The Xenopus laevis oocyte was examined as a model system for investigating insulin receptor function. The role of extracellular calcium on insulin-stimulated deoxyglucose uptake (ISDU) in the 𝘟𝘦𝘯𝘰𝘱𝘶𝘴 𝘭𝘢𝘦𝘷𝘪𝘴 oocyte was investigated. It was determined that removal of calcium from the medium did not alter the rate of ⁴⁵Ca²⁺ release from oocytes preloaded with ⁴⁵CaCl₂ In contrast to earlier reports using tissue explants and cultured cells, the insulin response in oocytes is not sensitive to a range of extracellular calcium concentrations from 1 μM to 10 mM. However, treatment of oocytes with 1 mM EGTA, in the absence of Ca²⁺, prior to, during or within 5 minutes of insulin addition resulted in a 2-4 fold inhibition of ISDU. To further investigate the event(s) in insulin signalling inhibited by EGTA, the number of receptors for insulin on the oocyte must be increased. To this end we have investigated the effects of the 5' and 3' untranslated regions as well as the coding region of mRNA on translational efficiency in reticulocyte lysate and oocytes. The results obtained in Xenopus oocytes are consistent with earlier cell-free data (Falcone and Andrews, 1991). We have demonstrated that replacing the cognate 5' UTR with the Xenopus beta globin 5' UTR appropriately linked to a consensus sequence for efficient translation initiation (ACCATGG) results in increased in translation in Xenopus oocytes. In vitro synthesized preprolactin transcript injected into oocytes was found to be functionally stable for several days (D. Andrews unpublished data). Stabilization of the preprolactin 3' transcript was localized to the UTR. Furthermore, inserting the preprolactin 3' UTR downstream of another coding region resulted in stabilization of the modified transcript. These results provided a basis for improving expression of cloned human insulin receptor in Xenopus oocytes. By optimizing the 5' and 3' UTR's of the insulin receptor clone we were successful in expressing high levels of insulin receptors in Xenopus oocytes. Effects of the coding region on translation were also investigated and we provide evidence that sequences in the coding region modulate translational efficiency. / Thesis / Master of Science (MSc)

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Early development of O2 chemoreception and hypoxic responses under normoxic (150 mmHg) and chronically hypoxic (110 mmHg) conditions were investigated in Xenopus laevis from hatching to 3 weeks post fertilization. Development, growth, O2 consumption, ventilatory and cardiac performance, and branchial neuroepithelial cells (NEC) density and size were determined. At 3 days post fertilization (dpf), larvae started gill ventilation at a rate of 28 ± 4 beats/min and showed increased frequency to 60 ± 2 beats/min at a PO2 of 30 mmHg. Also at 3 dpf, NECs were identified in the gill filament buds using immunohistochemical methods. Lung ventilation began at 5 dpf and exhibited a 3-fold increase in frequency from normoxia to a PO2 of 30 mmHg. Hypoxic tachycardia developed at 5 dpf, causing an increase of 20 beats/min in heart rate, which led to a 2-fold increase in mass-specific cardiac output at a PO2 of 70 mmHg. At 10 dpf, gill ventilatory sensitivity to hypoxia increased, which was associated with the increase in NEC density, from 15 ± 1 to 29 ± 2 cells/mm of filament at 5 and 10 dpf, respectively. Unlike the elevated rate, cardiac and ventilatory volumes were independent of acute hypoxia. Despite increased cardioventilatory frequency, larvae experienced an average of 80% depression in during acute hypoxia. Chronic hypoxia (PO2 of 110 mmHg) decreased mass-specific cardiac performance before 10 dpf. In older larvae (10 to 21 dpf), chronic hypoxia decreased acute branchial and pulmonary hypoxic hyperventilation and increased NEC size. Collectively, these data suggest that larvae exhibit strong O2-driven acute hypoxic responses post-hatching, yet are still O2 conformers. All acute hypoxic responses developed before 5 dpf, and then the effects of chronic hypoxia started to show between 7 and 21 dpf. Thus, the early formation of acute hypoxic responses is susceptible to the environment and can be shaped by the ambient PO2.

Memoria para optar al Título Profesional de Médico Veterinario / La regulación transcripcional negativa es un mecanismo importante de control de la expresión de genes que contribuyen en el fenotipo neuronal. El elemento represor de la transcripción REST/NRSF ha sido propuesto como un regulador negativo de muchos genes de diferenciación neuronal terminal, expresándose en células no neuronales, precursores neuronales y neuronas en diferenciación. El papel de REST in vivo durante la diferenciación del sistema nervioso aún se desconoce, dada, entre otras, la letalidad de la pérdida de función de REST en ratones. El laboratorio en el que se desarrolló esta memoria de título ha utilizado otro organismo modelo, Xenopus laevis, a partir del cual se han obtenido resultados compatibles con la participación de REST en procesos muy tempranos del desarrollo neural. La interpretación de estos resultados requiere del análisis de los patrones de expresión de la proteína REST, lo que origina el objetivo principal de esta memoria de título: generar anticuerpos policlonales contra REST/NRSF de Xenopus laevis, para luego ser probados en embriones de Xenopus en diferentes estadíos del desarrollo. La electrotransferencia de extractos de embriones, evidenció que el suero antiREST es inmunoreactivo a una proteína de ~200 KDa, la cual está presente en embriones en los estadíos de clivaje, blástula, gástrula, neurula y organogénesis. En embriones inyectados con un morfolino antisentido de REST, el suero antiREST no detectó ninguna proteína; a diferencia de los embriones control no inyectados, en los cuales reconoció una proteína de ~200 KDa. Estos resultados son compatibles con la idea de que el suero antiREST reconoce la proteína REST endógena de Xenopus laevis. Por otra parte, el suero antiREST no resultó ser de utilidad en el reconocimiento de la proteína REST en embriones y en cortes de ellos mediante inmunohistoquímica

