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

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.

Au cours de ma thèse, j'ai étudié l'impact des stress associés aux vols spatiaux sur l'immunité humorale du pleurodèle et de la souris. Chez le pleurodèle adulte, j'ai d'abord étudié l'utilisation des gènes VH lors de la synthèse des chaînes lourdes d'anticorps suite à une immunisation pendant 5 mois à bord de Mir (expérience Genesis en 1999). J'ai ensuite étudié le processus de maturation de l'affinité des anticorps chez ces mêmes animaux. Ce processus s'effectue par l'apparition d'hypermutations somatiques dans les segments variables des gènes d'anticorps. Ces travaux ont permis de montrer que les segments VH sont utilisés différemment sur Terre et dans Mir et que la fréquence des hypermutations est diminuée suite au vol. Ensuite, j'ai étudié l'impact des stress rencontrés lors d'un autre vol spatial sur la synthèse des premiers anticorps (IgM) chez le pleurodèle en développement (expérience AMPHIBODY en 2006). Le taux d'IgM étant modifié suite à cette expérience, nous avons recréé sur Terre chacun des stress rencontrés en vol (microgravité, hypergravité, choc thermique, radiations, perturbation du rythme circadien et confinement) afin de connaître le(s) stress responsable(s) de cette modification. Ainsi, seule la gravité modifiée affecte l'expression des IgM. Enfin, j'ai étudié l'impact de l'hypergravité (2G et 3G) sur la réponse au stress et le système immunitaire de la souris. Nous avons mis en évidence une réponse physiologique et comportementale au stress à 3G mais pas à 2G. Pourtant, des modifications du système immunitaire sont constatées dès 2G. Cela montre qu'une modification de la gravité, associée ou non à une réponse au stress, affecte le système immunitaire / During my PhD, I studied the impact of spaceflight-associated stresses on Pleurodeles waltl and Mus musculus humoral immunity. In adult P. waltl immunized during 5 months onboard the Mir space station (Genesis experiment in 1999), I first determined how individual VH genes are used. Then, I studied antibodies affinity maturation in these animals. This maturation implies the introduction of somatic hypermutations (SHM) in DNA encoding the variable segments of antibodies genes. These two pieces of work have shown that variable segments of heavy chain gene are differently used and that SHM frequency is reduced when immunization occurs in space. Then, I studied antibodies production during animal development onboard the international space station (ISS) (AMPHIBODY experiment in 2006). The antibodies production being increased in larvae that developed in the ISS, we recreated in the laboratory each stress encountered during the spaceflight (hypergravity, microgravity, heat shock associated to the re-entry in the atmosphere, radiations, perturbation of circadian rhythm and confinement) to determine their impact on IgM heavy chain transcription. This allowed to observe that only gravity changes affect this transcription. Finally, I studied the impact of hypergravity (2G and 3G) on the murine immune system. I observed physiological and behavioural stress responses in mice exposed to 3G but not in 2G mice. However, immune system alterations were observed in both the 2G and 3G groups, suggesting that gravity modifications, associated or not with stress responses, are responsible for immune system modifications

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.

