easily interrupted or altered. Therefore, inhibiting regeneration provides a means to interrogate the underlying cellular and molecular mechanisms regulating this complex event. Here we show that application of a relatively low concentration of beryllium nitrate solution (100mM) causes a delay in skin regeneration and severely alters normal limb regeneration. We provide evidence showing a beryllium-induced reduction in dermal fibroblast migration in vivo and in vitro. We link this phenomenon to delayed regeneration of the skin and abnormal blastema formation resulting in limb patterning defects during regeneration. Though our results show a slight reduction in fibroblast proliferation during the early stages of limb regeneration, we attribute this to an overall reduction in fibroblast presence at the site of injury. Keratinocytes appeared unresponsive to beryllium treatment with the rates of re-epithelialization and proliferation not significantly different between treatment and control groups. Taken together, these data reinforce a necessary role for fibroblasts during tissue regeneration and show that beryllium nitrate inhibits normal fibroblast behavior.

The axolotl (Ambystoma mexicanum) has been used as a model organism for studying development, genetics, and regeneration. Although the sensory hair cells of the lateral line of this species have been shown to be able to regenerate, it is not known whether this also occurs in the inner ear. In fact, little is known about the hearing capabilities of the axolotl or other salamander species. I recorded auditory evoked potentials (AEPs) of six axolotls at eleven frequencies (0.1, 0.25, 0.4, 0.6, 0.8, 1, 1.5, 2, 3, 4, and 6 kHz) in order to produce baseline audiograms of underwater pressure sensitivity. Individuals were then subjected to a 48-hour, 150 Hz sound exposure at approximately 170 dB (re 1 μPa). AEPs were then performed to measure hearing thresholds immediately after sound exposure and at 2, 4, and 8 days post-sound exposure (DPSE). In the baseline audiogram, axolotls were most sensitive at 600 Hz, with an additional peak of sensitivity at 3 kHz. Following sound exposure, axolotls experienced a 6 to 12 dB temporary threshold shift (TTS) after sound exposure, with TTS being greatest at low frequencies near the 150 Hz stimulus frequency (i.e., 100 and 250 Hz). Hearing sensitivity returned to control levels within 8 DPSE. This indicates that axolotls do possess the ability to recover hearing sensitivity after damage following acoustical trauma. This study is the first to document hearing loss in the axolotl. Future studies are needed to correlate this hearing loss and recovery to sensory hair cell loss and regeneration in the axolotl inner ear.

A robotic microscope was designed using a microcontroller to take time-lapse digital photographs of developing salamander embryos. The microcontroller operated three stepper motors to control three-axis movement accurately, and two six mega-pixel digital cameras to capture through-focus time-lapse digital pictures of six views of Ambystoma mexicanum embryos (axolotl, a salamander). The device is designed to take images every five minutes for 80 hours of early development, from fertilization to stage 20, when the neural tube closes to form the brain and spinal column. Techniques to enhance the embryo images were investigated including image fusion to get in-focus views from a stack of images. In the early embryo surface epithelial cells differentiate to form neural tissue and external skin tissue. Observing the whole embryo surface at cellular level will give a better idea of the stress and strain each cell undergoes and what physical forces are involved in cell differentiation.

A robotic microscope was designed using a microcontroller to take time-lapse digital photographs of developing salamander embryos. The microcontroller operated three stepper motors to control three-axis movement accurately, and two six mega-pixel digital cameras to capture through-focus time-lapse digital pictures of six views of Ambystoma mexicanum embryos (axolotl, a salamander). The device is designed to take images every five minutes for 80 hours of early development, from fertilization to stage 20, when the neural tube closes to form the brain and spinal column. Techniques to enhance the embryo images were investigated including image fusion to get in-focus views from a stack of images. In the early embryo surface epithelial cells differentiate to form neural tissue and external skin tissue. Observing the whole embryo surface at cellular level will give a better idea of the stress and strain each cell undergoes and what physical forces are involved in cell differentiation.

