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  • Wip1 phosphatase is associated with chromatin and dephosphorylates gammaH2AX to promote checkpoint inhibition. 20101220

    DNA double-stranded breaks (DSBs) elicit a checkpoint response that causes a delay in cell cycle progression. Early in the checkpoint response, histone H2AX is phosphorylated in the chromatin region flanking the DSB by ATM/ATR and DNA-PK kinases. The resulting foci of phosphorylated H2AX (gamma-H2AX) serve as a platform for recruitment and retention of additional components of the checkpoint-signaling cascade that enhance checkpoint signaling and DSB repair. Upon repair, both the assembled protein complexes and the chromatin modifications are removed to quench the checkpoint signal. In this study, we show that the DNA damage-responsive Wip1 phosphatase is bound to chromatin. Moreover, Wip1 directly dephosphorylates gamma-H2AX and cells depleted of Wip1 fail to dephosphorylate gamma-H2AX during checkpoint recovery. Conversely, premature activation of Wip1 leads to displacement of MDC1 from damage foci and prevents activation of the checkpoint. Taken together, our data show that Wip1 has an essential role in dephosphorylation of gamma-H2AX to silence the checkpoint and restore chromatin structure once DNA damage is repaired.
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  • The homeobox gene Gsx2 regulates the self-renewal and differentiation of neural stem cells and the cell fate of postnatal progenitors. 22242181

    The Genetic screened homeobox 2 (Gsx2) transcription factor is required for the development of olfactory bulb (OB) and striatal neurons, and for the regional specification of the embryonic telencephalon. Although Gsx2 is expressed abundantly by progenitor cells in the ventral telencephalon, its precise function in the generation of neurons from neural stem cells (NSCs) is not clear. Similarly, the role of Gsx2 in regulating the self-renewal and multipotentiality of NSCs has been little explored. Using retroviral vectors to express Gsx2, we have studied the effect of Gsx2 on the growth of NSCs isolated from the OB and ganglionic eminences (GE), as well as its influence on the proliferation and cell fate of progenitors in the postnatal mouse OB. Expression of Gsx2 reduces proliferation and the self-renewal capacity of NSCs, without significantly affecting cell death. Furthermore, Gsx2 overexpression decreases the differentiation of NSCs into neurons and glia, and it maintains the cells that do not differentiate as cycling progenitors. These effects were stronger in GESCs than in OBSCs, indicating that the actions of Gsx2 are cell-dependent. In vivo, Gsx2 produces a decrease in the number of Pax6+ cells and doublecortin+ neuroblasts, and an increase in Olig2+ cells. In summary, our findings show that Gsx2 inhibits the ability of NSCs to proliferate and self-renew, as well as the capacity of NSC-derived progenitors to differentiate, suggesting that this transcription factor regulates the quiescent and undifferentiated state of NSCs and progenitors. Furthermore, our data indicate that Gsx2 negatively regulates neurogenesis from postnatal progenitor cells.
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  • ApoA-II directs morphogenetic movements of zebrafish embryo by preventing chromosome fusion during nuclear division in yolk syncytial layer. 21212265

    The high density lipoprotein (HDL) represents a class of lipid- and protein-containing particles and consists of two major apolipoproteins apoA-I and apoA-II. ApoA-II has been shown to be involved in the pathogenesis of insulin resistance, adiposity, diabetes, and metabolic syndrome. In embryo, apoa2 mRNAs are abundant in the liver, brain, lung, placenta, and in fish yolk syncytial layer (YSL), suggesting that apoa2 may perform a function during embryonic development. Here we find out that apoa2 modulates zebrafish embryonic development by regulating the organization of YSL. Disruption of apoa2 function in zebrafish caused chromosome fusing, which strongly blocked YSL nuclear division, inducing disorders in YSL organization and finally disturbing the embryonic epiboly. Purified native human apoA-II was able specifically to rescue the defects and induced nuclear division in zebrafish embryos and in human HeLa cells. The C terminus of apoA-II was required for the proper chromosome separation during nuclear division of YSL in zebrafish embryos and in human HeLa cells. Our data indicate that organization of YSL is required for blastoderm patterning and morphogenesis and suggest that apolipoprotein apoA-II is a novel factor of nuclear division in YSL involved in the regulation of early zebrafish embryonic morphogenesis and in mammalian cells for proliferation.
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  • Effects of granulosa cell-specific deletion of Rb in Inha-alpha null female mice. 17510234

