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1.  RNAi-Mediated Gene Function Analysis in Skin 
We have recently developed a method for RNAi-mediated gene function analysis in skin (Beronja et al., Nat Med 16:821–827, 2010). It employs ultrasound-guided in utero microinjections of lentivirus into the amniotic cavity of embryonic day 9 mice, which result in rapid, efficient, and stable transduction into mouse skin. Our technique greatly extends the available molecular and genetic toolbox for comprehensive functional examination of outstanding problems in epidermal biology. In its simplest form, as a single-gene function analysis via shRNA-mediated gene knockdown, our technique requires no animal mating and may need as little as only a few days between manipulation and phenotypic analysis.
doi:10.1007/978-1-62703-227-8_23
PMCID: PMC4160826  PMID: 23325656
Lentivirus; Intraamniotic; In utero; In vivo RNAi; shRNA; RNA interference; Ultrasound-guided microinjection
2.  Direct in Vivo RNAi Screen Unveils Myosin IIa as a Tumor Suppressor of Squamous Cell Carcinomas 
Science (New York, N.Y.)  2014;343(6168):309-313.
Mining modern genomics for cancer therapies is predicated on weeding out “bystander” alterations (nonconsequential mutations) and identifying “driver” mutations responsible for tumorigenesis and/or metastasis. We used a direct in vivo RNA interference (RNAi) strategy to screen for genes that upon repression predispose mice to squamous cell carcinomas (SCCs). Seven of our top hits—including Myh9, which encodes nonmuscle myosin IIa—have not been linked to tumor development, yet tissue-specific Myh9 RNAi and Myh9 knockout trigger invasive SCC formation on tumor-susceptible backgrounds. In human and mouse keratinocytes, myosin IIa's function is manifested not only in conventional actin-related processes but also in regulating posttranscriptional p53 stabilization. Myosin IIa is diminished in human SCCs with poor survival, which suggests that in vivo RNAi technology might be useful for identifying potent but low-penetrance tumor suppressors.
doi:10.1126/science.1248627
PMCID: PMC4159249  PMID: 24436421
3.  RNAi screens in mice identify physiological regulators of oncogenic growth 
Nature  2013;501(7466):185-190.
Summary
Tissue growth is the multifaceted outcome of a cell’s intrinsic capabilities and its interactions with the surrounding environment. Decoding these complexities is essential for understanding human development and tumorigenesis. Here, we tackle this problem by carrying out the first genome-wide RNAi-mediated screens in mice. Focusing on skin development and oncogenic (HrasG12V-induced) hyperplasia, our screens uncover novel as well as anticipated regulators of embryonic epidermal growth. Among top oncogenic screen hits are Mllt6 and the Wnt effector β-catenin; they maintain HrasG12V-dependent hyperproliferation. We also expose β-catenin as an unanticipated antagonist of normal epidermal growth, functioning through Wnt-independent intercellular adhesion. Finally, we document physiological relevance to mouse and human cancers, thereby establishing the feasibility of in vivo mammalian genome-wide investigations to dissect tissue development and tumorigenesis. By documenting some oncogenic growth regulators, we pave the way for future investigations of other hits and raise promise for unearthing new targets for cancer therapies.
doi:10.1038/nature12464
PMCID: PMC3774280  PMID: 23945586
4.  An RNA interference screen uncovers a new molecule in stem cell self-renewal and long-term regeneration 
Nature  2012;485(7396):104-108.
Adult stem cells sustain tissue maintenance and regeneration throughout the lifetime of an animal1,2. These cells often reside in specific signalling niches that orchestrate the stem cell’s balancing act between quiescence and cell-cycle re-entry based on the demand for tissue regeneration2–4. How stem cells maintain their capacity to replenish themselves after tissue regeneration is poorly understood. Here we use RNA-interference-based loss-of-function screening as a powerful approach to uncover transcriptional regulators that govern the self-renewal capacity and regenerative potential of stem cells. Hair follicle stem cells provide an ideal model. These cells have been purified and characterized from their native niche in vivo and, in contrast to their rapidly dividing progeny, they can be maintained and passaged long-term in vitro5–7. Focusing on the nuclear proteins and/or transcription factors that are enriched in stem cells compared with their progeny5,6, we screened ~2,000 short hairpin RNAs for their effect on long-term, but not short-term, stem cell self-renewal in vitro. To address the physiological relevance of our findings, we selected one candidate that was uncovered in the screen: TBX1. This transcription factor is expressed in many tissues but has not been studied in the context of stem cell biology. By conditionally ablating Tbx1 in vivo, we showed that during homeostasis, tissue regeneration occurs normally but is markedly delayed. We then devised an in vivo assay for stem cell replenishment and found that when challenged with repetitive rounds of regeneration, the Tbx1-deficient stem cell niche becomes progressively depleted. Addressing the mechanism of TBX1 action, we discovered that TBX1 acts as an intrinsic rheostat of BMP signalling: it is a gatekeeper that governs the transition between stem cell quiescence and proliferation in hair follicles. Our results validate the RNA interference screen and underscore its power in unearthing new molecules that govern stem cell self-renewal and tissue-regenerative potential.
doi:10.1038/nature10940
PMCID: PMC3600643  PMID: 22495305
5.  A Breath of Fresh Air in Lung Regeneration 
Cell  2011;147(3):485-487.
