Sox2 -Deficient Müller Glia Disrupt the Structural and Functional Maturation of the Mammalian Retina

Müller glia (MG), the principal glial cells of the vertebrate retina, display quiescent progenitor cell characteristics. They express key progenitor markers, including the high mobility group box transcription factor SOX2 and maintain a progenitor-like morphology. In the embryonic and mature central nervous system, SOX2 maintains neural stem cell identity. However, its function in committed Müller glia has yet to be determined

Viral Single-Strand DNA Induces p53-Dependent Apoptosis in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication

Ground-State Transcriptional Requirements for Skin-Derived Precursors

Skin-derived precursors (SKPs) are an attractive stem cell model for cell-based therapies. SKPs can be readily generated from embryonic and adult mice and adult humans, exhibit a high degree of multipotency, and have the potential to serve as a patient autologous stem cell. The advancement of these cells toward therapeutic use depends on the ability to control precisely the self-renewal and differentiation of SKPs. Here we show that two well-known stem cell factors, Foxd3 and Sox2, are critical regulators of the stem cell properties of SKPs. Deletion of Foxd3 completely abolishes the sphere-forming potential of these cells. In the absence of Sox2, SKP spheres can be formed, but with reduced size and frequency. Our results provide entry points into the gene regulatory networks dictating SKP behavior, and pave the way for future studies on a therapeutically relevant stem cell

Human Embryonic Stem Cells Have Constitutively Active Bax at the Golgi and Are Primed to Undergo Rapid Apoptosis

Human embryonic stem (hES) cells activate a rapid apoptotic response after DNA damage but the underlying mechanisms are unknown. A critical mediator of apoptosis is Bax, which is reported to become active and translocate to the mitochondria only after apoptotic stimuli. Here we show that undifferentiated hES cells constitutively maintain Bax in its active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having pre-activated Bax. After DNA damage, active Bax rapidly translocated to the mitochondria by a p53-dependent mechanism. Interestingly, upon differentiation, Bax was no longer active and cells were not acutely sensitive to DNA damage. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development

Casz1 is required for cardiomyocyte G1-to-S phase progression during mammalian cardiac development

Organ growth occurs through the integration of external growth signals during the G1 phase of the cell cycle to initiate DNA replication. Although numerous growth factor signals have been shown to be required for the proliferation of cardiomyocytes, genetic studies have only identified a very limited number of transcription factors that act to regulate the entry of cardiomyocytes into S phase. Here, we report that the cardiac para-zinc-finger protein CASZ1 is expressed in murine cardiomyocytes. Genetic fate mapping with an inducible Casz1 allele demonstrates that CASZ1-expressing cells give rise to cardiomyocytes in the first and second heart fields. We show through the generation of a cardiac conditional null mutation that Casz1 is essential for the proliferation of cardiomyocytes in both heart fields and that loss of Casz1 leads to a decrease in cardiomyocyte cell number. We further report that the loss of Casz1 leads to a prolonged or arrested S phase, a decrease in DNA synthesis, an increase in phospho-RB and a concomitant decrease in the cardiac mitotic index. Taken together, these studies establish a role for CASZ1 in mammalian cardiomyocyte cell cycle progression in both the first and second heart fields

TAp63 Prevents Premature Aging by Promoting Adult Stem Cell Maintenance

The cellular mechanisms that regulate the maintenance of adult tissue stem cells are still largely unknown. We show here that the p53 family member, TAp63, is essential for maintenance of epidermal and dermal precursors and that, in its absence, these precursors senesce and skin ages prematurely. Specifically, we have developed a TAp63 conditional knockout mouse and used it to ablate TAp63 in the germline (TAp63−/−) or in K14-expressing cells in the basal layer of the epidermis (TAp63fl/fl;K14cre+). TAp63−/− mice age prematurely and develop blisters, skin ulcerations, senescence of hair follicle-associated dermal and epidermal cells, and decreased hair morphogenesis. These phenotypes are likely due to loss of TAp63 in dermal and epidermal precursors since both cell types show defective proliferation, early senescence, and genomic instability. These data indicate that TAp63 serves to maintain adult skin stem cells by regulating cellular senescence and genomic stability, thereby preventing premature tissue aging

Identification of novel pathways involved in the pathogenesis of human adamantinomatous craniopharyngioma

