Generation of stable Xenopus laevis transgenic lines expressing a transgene controlled by weak promoters.

International audienceCombining two existing protocols of trangenesis, namely the REMI and the I-SceI meganuclease methods, we generated Xenopus leavis expressing a transgene under the control of a promoter that presented a restricted pattern of activity and a low level of expression. This was realized by co-incubating sperm nuclei, the I-SceI enzyme and the transgene prior to transplantation into unfertilized eggs. The addition of the woodchuck hepatitis virus posttranscriptional regulatory element in our constructs further enhanced the expression of the transgene without affecting the tissue-specificity of the promoter activity. Using this combination of methods we produced high rates of fully transgenic animals that stably transmitted the transgene to the next generations with a transmission rate of 50% indicating a single integration event

Cyp19a1 (aromatase) expression in the Xenopus brain at different developmental stages.

International audienceCytochrome P450 aromatase (P450arom; aromatase) is a microsomal enzyme involved in the production of endogeneous sex steroids by converting testosterone into oestradiol. Aromatase is the product of the cyp19a1 gene and plays a crucial role in the sexual differentiation of the brain and in the regulation of reproductive functions. In the brain of mammals and birds, expression of cyp19a1 has been demonstrated in neuronal populations of the telencephalon and diencephalon. By contrast, a wealth of evidence established that, in teleost fishes, aromatase expression in the brain is restricted to radial glial cells. The present study investigated the precise neuroanatomical distribution of cyp19a1 mRNA during brain development in Xenopus laevis (late embryonic to juvenile stages). For this purpose, we used in situ hybridisation alone or combined with the detection of a proliferative (proliferating cell nuclear antigen), glial (brain lipid binding protein, Vimentin) or neuronal (acetylated tubulin; HuC/D; NeuroβTubulin) markers. We provide evidence that cyp19a1 expression in the brain is initiated from the very early larval stage and remains strongly detected until the juvenile and adult stages. At all stages analysed, we found the highest expression of cyp19a1 in the preoptic area and the hypothalamus compared to the rest of the brain. In these two brain regions, cyp19a1-positive cells were never detected in the ventricular layers. Indeed, no co-labelling could be observed with radial glial (brain lipid binding protein, Vimentin) or dividing progenitors (proliferating cell nuclear antigen) markers. By contrast, cyp19a1-positive cells perfectly matched with the distribution of post-mitotic neurones as shown by the use of specific markers (HuC/D, acetylated tubulin and NeuroβTubulin). These data suggest that, similar to that found in other tetrapods, aromatase in the brain of amphibians is found in post-mitotic neurones and not in radial glia as reported in teleosts

Expression of the cyp19a1 gene in the adult brain of Xenopus is neuronal and not sexually dimorphic

International audienceThe last step of oestrogen biosynthesis is catalyzed by the enzyme aromatase, the product of the cyp19a1 gene. In vertebrates, cyp19a1 is expressed in the brain resulting in a local oestrogen production that seems important not only for the control of reproduction-related circuits and sexual behaviour, but also for the regulation of neural development, synaptic plasticity and cell survival. In adult amphibians, the precise sites of expression of cyp19a1 in the brain have not been investigated which prevents proper understanding of its potential physiological functions. The present study aimed at examining the precise neuroanatomical distribution of cyp19a1 transcripts in adult brains of both male and female Xenopus. We found that cyp19a1 expression is highly regionalized in the brains of both sexes. The highest expression was found in the anterior part of the preoptic area and in the caudal hypothalamus, but significant levels of cyp19a1 transcripts were also found in the supraoptic paraventricular and suprachiasmatic areas, and in brain regions corresponding to the septum, bed nucleus of the stria terminalis and amygdala. Importantly, no obvious difference between male and female Xenopus was detected at the level of cyp19a1 transcripts. Additionally, in the brain of adult Xenopus, cyp19a1 transcripts were detected in neurons, and not in glial cells. These data and those available in other vertebrates on cyp19a1/aromatase expression suggest that, with the intriguing exception of teleost fishes, cyp19a1 was under strong evolutionary conservation with respect to its sites of expression and the nature of the cells in which it is expresse

Proliferation, migration and differentiation in juvenile and adult Xenopus laevis brains.