Sexual selection can influence the mating system of an organism through multiple mechanisms. These mechanisms result in variation in reproductive success among individuals, and include scramble competition, endurance rivalries, contests, mate choice and cryptic choice, and sperm competition. Understanding the mating system of a species requires the identification of which processes are occurring, and to what degree. In this thesis, I explored the influence of mate choice mechanisms on the mating system of the terrestrial red-legged salamander, Plethodon shermani. I also documented the potential for post-copulatory processes (such as sperm competition and cryptic choice) to influence mating system dynamics. The evolution of mate choice requires (among other factors) variation in the reproductive value of potential mates. This variation is made apparent to choosy individuals through cues. Most animals use multiples cues incorporating many modalities to assess the reproductive quality of potential mates. In Chapter 2, I tested the contribution of two cues (chemical and visual) to mate choice by female P. shermani. I found that a male visual cue ("foot-dancing") increased courtship success. In contrast, delivery of non-volatile pheromones during courtship did not influence courtship success in the laboratory setting, but did affect the duration of one of the courtship stages. In Chapter 4, I identified a tactile cue that was significantly correlated with male reproductive success. Thus, P. shermani females could use at least three modalities to assess the reproductive quality of potential mates. Mate choice can also evolve in males. In Chapter 3, I tested this possibility in P. shermani. I found that males vary the reproductive effort they invest in a particular courtship based on the reproductive value of their partner, indicating male mate choice is occurring. A male invested most when paired with a female with large, well developed ova, and invested less with females that were non-gravid or had small ova. In addition to documenting male mate choice, I showed that the male visual display ("foot-dancing") that affected female mate choice was correlated with male condition, implying foot-dancing may be an honest indicator of male quality. Finally, in Chapter 5, I explored the potential for post-copulatory processes to influence the P. shermani mating system. The opportunity for sperm from multiple males to overlap in the female reproductive tract (i.e., the opportunity for females to mate multiply) is necessary for post-copulatory processes such as cryptic choice and sperm competition. The capacity for long-term sperm storage by females can increase the likelihood that this overlap in sperm from multiple males will occur. I found that females can store viable sperm for at least 9 months and in some cases beyond oviposition. In addition, I documented one female with sperm in her sperm storage organ from a mating that occurred 17 months earlier. Such lengthy sperm storage allows the possibility of sperm from one breeding season to interact with sperm from a subsequent season. Thus, the potential for post-copulatory sexual selection within this salamander system is high. / Graduation date: 2012

Frogs, toads, and salamanders are well known for harboring an array of distasteful (and poisonous) secondary metabolites, presumably as antipredator defenses; yet few experiments have rigorously demonstrated the efficacy of amphibian chemical defenses against ecologically relevant consumers. For example, despite an absence of rigorous statistical evidence showing their distastefulness to predators, eastern newts (Notophthalmus viridescens (Rafinesque))--a common salamander in lentic North American habitats--are assumed to tolerate diverse predator assemblages because newts secrete tetrodotoxin (TTX), a neurotoxin. Here we combine laboratory and field-based ecology with bioassay-guided separation of chemical extracts to show that eastern newts--although chemically protected against ecologically important consumers in lentic systems--nonetheless suffer substantial predation when tethered in the field. When offered newts with alternative prey (paedomorphic Ambystoma talpoideum), red swamp crayfish (Procambarus clarkii) and largemouth bass (Micropterus salmoides) were 9-10x as likely to feed on A. talpoideum as newts. Additionally, juvenile bluegill (Lepomis machrochirus) were 70% less likely to consume newt eggs compared to control food pellets. We also show that different newt tissues were differentially palatable to predatory fish. All bluegill tested consumed a palatable control food, but only 20% consumed dorsal skin, only 35% ate ventral skin, but 75% fed on newt viscera, suggesting that deterrent metabolites are concentrated in the skin. Bioassay-guided fractionation revealed that crude and water-soluble newt chemical extracts inhibited bluegill feeding, definitively establishing the chemical nature of newt antipredator defenses, although we were unsuccessful at isolating the chemical compounds responsible for unpalatability. Yet, deterrent activity in the polar but not the lipophilic chemical fraction and bioassay results demonstrating that naıve predators rapidly learn to avoid natural concentrations of TTX support the possible role of TTX in suppressing predation on newts. However, when tethered in the field, newt mortality was 55% higher in ponds with predatory fishes than in ponds lacking fishes (62% vs. 40% respectively), indicating the possible existence of other predators that are resistant to (or tolerant of) newt chemical defenses. Together, these results stress the importance of rigorous, ecologically relevant, and hypothesis-driven experimentation to better understand the complexity of chemically- mediated predator-prey interactions, even for well-studied species like N. viridescens.

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.