Les capacités de guérison humaine étant limitées et grandement associées à la fibrose, la possibilité de régénérer tous tissus contribueraient grandement à l’amélioration de la santé des patients. Dans le cadre de ce projet de doctorat, nous avons publié un article montrant les limitations de certains modèles de recherche en ce qui a trait à la guérison des plaies. Ces limitations sont d’autant plus importantes lorsque la recherche traite de régénération tissulaire. Aussi, cette publication positionne l’axolotl (Ambystoma mexicanum) comme un excellent modèle pour étudier le processus de régénération épimorphique ainsi que l’importance de la signalisation TGF-β. La cytokine multifonctionnelle TGF-β est impliquée dans la guérison, l’induction des cicatrices, la différenciation, la croissance et la migration cellulaire. Cette cytokine est responsable de la guérison quasi parfaite des muqueuses buccales chez les mammifères, mais est aussi liée à la cicatrisation de plusieurs autres types tissulaires. La famille des TGF-β est aussi impliquée dans la régénération épimorphique chez l’échinoderme, ainsi que dans la régénération hépatique (hyperplasie compensatoire), ce qui confirme son rôle régulateur de la guérison parfaite. Des travaux précédents ont montré que l’utilisation d’un inhibiteur spécifique de la signalisation des TGF-β (SB-431542) empêche la régénération (Lévesque et al., 2007). Comme la voie canonique de signalisation de TGF-β s’opère via les protéines Smads (Smad2 & 3), l’étude de ces deux protéines est au cœur du second article. Lors du processus de régénération, Smad2 est phosphorylé entre 6h et 48h post-amputation (pa), ce qui correspond à la phase de migration cellulaire et au début de la prolifération. D’un autre côté, Smad3 est phosphorylé plus tôt, entre 3h et 6h pa, alors que la quantité de protéine totale diminue lors de la phase de préparation. L’administration de l’inhibiteur SB-431542 au moment de l’amputation bloque l’activation de Smad2 et de Smad3. Aucun blastème ne se forme, bien que la plaie ferme normalement. L’utilisation des inhibiteurs SIS3 et Naringenin (spécifique à Smad3) réduisent la phosphorylation de Smad3 d’environ 50 % (lorsque mesurée par immunobuvardage). Le processus de régénération ne semble toutefois pas affecté. La régulation différentielle des Smads est donc centrale au processus de régénération de l’axolotl. Dans le cadre de ce projet, nous avons aussi tenté de bloquer spécifiquement l’expression, ainsi que l’activation de Smad2. J’ai premièrement établi que Smad2 et Smad3 étaient présents dans la lignée cellulaire AL-1 et qu’ils peuvent être phosphorylés. J’ai ensuite tenté, par différentes techniques, de réduire l’activation spécifique de Smad2, sans succès. D’autre part, plusieurs expériences complémentaires confirment que l’activation de Smad3 est difficilement détectable et est peu importante pour la formation du blastème. La capacité exceptionnelle de régénération de l’axolotl est intimement liée à une activation différentielle des protéines Smad2 et Smad3. L’activation de Smad2 est associée à une prolifération cellulaire importante. D’autre part, l’absence de fibrose est potentiellement due à la faible activation de Smad3 au cours du processus de régénération. / Since wound healing in human is imperfect and associated with fibrosis, understanding how regeneration works would be a great asset to improve patient’s health. During this PhD project, we have published a paper exposing the weaknesses of certain research models when studying wound healing. Those limitations are even more striking when studying regeneration. This publication sets the stage for the use of the axolotl (Ambystoma mexicanum) as an excellent model to study regeneration and the importance of TGF- for the process. The multifunctional cytokine TGF-β is involved in healing, scarring, cellular differentiation, growth and migration. This cytokine is associated with the near perfect healing of oral tissues in humans, but is also associated with scarring of multiple tissue types. TGF-β is also associated with epimorphic regeneration in echinoderm and liver hyperplasia. Previous work had shown that treatment of regenerating axolotl limbs with a specific inhibitor of TGF-β canonical signalling (SB-431542) prevents regeneration (Lévesque et al., 2007). Since canonical signaling goes through Smad2 and Smad3, those two proteins are at the center of the second publication. During limb regeneration, Smad2 is phosphorylated at 6h-48h post-amputation (pa), which corresponds to the cellular migration phase and the beginning of the proliferative phase. On the other hand, Smad3 phosphorylation happens earlier (3h-6h pa), while the total protein expression is lower. Treatment with SB-421543 blocks the phosphorylation of both Smad2 and Smad3. No blastema is formed, but the wound closes at the same rate. Treatment with other inhibitors, SIS3 or Naringenin (specifically targeting Smad3), blocks approximately 50% of Smad3 phosphorylation (as determined by western blotting), but regeneration is not affected. Differential regulation of Smads is essential for proper regeneration to occur. Lastly, we have tried multiple approaches to diminish specifically the activation of Smad2. Using the only axolotl cell line available (AL-1), we have tried inhibition with LNA molecules, long antisense and overexpression of a competitor. None of these approaches specifically reduced the levels of Smad2. In addition, other experiments confirmed that activation of Smad3 during the regeneration process is limited. The extraordinary ability to regenerate that the axolotl possesses is tightly linked to a differential activation of Smad2 and Smad3 proteins. Smad2 phosphorylation is associated with cellular proliferation and migration, hence blastema formation, while the apparent lack of Smad3 activity might partly explain why these animals do not form scar tissues.

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