RESUMEN
All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.
Asunto(s)
Competencia Celular , Células Clonales/patología , Leucemia Mieloide Aguda/patología , Análisis de la Célula Individual , Animales , Competencia Celular/efectos de los fármacos , Línea Celular , Linaje de la Célula/efectos de los fármacos , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Femenino , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Ratones , Ratones Endogámicos C57BL , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismoRESUMEN
BACKGROUND: Cellular barcoding is a recently developed biotechnology tool that enables the familial identification of progeny of individual cells in vivo. In immunology, it has been used to track the burst-sizes of multiple distinct responding T cells over several adaptive immune responses. In the study of hematopoiesis, it revealed fate heterogeneity amongst phenotypically identical multipotent cells. Most existing approaches rely on ex vivo viral transduction of cells with barcodes followed by adoptive transfer into an animal, which works well for some systems, but precludes barcoding cells in their native environment such as those inside solid tissues. RESULTS: With a view to overcoming this limitation, we propose a new design for a genetic barcoding construct based on the Cre Lox system that induces randomly created stable barcodes in cells in situ by exploiting inherent sequence distance constraints during site-specific recombination. We identify the cassette whose provably maximal code diversity is several orders of magnitude higher than what is attainable with previously considered Cre Lox barcoding approaches, exceeding the number of lymphocytes or hematopoietic progenitor cells in mice. CONCLUSIONS: Its high diversity and in situ applicability, make the proposed Cre Lox based tagging system suitable for whole tissue or even whole animal barcoding. Moreover, it can be built using established technology.
Asunto(s)
Ingeniería Genética/métodos , Integrasas/metabolismo , Recombinación Genética , Animales , Linfocitos T CD8-positivos/metabolismo , Variación Genética , Secuencias Invertidas Repetidas/genética , RatonesRESUMEN
Sexual development is initiated through differentiation of testicular Sertoli cells or ovarian granulosa cells. Although these supporting cells are considered to develop from common bipotential precursors, recent evidence suggests that distinct supporting cell populations are present in the ovary, with one providing granulosa cells of the medullary follicles and the other providing granulosa cells of the cortical follicles, the latter of which support lifelong fertility. Here, we demonstrate that XX fetal gonads contain GATA4 expressing supporting cells that either enter mitotic arrest, or remain proliferative. Blocking WNT signalling reduces XX supporting cell proliferation, while stabilising ß-catenin signalling promotes proliferation, indicating that the renewal of pre-granulosa cells is dependent on WNT/ß-catenin signalling in the proliferative supporting cell population. In contrast, XX supporting cells express p27 and FOXL2 and are maintained in mitotic arrest. Although FOXL2 is required for maintaining high levels of p27 expression, it is dispensable for entry and maintenance of mitotic arrest in XX supporting cells. Combined our data suggest that both medullary and cortical precursors arise from a common GATA4 expressing cell type. In addition, this work indicates that a balance between supporting cell self-renewal and differentiation is maintained in the developing ovary by relative WNT/ß-catenin and p27/FOXL2 activities. This study provides significant new insights into the origin and formation of ovarian follicles and evidence supporting a common fetal origin of medullary and cortical granulosa cells.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Factores de Transcripción Forkhead/metabolismo , Ovario/metabolismo , Proteína Wnt4/metabolismo , beta Catenina/metabolismo , Animales , Puntos de Control del Ciclo Celular , Diferenciación Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteína Forkhead Box L2 , Factores de Transcripción Forkhead/genética , Células de la Granulosa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones de la Cepa 129 , Ratones Noqueados , Ratones Transgénicos , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Ovario/citología , Ovario/embriologíaRESUMEN
EZH2 is frequently overexpressed in glioblastoma (GBM), suggesting an oncogenic function that could be a target for therapeutic intervention. However, reduced EZH2 activity can also promote tumorigenesis, leading to concerns about the use of EZH2 inhibitors. Here, we provide further insight about the effects of prolonged Ezh2 inhibition in glioblastoma using preclinical mouse models and primary tumor-derived human GBM cell lines. Using doxycycline-inducible shRNAs that mimic the effects of a selective EZH2 inhibitor, we demonstrate that prolonged Ezh2 depletion causes a robust switch in cell fate, including significantly enhanced proliferation, DNA damage repair, and activation of part of the pluripotency network, resulting in altered tumor cell identity and tumor progression. Short-term Ezh2 depletion significantly improved survival without the tumor progression observed upon prolonged Ezh2 depletion, suggesting that precise dosing regiments are very important. These results could be of high clinical relevance with regard to how glioblastomas should be treated with epigenetic therapies.