Circuits underlying motor patterns of closely related species provide an ideal framework in which to study how evolution shapes behavioral variation. Male African clawed frogs (Xenopus and Silurana) advertisement call to attract female mates and silence male rivals. Males of each species produce a unique vocal pattern that serves as a species-identifier. Xenopus laevis is the most well-studied species in terms of its vocal behavior and underlying anatomy and physiology. The clade that includes X. laevis, or X. laevis senso lato, also includes 3 other species that diverged ~8.5 million years ago. All 4 of these species produce advertisement calls that include fast trills – trains of fast rate (~60 Hz) sound pulses. However, their calls differ substantially between species in measures of trill duration and period. I examined the premotor circuit underlying vocal patterning in three of these species: X. laevis, X. petersii, and X. victorianus. I used extracellular recordings to find that a premotor nucleus, DTAM, which is part of the vocal central pattern generator, is the likely source of species-variation of vocal patterns. Species-specific trill duration and period are intrinsic to the region of the hindbrain that includes DTAM. Next, I used blind whole-cell patch recordings in DTAM of X. laevis and X. petersii to examine the cells that encode trill duration and period. I identified homologous populations of premotor vocal cells in both species that code for trill duration and period in a species-specific manner. Together, these results support an autonomous role of the DTAM circuit for generation of species variation in call duration and period.

The vertebrate planar cell polarity (PCP) pathway has been previously found to control polarized cell behaviors rather than cell fate. We report here that disruption of Xenopus laevis orthologs of the Drosophila melanogaster PCP genes Xint or Xfy affected not only PCP-dependent convergent extension but also caused embryonic phenotypes consistent with defective Hedgehog signaling. These defects in Hedgehog signaling resulted from a broad requirement for Inturned and Fuzzy in ciliogenesis. We show that these proteins are necessary for the formation of both primary cilium in the neural tube and multi-cilia in the epidermis. Also, using Xenopus muco-ciliary epidermis, we demonstrated that one of the core PCP genes Dishevelled performs dual functions in ciliogenesis, basal body docking and planar polarization of ciliary beating. To this end, we showed that Dishevelled works in concert with the PCP effector protein Inturned and Rho GTPase to mediate the docking of basal bodies to the apical cell surface. We suggest that this docking involves a Dvl-dependent association of basal bodies with vesicles, and with the vesicle-trafficking protein Sec8. Finally, we showed that independent of their roles in apical docking, Dvl/PCP signaling is required again for directional ciliary beating. For the first time, this study uncovered the mechanism for controlling the apical docking of basal bodies. Moreover, the results suggest that the same Dvl/PCP signaling is also important for the planar polarization of ciliary beating in a vertebrate muco-ciliary epithelium. / text

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La voie de biosynthèse des purines est une voie métabolique conservée et essentielle. Chez l’Homme, des mutations dans plusieurs gènes impliqués dans cette voie provoquent de sévères maladies neuro-musculaires à composante développementale. Cependant, le lien entre génotypes et phénotypes n’est pas connu. Afin de mieux comprendre le rôle des gènes de la voie des purines au cours du développement, nous avons utilisé Xenopus laevis comme modèle vertébré. Les principaux gènes de la voie des purines du xénope n’étaient pas connus, ils ont donc tout d’abord été identifiés in sillico, puis les fonctions enzymatiques pour lesquels ils codent ont été validées in vivo en système hétérologue chez S. cerevisiae. Des analyses d’expression spatiotemporelle chez l’embryon de xénope ont montré que ces gènes sont exprimés tout au long du développement et en particulier dans les tissus neuro-musculaires, suggérant un rôle dans le développement de ces tissus. Le knock-down des gènes, ppat, hprt ou adsl, trois gènes clés de la voie des purines, conduit dans chaque cas à de sévères altérations des muscles squelettiques et en particulier des somites et des muscles hypaxiaux des embryons. Ces phénotypes musculaires sont la conséquence d’une altération précoce de l’expression des gènes MRF (Myogenic Regulatory Factors) myoD et myf5. Un défaut de migration des myoblastes précurseurs des muscles hypaxiaux a également été mis en évidence. Pour conclure, X. laevis est un modèle pertinent qui apporte de nouvelles connaissances permettant de mieux comprendre la cause des altérations musculaires développementales associées aux déficiences en purines. / The purine biosynthesis pathway is a conserved metabolic pathway essential for many cell functions. In Human, several mutations in genes involved in this pathway lead to severe neuromuscular diseases, which are at least in part caused by unknown developmental impairments. We established a Xenopus laevis model to decipher the role of the purine biosynthesis genes during vertebrate development. As no data was available regarding this pathway, the main Xenopus purine genes were first identified in silico and functionally validated in vivo using the yeast Saccharomyces cerevisiae as a heterologous system. Spatio-temporal analyses revealed that these genes are expressed all along the development, especially in neuromuscular tissues, suggesting an important role during their formation. The knock-down of ppat, adsl or hprt, three key purine genes, leads in each case to severe defects in skeletal muscles embryonic defects, in particular in somites and hypaxial muscles. These muscular phenotypes are the consequence of an early alteration in expression of some crucial Myogenic Regulatory Factors (MRF), such as myoD and myf5. Moreover, an alteration of the hypaxial muscles precursors was observed. In conclusion our results establish X. laevis as an ideal model to get new insights into the neuromuscular developmental alterations associated to purine deficiencies.

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