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

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.

Developmental thyroid disruption permanently affects the neuroglial output in the murine subventricular zone.

Neural stem cells (NSCs) in the adult brain are a source of neural cells for brain injury repair. We investigated whether their capacity to generate new neurons and glia is determined by thyroid hormone (TH) during development because serum levels peak during postnatal reorganization of the main NSC niche, the subventricular zone (SVZ). Re-analysis of mouse transcriptome data revealed increased expression of TH transporters and deiodinases in postnatal SVZ NSCs, promoting local TH action, concomitant with a burst in neurogenesis. Inducing developmental hypothyroidism reduced NSC proliferation, disrupted expression of genes implicated in NSC determination and TH signaling, and altered the neuron/glia output in newborns. Three-month-old adult mice recovering from developmental hypothyroidism had fewer olfactory interneurons and underperformed on short-memory odor tests, dependent on SVZ neurogenesis. Our data provide readouts permitting comparison with adverse long-term events following thyroid disruptor exposure and ideas regarding the etiology of prevalent neurodegenerative diseases in industrialized countries.

Absence of Both Thyroid Hormone Transporters MCT8 and OATP1C1 Impairs Neural Stem Cell Fate in the Adult Mouse Subventricular Zone.

Adult neural stem cell (NSC) generation in vertebrate brains requires thyroid hormones (THs). How THs enter the NSC population is unknown, although TH availability determines proliferation and neuronal versus glial progenitor determination in murine subventricular zone (SVZ) NSCs. Mice display neurological signs of the severely disabling human disease, Allan-Herndon-Dudley syndrome, if they lack both MCT8 and OATP1C1 transporters, or MCT8 and deiodinase type 2. We analyzed the distribution of MCT8 and OATP1C1 in adult mouse SVZ. Both are strongly expressed in NSCs and at a lower level in neuronal cell precursors but not in oligodendrocyte progenitors. Next, we analyzed Mct8/Oatp1c1 double-knockout mice, where brain uptake of THs is strongly reduced. NSC proliferation and determination to neuronal fates were severely affected, but not SVZ-oligodendroglial progenitor generation. This work highlights how tight control of TH availability determines NSC function and glial-neuron cell-fate choice in adult brains.

Gender-specific effects of transthyretin on neural stem cell fate in the subventricular zone of the adult mouse.

Choroid plexus epithelial cells produce and secrete transthyretin (TTR). TTR binds and distributes thyroid hormone (TH) to brain cells via the cerebrospinal fluid. The adult murine subventricular zone (SVZ) is in close proximity to the choroid plexus. In the SVZ, TH determines neural stem cell (NSC) fate towards a neuronal or a glial cell. We investigated whether the loss of TTR also disrupted NSC fate choice. Our results show a decreased neurogenic versus oligodendrogenic balance in the lateroventral SVZ of Ttr knockout mice. This balance was also decreased in the dorsal SVZ, but only in Ttr knockout male mice, concomitant with an increased oligodendrocyte precursor density in the corpus callosum. Quantitative RTqPCR analysis following FACS-dissected SVZs, or marked-coupled microbeads sorting of in vitro neurospheres, showed elevated Ttr mRNA levels in neuronal cells, as compared to uncommitted precursor and glial cells. However, TTR protein was undetectable in vivo using immunostaining, and this despite the presence of Ttr mRNA-expressing SVZ cells. Altogether, our data demonstrate that TTR is an important factor in SVZ neuro- and oligodendrogenesis. They also reveal important gender-specific differences and spatial heterogeneity, providing new avenues for stimulating endogenous repair in neurodegenerative diseases.

Expression of the inactivating deiodinase, Deiodinase 3, in the pre-metamorphic tadpole retina.

Thyroid hormone (TH) orchestrates amphibian metamorphosis. Thus, this developmental phase is often used to study TH-dependent responses in specific tissues. However, TH signaling appears early in development raising the question of the control of TH availability in specific cell types prior to metamorphosis. TH availability is under strict temporal and tissue-specific control by deiodinases. We examined the expression of the TH-inactivating enzyme, deiodinase type 3 (D3), during early retinal development. To this end we created a Xenopus laevis transgenic line expressing GFP from the Xenopus dio3 promoter region (pdio3) and followed pdio3-GFP expression in pre-metamorphic tadpoles. To validate retinal GFP expression in the transgenic line as a function of dio3 promoter activity, we used in situ hybridization to compare endogenous dio3 expression to reporter-driven GFP activity. Retinal expression of dio3 increased during pre-metamorphosis through stages NF41, 45 and 48. Both sets of results show dio3 to have cell-specific, dynamic expression in the pre-metamorphic retina. At stage NF48, dio3 expression co-localised with markers for photoreceptors, rods, Opsin-S cones and bipolar neurons. In contrast, in post-metamorphic juveniles dio3 expression was reduced and spatially confined to certain photoreceptors and amacrine cells. We compared dio3 expression at stages NF41 and NF48 with TH-dependent transcriptional responses using another transgenic reporter line: THbZIP-GFP and by analyzing the expression of T3-regulated genes in distinct TH availability contexts. At stage NF48, the majority of retinal cells expressing dio3 were negative for T3 signaling. Notably, most ganglion cells were virtually both dio3-free and T3-responsive. The results show that dio3 can reduce TH availability at the cellular scale. Further, a reduction in dio3 expression can trigger fine-tuned T3 action in cell-type specific maturation at the right time, as exemplified here in photoreceptor survival in the pre-metamorphic retina.

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 frog's 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-X(L) 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.

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 RARbeta-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 1(st) PA by CNCCs. We provide evidence showing that RA acts on the signalling epithelium of the 1(st) 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.

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.

Autonomous regulation of muscle fibre fate during metamorphosis in Xenopus tropicalis.

A key event in metamorphosis of anuran amphibians is tail resorption. This composite structure includes epidermal cells, spinal cord, muscle fibres and connective tissue. It is unclear how resorption proceeds and to what extent the signals for the death process are transmitted between cells. We determined the kinetics of metamorphosis, apoptosis, and tail regression in the diploid anuran, Xenopus tropicalis, a species more suited to genetic analysis than the pseudotetraploid, Xenopus laevis. Metamorphosis was found to proceed at a regular and predictable rate in X. tropicalis but not in X. laevis. Caspase 3 activity and mRNA levels were correlated with TdT-mediated dUTP nick end-labeling (TUNEL) signalling and most markedly increased in tail muscle and spinal cord. It has been proposed that muscles die as a result of loss of connectivity with the surrounding matrix. To test this hypothesis, we used direct DNA injection in trunk and tail muscle to overexpress Xenopus Bcl-X(L) (xR11), an anti-apoptotic gene, along with a marker gene (luciferase or GFP). xR11 significantly inhibited the cell death process in both trunk and tail muscle. This protection was functional even up to stage 64 on completion of tail regression. We conclude that (1) somatic gene transfer can be applied to analyse cell fate in X. tropicalis, and (2) that muscle death can be abrogated despite extracellular matrix loss.