International audienceIn contrast to mammals, the brain of adult non-mammalian vertebrates exhibits a higher proliferative and/or neurogenic activity. To provide new models on this issue, we have examined origin, distribution and fate of proliferating cells in the entire brain of juvenile and adult Xenopus laevis. Using immunohistochemistry for the Proliferation Cell Nuclear Antigen (PCNA), and/or the thymidine analog, 5-Bromo-2' deoxyUridine (BrdU), the labeled cells are located in ventricular zones of the olfactory bulbs, cerebral hemispheres, preoptic region, ventral hypothalamus and cerebellum. Qualitatively, the highest level of proliferative cells was found in the telencephalic ventricles. By using in situ hybridization/immunocytochemistry double-labeling techniques, we demonstrate for the first time in post-metamorphic frog brain that the proliferative cells are localized in very close vivinity to the radial glial cells, progenitor cells that we have also identified in the ventricular layer using classical molecular markers (BLBP, Vimentin). In addition, after long post-BrdU administration survival times ranging between 14 and 28days, BrdU labeling combined with immunohistochemistry for markers of cell migration (DoubleCortin) or radial glial cells (BLBP), reveals that the proliferative cells are able to migrate from the ventricular zone into the brain parenchyma, most likely by migrating along the radial processes. Finally, at survival time of 28days and by using a combination of BrdU labeling and in situ hybridization for markers of differentiation states (Neuro-β-tubulin, Proteolipid Protein), we demonstrate that newborn cells can differentiate in large portion into either neurons or oligodendrocytes

The neurogenic factor NeuroD1 is expressed in post-mitotic cells during juvenile and adult Xenopus neurogenesis and not in progenitor or radial glial cells.

In contrast to mammals that have limited proliferation and neurogenesis capacities, the Xenopus frog exhibit a great potential regarding proliferation and production of new cells in the adult brain. This ability makes Xenopus a useful model for understanding the molecular programs required for adult neurogenesis. Transcriptional factors that control adult neurogenesis in vertebrate species undergoing widespread neurogenesis are unknown. NeuroD1 is a member of the family of proneural genes, which function during embryonic neurogenesis as a potent neuronal differentiation factor. Here, we study in detail the expression of NeuroD1 gene in the juvenile and adult Xenopus brains by in situ hybridization combined with immunodetections for proliferation markers (PCNA, BrdU) or in situ hybridizations for cell type markers (Vimentin, Sox2). We found NeuroD1 gene activity in many brain regions, including olfactory bulbs, pallial regions of cerebral hemispheres, preoptic area, habenula, hypothalamus, cerebellum and medulla oblongata. We also demonstrated by double staining NeuroD1/BrdU experiments, after long post-BrdU administration survival times, that NeuroD1 gene activity was turned on in new born neurons during post-metamorphic neurogenesis. Importantly, we provided evidence that NeuroD1-expressing cells at this brain developmental stage were post-mitotic (PCNA-) cells and not radial glial (Vimentin+) or progenitors (Sox2+) cells

Structural origin of the drastic modification of second harmonic generation intensity pattern occurring in tail muscles of climax stages xenopus tadpoles.

International audienceSecond harmonic generation (SHG) microscopy is a powerful tool for studying submicron architecture of muscles tissues. Using this technique, we show that the canonical single frequency sarcomeric SHG intensity pattern (SHG-IP) of premetamorphic xenopus tadpole tail muscles is converted to double frequency (2f) sarcomeric SHG-IP in metamorphic climax stages due to massive physiological muscle proteolysis. This conversion was found to rise from 7% in premetamorphic muscles to about 97% in fragmented muscular apoptotic bodies. Moreover a 66% conversion was also found in non-fragmented metamorphic tail muscles. Also, a strong correlation between predominant 2f sarcomeric SHG-IPs and myofibrillar misalignment is established with electron microscopy. Experimental and theoretical results demonstrate the higher sensitivity and the supra resolution power of SHG microscopy over TPEF to reveal 3D myofibrillar misalignment. From this study, we suggest that 2f sarcomeric SHG-IP could be used as signature of triad defect and disruption of excitation–contraction coupling. As the mechanism of muscle proteolysis is similar to that found in mdx mouse muscles, we further suggest that xenopus tadpole tail resorption at climax stages could be used as an alternative or complementary model of Duchene muscular dystrophy

Impacts of bisphenol A analogues on zebrafish post-embryonic brain

International audienceBisphenol A (BPA) is a widely studied and well-recognised endocrine-disrupting chemical, and one of the current issues is its safe replacement by various analogues. Using larva zebrafish as a model, the present study reveals that moderate and chronic exposure to BPA analogues such as bisphenol S, bisphenol F and bisphenol AF may also affect vertebrate neurodevelopment and locomotor activity. Several parameters of embryo-larval development were investigated, such as mortality, hatching, number of mitotically active cell, as defined by 5-bromo-2'-deoxyuridine incorporation and proliferative cell nuclear antigen labelling, aromatase B protein expression in radial glial cell and locomotor activity. Our results show that exposure to several bisphenol analogues induced an acceleration of embryo hatching rate. At the level of the developing brain, a strong up-regulation of the oestrogen-sensitive Aromatase B was also detected in the hypothalamic region. This up-regulation was not associated with effects on the numbers of mitotically active progenitors nor differentiated neurones in the preoptic area and in the nuclear recessus posterior of the hypothalamus zebrafish larvae. Furthermore, using a high-throughput video tracking system to monitor locomotor activity in zebrafish larvae, we show that some bisphenol analogues, such as bisphenol AF, significantly reduced locomotor activity following 6 days of exposure. Taken together, our study provides evidence that BPA analogues can also affect the neurobehavioural development of zebrafish