Enhancing the ability of the lungs to regenerate following injury could revolutionize the treatment of a wide range of different diseases. In this issue, Kumar et al. (2011) and Ding et al. (2011) dissect the cellular and molecular mechanisms of murine lung regeneration following injury and provide insights into the basic biology of the organ with implications for development of future therapeutic approaches.
doi:10.1016/j.cell.2011.10.008
PMCID: PMC3289402  PMID: 22036554
6.  Asymmetric Cell Divisions Promote Notch-Dependent Epidermal Differentiation 
Nature  2011;470(7334):353-358.
Summary
Stem and progenitor cells utilize asymmetric cell divisions to balance proliferation and differentiation. Evidence from lower eukaryotes shows that this process is regulated by proteins asymmetrically distributed at the cell cortex during mitosis: (1) Par3-Par6-aPKC, conferring polarity; (2) Gαi-LGN/AGS3-NuMA-p150glued, governing spindle positioning. Here, we focus on developing mouse skin, where progenitors execute a switch from predominantly symmetric to asymmetric divisions concomitant with stratification. Using in vivo skin-specific lentiviral RNAi, we investigate spindle orientation regulation and provide direct evidence that LGN, Numa1 and Dctn1 are involved. In compromising asymmetric cell divisions, we uncover profound defects in stratification, differentiation and barrier formation, and implicate Notch signalling as an important effector. Our study demonstrates the efficacy of applying RNAi in vivo to mammalian systems, and the ease of uncovering complex genetic interactions, here to gain insights into how changes in spindle orientation are coupled to establishing proper tissue architecture during skin development.
doi:10.1038/nature09793
PMCID: PMC3077085  PMID: 21331036
7.  Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos 
Nature medicine  2010;16(7):821-827.
Using ultrasound-guided in utero infections of fluorescently traceable lentiviruses carrying RNAi or Cre recombinase into mouse embryos, we have demonstrated noninvasive, highly efficient selective transduction of surface epithelium, in which progenitors stably incorporate and propagate the desired genetic alterations. We achieved epidermal-specific infection using small generic promoters of existing lentiviral short hairpin RNA libraries, thus enabling rapid assessment of gene function as well as complex genetic interactions in skin morphogenesis and disease in vivo. We adapted this technology to devise a new quantitative method for ascertaining whether a gene confers a growth advantage or disadvantage in skin tumorigenesis. Using α1-catenin as a model, we uncover new insights into its role as a widely expressed tumor suppressor and reveal physiological interactions between Ctnna1 and the Hras1-Mapk3 and Trp53 gene pathways in regulating skin cell proliferation and apoptosis. Our study illustrates the strategy and its broad applicability for investigations of tissue morphogenesis, lineage specification and cancers.
doi:10.1038/nm.2167
PMCID: PMC2911018  PMID: 20526348
8.  The FERM Protein Yurt Is a Negative Regulatory Component of the Crumbs Complex that Controls Epithelial Polarity and Apical Membrane Size 
Developmental cell  2006;11(3):363-374.
Summary
The Crumbs (Crb) complex is a key regulator of epithelial cell architecture where it promotes apical membrane formation. Here, we show that binding of the FERM protein Yurt to the cytoplasmic domain of Crb is part of a negative-feedback loop that regulates Crb activity. Yurt is predominantly a basolateral protein but is recruited by Crb to apical membranes late during epithelial development. Loss of Yurt causes an expansion of the apical membrane in embryonic epithelia and photoreceptor cells similar to Crb overexpression and in contrast to loss of Crb. Analysis of yurt crb double mutants suggests that these genes function in one pathway and that yurt negatively regulates crb. We also show that the mammalian Yurt orthologs YMO1 and EHM2 bind to mammalian Crb proteins. We propose that Yurt is part of an evolutionary conserved negative-feedback mechanism that restricts Crb complex activity in promoting apical membrane formation.
doi:10.1016/j.devcel.2006.06.001
PMCID: PMC2834949  PMID: 16950127
9.  Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells 
The Journal of Cell Biology  2005;169(4):635-646.
Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.
doi:10.1083/jcb.200410081
PMCID: PMC2171699  PMID: 15897260

Results 1-9 (9)