ABSTRACT
Transcriptional control plays a key role in regulating epidermal proliferation and differentiation. Although ample information has been obtained on how epidermal homeostasis is controlled in adult skin, less is known about the control of proliferation/differentiation of epidermal stem/progenitor cells in the developing embryo. Ovol1, encoding a zinc finger protein homologous to Drosophila melanogaster Ovo, is expressed in embryonic epidermal progenitor cells that are transiting from proliferation to terminal differentiation. In this study, we demonstrate a function for Ovol1 in interfollicular epidermal development. In its absence, developing epidermis fails to properly restrict the proliferative potential of progenitor cells, and cultured keratinocytes fail to efficiently undergo growth arrest in response to extrinsic growth-inhibitory signals. We present molecular evidence that c-myc expression is up-regulated in Ovol1-deficient suprabasal cells and that Ovol1 represses c-myc transcription by directly binding to its promoter. Collectively, our findings indicate that Ovol1 is required for proliferation exit of committed epidermal progenitor cells and identify c-myc as an Ovol1 target.
Subject(s)
DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Epidermal Cells , Stem Cells/metabolism , Transcription Factors/metabolism , Transcription Factors/physiology , Transcription, Genetic , Animals , Base Sequence , Cell Line , Cell Proliferation , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/pharmacology , Down-Regulation , Epidermis/embryology , Female , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Mice , Mice, Transgenic , Models, Biological , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , Salivary alpha-Amylases , Stem Cells/cytology , Transcription Factors/drug effects , Transcription Factors/genetics , Transcription Factors/pharmacologyABSTRACT
BACKGROUND: An estimated 70.8 million people are forcibly displaced worldwide, 75% of whom are women and children. Prioritizing a global research agenda to inform guidance, service delivery, access to and quality of services is essential to improve the survival and health of women, children and adolescents in humanitarian settings. METHOD: A mixed-methods design was adapted from the Child Health and Nutrition Research Initiative (CHNRI) methodology to solicit priority research questions across the sexual, reproductive, maternal, newborn, child and adolescent health (SRMNCAH) domains in humanitarian settings. The first step (CHNRI) involved data collection and scoring of perceived priority questions, using a web-based survey over two rounds (first, to generate the questions and secondly, to score them). Over 1000 stakeholders from across the globe were approached; 177 took part in the first survey and 69 took part in the second. These research questions were prioritized by generating a research prioritization score (RPP) across four dimensions: answerability, program feasibility, public health relevance and equity. A Delphi process of 29 experts followed, where the 50 scored and prioritized CHRNI research questions were shortlisted. The top five questions from the CHNRI scored list for each SRMNCAH domain were voted on, rendering a final list per domain. RESULTS: A total of 280 questions were generated. Generated questions covered sexual and reproductive health (SRH) (n = 90, 32.1%), maternal health (n = 75, 26.8%), newborn health (n = 42, 15.0%), child health (n = 43, 15.4%), and non-SRH aspects of adolescent health (n = 31, 11.1%). A shortlist of the top ten prioritized questions for each domain were generated on the basis of the computed RPPs. During the Delphi process, the prioritized questions, based on the CHNRI process, were further refined. Five questions from the shortlist of each of the SRMNCAH domain were formulated, resulting in 25 priority questions across SRMNCAH. For example, one of the prioritized SRH shortlisted and prioritized research question included: "What are effective strategies to implement good quality comprehensive contraceptive services (long-acting, short-acting and EC) for women and girls in humanitarian settings?" CONCLUSION: Data needs, effective intervention strategies and approaches, as well as greater efficiency and quality during delivery of care in humanitarian settings were prioritized. The findings from this research provide guidance for researchers, program implementers, as well as donor agencies on SRMNCAH research priorities in humanitarian settings. A global research agenda could save the lives of those who are at greatest risk and vulnerability as well as increase opportunities for translation and innovation for SRMNCAH in humanitarian settings.
ABSTRACT
PURPOSE: We introduce and evaluate a high throughput biodosimetry test system (REDI-Dx) suitable for testing of thousands of potential radiation victims following a mass scale nuclear event caused by detonation of a nuclear device or a nuclear accident, as part of an overall strategy for effective medical management of the crisis. MATERIALS AND METHODS: The performance of a high throughput biodosimetry test was evaluated by collecting samples of both non-irradiated presumed healthy donors as well as irradiated subjects collected as part of either cancer treatment regimens or banked from previous studies. The test measures the gene expression of a set of radiation responsive genes based on the DxDirect® genomic platform. The potential diagnostic accuracy of REDI-Dx was evaluated as a predictor of actual dose of radiation. While the REDI-Dx test has been calibrated to provide a quantitative measure of actual absorbed dose, we compared the performance of the REDI-Dx test (sensitivity and specificity) as a qualitative result at the most commonly applied thresholds 2.0 Gy and 6.0 Gy. RESULTS: The test demonstrated high specificity and lack of effect of medical conditions. Using receiver operating characteristic (ROC) curve analysis, REDI-Dx was shown to be a good predictor of actual dose for determining treatment category based on either 2.0 or 6.0 Gy, with a 98.5% sensitivity and 90% specificity for 2.0 Gy, and 92% sensitivity and 84% specificity for 6.0 Gy. Results were reproducible between clinical laboratories with an SD of 0.2 Gy for samples ≤2.0 Gy and a CV of 10.3% for samples from 2.0 to 10.0 Gy. CONCLUSIONS: Use of a biodosimetry test, like REDI-Dx test system would provide valuable information that would improve the ability to assign patients to the correct treatment category when combined with currently available biodosimetry tools, as compared to the use of existing tools alone. The REDI-Dx biodosimetry test system is for investigational use only in the U.S.A. The performance characteristics of this product have not been established.
