Laurent Coen

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.

Xenopus Bcl-X(L) selectively protects Rohon-Beard neurons from metamorphic degeneration.

Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-XL plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-XL homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal β-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.

In Vivo Neuronal Tracing with GFP-TTC Gene Delivery

The retrograde transport and transynaptic transfer properties of the nontoxic tetanus toxin C-fragment (TTC) can be used to visualize specific neural pathways or to deliver biomolecules in the central nervous system (CNS). Here we tested different delivery techniques to explore the potential use of a new GFP-TTC fusion construct for use as a genetic tracer in vivo. Plasmids encoding GFP-TTC were targeted to brain regions using intracerebral grafted transfected cells or adenoviral transduction. Transport was monitored using GFP fluorescence. We show that following GFP-TTC synthesis in grafted transfected cells, the TTC fragment alone, with no signal peptide, is necessary and sufficient to provide secretion and uptake of the fusion protein into neighboring neurons around the injection site. Using an adenoviral vector to express the fusion protein into brain neurons, we show that transduced neurons can deliver the fusion protein specifically into connected neurons, demonstrating that synaptic transfer in the CNS can be visualized with GFP-TTC.

Molecular dynamics of retinoic acid-induced craniofacial malformations: implications for the origin of gnathostome jaws.

Background Intake of retinoic acid (RA) or of its precursor, vitamin A, during early pregnancy is associated with increased incidence of craniofacial lesions. The origin of these teratogenic effects remains enigmatic as in cranial neural crest cells (CNCCs), which largely contribute to craniofacial structures, the RA-transduction pathway is not active. Recent results suggest that RA could act on the endoderm of the first pharyngeal arch (1stPA), through a RARß-dependent mechanism. Methodology/Principal Findings Here we show that RA provokes dramatically different craniofacial malformations when administered at slightly different developmental times within a narrow temporal interval corresponding to the colonization of the 1st PA by CNCCs. We provide evidence showing that RA acts on the signalling epithelium of the 1st PA, gradually reducing the expression of endothelin-1 and Fgf8. These two molecular signals are instrumental in activating Dlx genes in incoming CNCCs, thereby triggering the morphogenetic programs, which specify different jaw elements. Conclusions/Significance The anatomical series induced by RA-treatments at different developmental times parallels, at least in some instances, the supposed origin of modern jaws (e.g., the fate of the incus). Our results might provide a conceptual framework for the rise of jaw morphotypes characteristic of gnathostomes.

Ventx factors function as Nanog-like guardians of developmental potential in Xenopus.

Vertebrate development requires progressive commitment of embryonic cells into specific lineages through a continuum of signals that play off differentiation versus multipotency. In mammals, Nanog is a key transcription factor that maintains cellular pluripotency by controlling competence to respond to differentiation cues. Nanog orthologs are known in most vertebrates examined to date, but absent from the Anuran amphibian Xenopus. Interestingly, in silico analyses and literature scanning reveal that basal vertebrate ventral homeobox (ventxs) and mammalian Nanog factors share extensive structural, evolutionary and functional properties. Here, we reassess the role of ventx activity in Xenopus laevis embryos and demonstrate that they play an unanticipated role as guardians of high developmental potential during early development. Joint over-expression of Xenopus ventx1.2 and ventx2.1-b (ventx1/2) counteracts lineage commitment towards both dorsal and ventral fates and prevents msx1-induced ventralization. Furthermore, ventx1/2 inactivation leads to down-regulation of the multipotency marker oct91 and to premature differentiation of blastula cells. Finally, supporting the key role of ventx1/2 in the control of developmental potential during development, mouse Nanog (mNanog) expression specifically rescues embryonic axis formation in ventx1/2 deficient embryos. We conclude that during Xenopus development ventx1/2 activity, reminiscent of that of Nanog in mammalian embryos, controls the switch of early embryonic cells from uncommitted to committed states.

Apoptosis of Tail Muscle During Amphibian Metamorphosis Involves a Caspase 9- Dependent Mechanism

The climax of amphibian metamorphosis is marked by thyroid hormone‐dependent tadpole tail resorption, implicating apoptosis of multiple cell types, including epidermal cells, fibroblasts, nerve cells, and muscles. The molecular cascades leading to and coordinating the death of different cell types are not fully elucidated. It is known that the mitochondrial pathway, and in particular the Bax and XR11 genes, regulates the balance between apoptosis and survival in muscle. However, the down‐stream factors modulated by changes in mitochondrial permeability have not been studied in a functional context. To investigate further the mitochondrial‐dependent pathway, we analyzed the regulation and the role of caspase 9 in Xenopus tadpoles. We report that caspase 9 mRNA is expressed in the tail before metamorphosis and increases before and during climax. Similarly, at the protein level, the production of active forms of caspase 9 increases in muscle tissue as metamorphosis progresses. To assess the functional role of caspase 9, we designed a dominant‐negative protein. Overexpression of this dominant‐negative abrogates both Bax‐induced cell death in vitro and muscle apoptosis in vivo during natural metamorphosis. These findings consolidate a model of metamorphic muscle death that directly implicates the mitochondrial pathway and the apoptosome. Developmental Dynamics 233:76–87, 2005.