Activating mutations in the gene encoding β-catenin have been identified in the paediatric form of human craniopharyngioma (adamantinomatous craniopharyngioma, ACP), a histologically benign but aggressive pituitary tumour accounting for up to 10% of paediatric intracranial tumours. Recently, we generated an ACP mouse model and revealed that, as in human ACP, nucleocytoplasmic accumulation of β-catenin (β-catnc) and over-activation of the Wnt/β-catenin pathway occurs only in a very small proportion of cells, which form clusters. Here, combining mouse genetics, fluorescence labelling and flow-sorting techniques, we have isolated these cells from tumorigenic mouse pituitaries and shown that the β-catnc cells are enriched for colony-forming cells when cultured in stem cell-promoting media, and have longer telomeres, indicating shared properties with normal pituitary progenitors/stem cells (PSCs). Global gene profiling analysis has revealed that these β-catnc cells express high levels of secreted mitogenic signals, such as members of the SHH, BMP and FGF family, in addition to several chemokines and their receptors, suggesting an important autocrine/paracrine role of these cells in the pathogenesis of ACP and a reciprocal communication with their environment. Finally, we highlight the clinical relevance of these findings by showing that these pathways are also up-regulated in the β-catnc cell clusters identified in human ACP. As well as providing further support to the concept that pituitary stem cells may play an important role in the oncogenesis of human ACP, our data reveal novel disease biomarkers and potential pharmacological targets for the treatment of these devastating childhood tumours.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-012-0957-9) contains supplementary material, which is available to authorized users

Sox2 is required for development of taste bud sensory cells

Sox2 is expressed in basal epithelial cells of the tongue, with high levels in taste bud placodes, fungiform papillae, and mature taste cells, and low levels in filiform papillae. High Sox2 expression appears to lie downstream from canonical Wnt signaling. In hypomorphic Sox2(EGFP/LP) embryos, placodes form but no mature taste buds develop. In contrast, transgenic overexpression of Sox2 in the basal cells inhibits differentiation of filiform keratinocytes. Together, our loss-of-function and gain-of-function studies suggest that Sox2 functions in a dose-dependent manner to regulate the differentiation of endodermal progenitor cells of the tongue into taste bud sensory cells versus keratinocytes

Establishment of the neurogenic boundary of the mouse retina requires cooperation of SOX2 and WNT signaling

BackgroundEye development in vertebrates relies on the critical regulation of SOX2 expression. Humans with mutations in SOX2 often suffer from eye defects including anophthalmia (no eye) and microphthalmia (small eye). In mice, deletion of Sox2 in optic cup progenitor cells results in loss of neural competence and cell fate conversion of the neural retina to a non-neurogenic fate, specifically the acquisition of fate associated with progenitors of the ciliary epithelium. This fate is also promoted with constitutive expression of stabilized β-Catenin in the optic cup, where the WNT pathway is up-regulated. We addressed whether SOX2 co-ordinates the neurogenic boundary of the retina through modulating the WNT/β-Catenin pathway by using a genetic approach in the mouse.ResultsUpon deletion of Sox2 in the optic cup, response to WNT signaling was expanded, correlating with loss of neural competence, cell fate conversion of the neural retina to ciliary epithelium primordium and, in addition, increased cell cycle time of optic cup progenitors. Removal of Ctnnb1 rescued the cell fate conversion; however, the loss of neural competence and the proliferation defect resulting from lack of SOX2 were not overcome. Lastly, central Sox2-deficient optic cup progenitor cells exhibited WNT-independent up-regulation of D-type Cyclins.ConclusionWe propose two distinct roles for SOX2 in the developing retina. Our findings suggest that SOX2 antagonizes the WNT pathway to maintain a neurogenic fate and, in contrast, regulates cycling of optic cup progenitors in a WNT-independent manner. Given that WNT signaling acting upstream of SOX2 has been implicated in the tumorigenicity of embryonic stem cell-derived retinal progenitor cells, our results distinguish the endogenous role of WNT signaling in early optic cup patterning and support a WNT-independent role for SOX2 in maintaining retinal progenitor cell proliferation.Electronic supplementary materialThe online version of this article (doi:10.1186/1749-8104-9-27) contains supplementary material, which is available to authorized users