Subject(s)
Patient Selection , Radiation Injuries/therapy , Radioactive Hazard Release , Dose-Response Relationship, Radiation , Humans , Lymphocytes/radiation effects , Radiation Injuries/complications , Radiation Injuries/etiology , Radiometry , Vomiting/complicationsABSTRACT
Mammalian spermiogenesis, a process where haploid male germ cells differentiate to become mature spermatozoa, entails dramatic morphological and biochemical changes including remodeling of the germ cell chromatin. Proteins that contain one or more plant homeodomain (PHD) fingers have been implicated in the regulation of chromatin structure and function. Pygopus 2 (Pygo2) belongs to a family of evolutionarily conserved PHD finger proteins thought to act as co-activators of Wnt signaling effector complexes composed of beta-catenin and LEF/TCF transcription factor. Here we analyze mice containing hypomorphic alleles of pygopus 2 (Pygo2 or mpygo2) and uncover a beta-catenin-independent involvement of the Pygo2 protein in spermiogenesis. Pygo2 is expressed in elongating spermatids at stages when chromatin remodeling occurs, and block of Pygo2 function leads to spermiogenesis arrest and consequent infertility. Analysis of spermiogenesis in Pygo2 mutants reveals reduced expression of select post-meiotic genes including protamines, transition protein 2, and H1fnt, all of which are required for germ cell chromatin condensation, and drastically altered pattern of histone H3 hyperacetylation. These findings suggest that Pygo2 is involved in the chromatin remodeling events that lead to nuclear compaction of male germ cells.
Subject(s)
Chromatin Assembly and Disassembly , Histones/metabolism , Intracellular Signaling Peptides and Proteins/physiology , Spermatogenesis , Acetylation , Animals , Cell Nucleus , Male , Mice , Spermatids/chemistryABSTRACT
Ovol1 belongs to a family of evolutionarily conserved zinc finger proteins that act downstream of key developmental signaling pathways such as Wnt and TGF-beta/BMP. It plays important roles in epithelial and germ cell development, particularly by repressing c-Myc and Id2 genes and modulating the balance between proliferation and differentiation of progenitor cells. In this study, we show that Ovol1 negatively regulates its own expression by binding to and repressing the activity of its promoter. We further demonstrate that Ovol1 uses both passive and active repression mechanisms to auto-repress: (1) it antagonizes transcriptional activation of c-Myb, a known positive regulator of proliferation, by competing for DNA binding; (2) it recruits histone deacetylase activity to the promoter via an N-terminal SNAG repressor domain. At Ovol1 cognate sites in the endogenous Ovol1 promoter, c-Myb binding correlates with increased histone acetylation, whereas the expression of Ovol1 correlates with a displacement of c-Myb from the DNA and decreased histone acetylation. Collectively, our data suggest that Ovol1 restricts its own expression by counteracting c-Myb activation and histone acetylation of the Ovol1 promoter.
Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation , Histone Deacetylases/metabolism , Proto-Oncogene Proteins c-myb/antagonists & inhibitors , Repressor Proteins/genetics , Trans-Activators/antagonists & inhibitors , Transcription Factors/genetics , Acetylation , Animals , Binding Sites , Binding, Competitive , Cell Line , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Histones/metabolism , Mice , Promoter Regions, Genetic , Protein Structure, Tertiary , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Ovol1 encodes a zinc finger transcriptional repressor that is downstream of the LEF1/beta-catenin complex, nuclear effectors of canonical Wnt signaling. Previous gene knockout studies performed in a 129SvxC57BL/6 mixed genetic background revealed that Ovol1-deficient mice survive to adulthood but display multiple tissue defects. In this study, we describe a C57BL/6 strain-specific reduction in perinatal survival of Ovol1 mutant mice. The perinatal lethality is accompanied by kidney epithelial cysts of embryonic onset and delayed skin barrier acquisition. Genetic analysis suggests a partial functional compensation by Ovol2 for the loss of Ovol1. The expression of Ovol2 was up-regulated in Ovol1-deficient epidermis, and Ovol1 represses the activity of Ovol2 promoter in a DNA binding-dependent manner. Collectively, these studies uncover novel functions of Ovol1 in mouse development and identify Ovol2 as a downstream target of Ovol1.