Caspase-9 regulates apoptosis/proliferation balance during metamorphic brain remodeling in Xenopus

During anuran metamorphosis, the tadpole brain is transformed producing the sensorial and motor systems required for the frogs predatory lifestyle. Nervous system remodeling simultaneously implicates apoptosis, cell division, and differentiation. The molecular mechanisms underlying this remodeling have yet to be characterized. Starting from the observation that active caspase-9 and the Bcl-XL homologue, XR11 are highly expressed in tadpole brain during metamorphosis, we determined their implication in regulating the balance of apoptosis and proliferation in the developing tadpole brain. In situ hybridization showed caspase-9 mRNA to be expressed mainly in the ventricular area, a site of neuroblast proliferation. To test the functional role of caspase-9 in equilibrating neuroblast production and elimination, we overexpressed a dominant-negative caspase-9 protein, DN9, in the tadpole brain using somatic gene transfer and germinal transgenesis. In both cases, abrogating caspase-9 activity significantly decreased brain apoptosis and increased numbers of actively proliferating cells in the ventricular zone. Moreover, overexpression of XR11 with or without DN9 was also effective in decreasing apoptosis and increasing cell division in the tadpole brain. We conclude that XR11 and caspase-9, two key members of the mitochondrial death pathway, are implicated in controlling the proliferative status of neuroblasts in the metamorphosing Xenopus brain. Modification of their expression during the critical period of metamorphosis alters the outcome of metamorphic neurogenesis, resulting in a modified brain phenotype in juvenile Xenopus.

Assessment of estrogenic endocrine-disrupting chemical actions in the brain using in vivo somatic gene transfer.

Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element–thymidine kinase–luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17β-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 μg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo.

Non-viral expression of mouse Oct4, Sox2 and Klf4 factors efficiently reprograms tadpole muscle fibers in vivo

Background: Non-viral generation of induced pluripotent stem cells (iPSCs) in vitro is generally of low efficiency. Results: In vivo expression of non-integrated transgenes Oct4, Sox2, and Klf4 efficiently reprograms muscle cells. Conclusion: Reprogrammed, undifferentiated cells can be reliably and rapidly produced using naked DNA, exploiting synergy between muscle repair and reprogramming. Significance: In vivo approach throws light on the molecular networks underlying reprogramming, suggesting alternate iPSC generation strategies. Adult mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by a limited combination of transcription factors. To date, most current iPSC generation protocols rely on viral vector usage in vitro, using cells removed from their physiological context. Such protocols are hindered by low derivation efficiency and risks associated with genome modifications of reprogrammed cells. Here, we reprogrammed cells in an in vivo context using non-viral somatic transgenesis in Xenopus tadpole tail muscle, a setting that provides long term expression of non-integrated transgenes in vivo. Expression of mouse mOct4, mSox2, and mKlf4 (OSK) led rapidly and reliably to formation of proliferating cell clusters. These clusters displayed the principal hallmarks of pluripotency: alkaline phosphatase activity, up-regulation of key epigenetic and chromatin remodeling markers, and reexpression of endogenous pluripotent markers. Furthermore, these clusters were capable of differentiating into derivatives of the three germ layers in vitro and into neurons and muscle fibers in vivo. As in situ reprogramming occurs along with muscle tissue repair, the data provide a link between these two processes and suggest that they act synergistically. Notably, every OSK injection resulted in cluster formation. We conclude that reprogramming is achievable in an anamniote model and propose that in vivo approaches could provide rapid and efficient alternative for non-viral iPSC production. The work opens new perspectives in basic stem cell research and in the longer term prospect of regenerative medicine protocols development.

Non-viral Expression of Mouse Oct4, Sox2, and Klf4 Transcription Factors Efficiently Reprograms Tadpole Muscle Fibers in Vivo

Abstract Adult mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by a limited combination of transcription factors. To date, most current iPSC generation protocols rely on viral vector usage in vitro, using cells removed from their physiological context. Such protocols are hindered by low derivation efficiency and risks associated with genome modifications of reprogrammed cells. Here, we reprogrammed cells in an in vivo context using non-viral somatic transgenesis in Xenopus tadpole tail muscle, a setting that provides long term expression of non-integrated transgenes in vivo. Expression of mouse mOct4, mSox2, and mKlf4 (OSK) led rapidly and reliably to formation of proliferating cell clusters. These clusters displayed the principal hallmarks of pluripotency: alkaline phosphatase activity, up-regulation of key epigenetic and chromatin remodeling markers, and reexpression of endogenous pluripotent markers. Furthermore, these clusters were capable of differentiating into derivatives of the three germ layers in vitro and into neurons and muscle fibers in vivo. As in situ reprogramming occurs along with muscle tissue repair, the data provide a link between these two processes and suggest that they act synergistically. Notably, every OSK injection resulted in cluster formation. We conclude that reprogramming is achievable in an anamniote model and propose that in vivo approaches could provide rapid and efficient alternative for non-viral iPSC production. The work opens new perspectives in basic stem cell research and in the longer term prospect of regenerative medicine protocols development.