    Our laboratory is interested in the gonadal growth regulatory properties of inhibins, members of the TGFbeta superfamily. We have previously shown that female mice lacking inhibins (Inha(-/-)) develop granulosa cell tumors and that concurrent loss of p27 accelerates tumor development. It has also been shown that the retinoblastoma protein RB regulates the G(1) to S phase transition of the cell cycle by controlling the activity of transcription factors and stabilizing the levels of the cell cycle inhibitor P27. Based on these data, we hypothesized that concurrent loss of Rb and inhibins in the ovary will exacerbate tumor formation. To test this hypothesis, we generated an ovarian granulosa cell conditional knockout (cKO) of Rb using the Cre/lox recombination system in the background of Inha(-/-) mice. Inha(-/-)/Rb cKO females show a modest increase in mortality rates compared with Inha(-/-) females. Although histologically similar to Inha(-/-) ovarian tumors, tumors from Inha(-/-)/Rb cKO females show increased number of mitotic figures and apoptotic rates. Interestingly, P27 levels are decreased in Inha(-/-)/Rb cKO ovarian tumors, likely due to the combined effect of Rb loss and increased Skp2 expression, which targets P27 to the proteosome. We propose that Rb loss may cause cell cycle delay or arrest, followed by apoptosis and that increases in p107 and p130 levels may compensate for Rb loss. These findings confirm the importance of P27 as a cell cycle regulator in granulosa cells and suggest functional compensation between RB-like proteins in ovarian tumorigenesis.
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  • Cyclin-dependent kinase suppression by WEE1 kinase protects the genome through control of replication initiation and nucleotide consumption. 22907750

    Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage.
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  • Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. 21828091

    Muscle regeneration requires the coordinated interaction of multiple cell types. Satellite cells have been implicated as the primary stem cell responsible for regenerating muscle, yet the necessity of these cells for regeneration has not been tested. Connective tissue fibroblasts also are likely to play a role in regeneration, as connective tissue fibrosis is a hallmark of regenerating muscle. However, the lack of molecular markers for these fibroblasts has precluded an investigation of their role. Using Tcf4, a newly identified fibroblast marker, and Pax7, a satellite cell marker, we found that after injury satellite cells and fibroblasts rapidly proliferate in close proximity to one another. To test the role of satellite cells and fibroblasts in muscle regeneration in vivo, we created Pax7(CreERT2) and Tcf4(CreERT2) mice and crossed these to R26R(DTA) mice to genetically ablate satellite cells and fibroblasts. Ablation of satellite cells resulted in a complete loss of regenerated muscle, as well as misregulation of fibroblasts and a dramatic increase in connective tissue. Ablation of fibroblasts altered the dynamics of satellite cells, leading to premature satellite cell differentiation, depletion of the early pool of satellite cells, and smaller regenerated myofibers. Thus, we provide direct, genetic evidence that satellite cells are required for muscle regeneration and also identify resident fibroblasts as a novel and vital component of the niche regulating satellite cell expansion during regeneration. Furthermore, we demonstrate that reciprocal interactions between fibroblasts and satellite cells contribute significantly to efficient, effective muscle regeneration.
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  • Role of noggin as an upstream signal in the lack of neuronal derivatives found in the avian caudal-most neural crest. 19369402

    Neural crest cells (NCCs) arising from trunk neural tube (NT) during primary and secondary neurulation give rise to melanocytes, glia and neurons, except for those in the caudal-most region during secondary neurulation (somites 47 to 53 in the chick embryo), from which no neurons are formed, either in vivo or in vitro. To elucidate this discrepancy, we have specifically analyzed caudal-most NCC ontogeny. In this region, NCCs emerge at E5/HH26, one day after full cavitation of the NT and differentiation of flanking somites. The absence of neurons does not seem to result from a defect in NCC specification as all the usual markers, with the exception of Msx1, are expressed in the dorsal caudal-most NT as early as E4/HH24. However, Bmp4-Wnt1 signaling, which triggers trunk NCC delamination, is impaired in this region due to persistence of noggin (Nog) expression. Concomitantly, a spectacular pattern of apoptosis occurs in the NT dorsal moiety. Rostral transplantation of either the caudal-most somites or caudal-most NT reveals that the observed features of caudal-most NCCs relate to properties intrinsic to these cells. Furthermore, by forced Nog expression in the trunk NT, we can reproduce most of these particular features. Conversely, increased Bmp4-Wnt1 signaling through Nog inhibition in the caudal-most NT at E4/HH24 induces proneurogenic markers in migratory NCCs, suggesting that noggin plays a role in the lack of neurogenic potential characterizing the caudal-most NCCs.
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  • MID1 and MID2 are required for Xenopus neural tube closure through the regulation of microtubule organization. 20534674

    Closure of the neural tube requires both the change and maintenance of cell shape. The change occurs mainly through two coordinated morphogenetic events: cell elongation and apical constriction. How cytoskeletal elements, including microtubules, are regulated in this process in vivo is largely unknown. Here, we show that neural tube closure in Xenopus depends on orthologs of two proteins: MID1, which is responsible for Opitz G/BBB syndrome in humans, and its paralog MID2. Depletion of the Xenopus MIDs (xMIDs) by morpholino-mediated knockdown disrupted epithelial morphology in the neural plate, leading to neural tube defects. In the xMID-depleted neural plate, the normal epithelial organization was perturbed without affecting neural fate. Furthermore, the xMID knockdown destabilized and caused the disorganization of microtubules, which are normally apicobasally polarized, accounting for the abnormal phenotypes. We also found that the xMIDs and their interacting protein Mig12 were coordinately required for microtubule stabilization during remodeling of the neural plate. Finally, we showed that the xMIDs are required for the formation of multiple epithelial organs. We propose that similar MID-governed mechanisms underlie the normal morphogenesis of epithelial tissues and organs, including the tissues affected in patients with Opitz G/BBB syndrome.
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