Subject(s)
DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Epidermis/pathology , Fetal Death/genetics , Kidney Diseases, Cystic/genetics , Transcription Factors/deficiency , Transcription Factors/genetics , Animals , Base Sequence , Coloring Agents/pharmacokinetics , DNA-Binding Proteins/pharmacology , Epidermis/abnormalities , Epidermis/metabolism , Genes, Lethal , Germ-Line Mutation , Kidney Diseases, Cystic/pathology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Molecular Sequence Data , Transcription Factors/drug effects , Transcription Factors/metabolism , Transcription Factors/pharmacology , Transcription, Genetic , Up-RegulationABSTRACT
Previous studies have shown that a targeted deletion of Ovol1 (previously known as movo1), encoding a member of the Ovo family of zinc-finger transcription factors, leads to germ cell degeneration and defective sperm production in adult mice. To explore the cellular and molecular mechanism of Ovol1 function, we have examined the mutant testis phenotype during the first wave of spermatogenesis in juvenile mice. Consistent with the detection of Ovol1 transcripts in pachytene spermatocytes of the meiotic prophase, Ovol1-deficient germ cells were defective in progressing through the pachytene stage. The pachytene arrest was accompanied by an inefficient exit from proliferation, increased apoptosis and an abnormal nuclear localization of the G2-M cell cycle regulator cyclin B1, but was not associated with apparent chromosomal or recombination defects. Transcriptional profiling and northern blot analysis revealed reduced expression of pachytene markers in the mutant, providing molecular evidence that pachytene differentiation was defective. In addition, the expression of Id2 (inhibitor of differentiation 2), a known regulator of spermatogenesis, was upregulated in Ovol1-deficient pachytene spermatocytes and repressed by Ovol1 in reporter assays. Taken together, our studies demonstrate a role for Ovol1 in regulating pachytene progression of male germ cells, and identify Id2 as a Ovol1 target.
Subject(s)
DNA-Binding Proteins/metabolism , Pachytene Stage/physiology , Repressor Proteins/metabolism , Spermatogenesis/physiology , Transcription Factors/metabolism , Animals , Biomarkers , DNA-Binding Proteins/genetics , Gene Expression Profiling , Genes, Reporter , Inhibitor of Differentiation Protein 2 , Male , Mice , Mutation , Pachytene Stage/genetics , Repressor Proteins/genetics , Spermatogenesis/genetics , Testis/metabolism , Transcription Factors/geneticsABSTRACT
The ovo gene family consists of evolutionarily conserved genes including those cloned from Caenorhabditis elegans, Drosophila melanogaster, mouse, and human. Here we report the isolation and characterization of mouse Ovol2 (also known as movol2 or movo2) and provide evidence supporting the existence of multiple Ovol2 transcripts. These transcripts are produced by alternative promoter usage and alternative splicing and encode long and short OVOL2 protein isoforms, whose sequences differ from those previously reported. Mouse and human OVOL2 genes are expressed in overlapping tissues including testis, where Ovol2 expression is developmentally regulated and correlates with the meiotic/postmeiotic stages of spermatogenesis. Mouse Ovol2 maps to chromosome 2 in a region containing blind-sterile (bs), a spontaneous mutation that causes spermatogenic defects and germ cell loss. No mutation has been detected in the coding region of Ovol2 from bs mice, but Ovol2 transcription was dramatically reduced in testes from these mice, suggesting that Ovol2 is expressed in male germ cells.
Subject(s)
Chromosome Mapping , DNA-Binding Proteins/genetics , Drosophila Proteins , Drosophila/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , Base Sequence , Blindness/genetics , DNA-Binding Proteins/biosynthesis , Humans , Infertility/genetics , Keratinocytes/metabolism , Male , Mice , Molecular Sequence Data , Sequence Analysis, DNA , Testis/metabolism , Transcription Factors/biosynthesisABSTRACT
Drosophila ovo/svb (dovo) is required for epidermal cuticle/denticle differentiation and is genetically downstream of the wg signaling pathway. Similarly, a mouse homolog of dovo, movo1, is required for the proper formation of hair, a mammalian epidermal appendage. Here, we provide biochemical evidence that movo1 encodes a nuclear DNA binding protein (mOvo1a) that binds to DNA sequences similar to those that dOvo binds to, further supporting the notion that mOvo1a and dOvo are genetically and biochemically homologous proteins. Additionally, we show that the movo1 promoter is activated by the lymphoid enhancer factor 1 (LEF1)/beta-catenin complex, a transducer of wnt signaling. Collectively, our findings suggest that movo1 is a developmental target of wnt signaling during hair morphogenesis in mice, and that the wg/wnt-ovo link in epidermal appendage regulatory pathways has been conserved between mice and flies.