RESUMEN
BACKGROUND: Germ cell development involves formation of the spermatogenic or oogenic lineages from the bipotential primordial germ cells. Signaling mechanisms in the fetal testis and ovary determine whether germ cells enter the male or female developmental pathway, respectively. These signaling processes underpin an important phase of germ cell development, disruption of which can lead to failed germ cell function resulting in infertility or the formation of germ cell tumours. FINDINGS: In this study we have developed a small molecule screening protocol combined with flow cytometry to identify signaling pathways that direct male-specific development of germ cells. Here we provide a detailed method for this screening protocol, which we have used to identify signaling pathways important for male germ cell development. CONCLUSION: This method will be of particular use in screening inhibitors of signaling pathways, endocrine disruptors or other chemicals for their ability to disrupt testis and germ cell development, thereby providing insight into testicular dysgenesis and factors underlying poor male reproductive health.
Asunto(s)
Bibliotecas de Moléculas Pequeñas , Espermatozoides/efectos de los fármacos , Animales , Femenino , Técnicas In Vitro , Masculino , Ratones , Ratones Transgénicos , Espermatozoides/citologíaRESUMEN
The developing testis provides an environment that nurtures germ cell development, ultimately ensuring spermatogenesis and fertility. Impacts on this environment are considered to underlie aberrant germ cell development and formation of germ cell tumour precursors. The signaling events involved in testis formation and male fetal germ cell development remain largely unknown. Analysis of knockout mice lacking single Tgfß family members has indicated that Tgfß's are not required for sex determination. However, due to functional redundancy, it is possible that additional functions for these ligands in gonad development remain to be discovered. Using FACS purified gonadal cells, in this study we show that the genes encoding Activin's, TGFß's, Nodal and their respective receptors, are expressed in sex and cell type specific patterns suggesting particular roles in testis and germ cell development. Inhibition of signaling through the receptors ALK4, ALK5 and ALK7, and ALK5 alone, demonstrated that TGFß signaling is required for testis cord formation during the critical testis-determining period. We also show that signaling through the Activin/NODAL receptors, ALK4 and ALK7 is required for promoting differentiation of male germ cells and their entry into mitotic arrest. Finally, our data demonstrate that Nodal is specifically expressed in male germ cells and expression of the key pluripotency gene, Nanog was significantly reduced when signaling through ALK4/5/7 was blocked. Our strategy of inhibiting multiple Activin/NODAL/TGFß receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFß and Activin signaling during testis formation and male germ cell development.
Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Células Germinativas/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Testículo/citología , Testículo/metabolismo , Receptores de Activinas Tipo I/genética , Animales , Técnica del Anticuerpo Fluorescente , Células Germinativas/metabolismo , Immunoblotting , Masculino , Ratones , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
The spermatogenic and oogenic lineages originate from bipotential primordial germ cells in response to signalling in the foetal testis or ovary, respectively. The signals required for male germ cell commitment and their entry into mitotic arrest remain largely unknown. Recent data show that the ligand GDNF is up regulated in the foetal testis indicating that it may be involved in male germ cell development. In this study genetic analysis of GDNF-RET signalling shows that RET is required for germ cell survival. Affected germ cells in Ret-/- mice lose expression of key germ cell markers, abnormally express cell cycle markers and undergo apoptosis. Surprisingly, a similar phenotype was not detected in Gdnf-/- mice indicating that either redundancy with a Gdnf related gene might compensate for its loss, or that RET operates in a GDNF independent manner in mouse foetal germ cells. Either way, this study identifies the proto-oncogene RET as a novel component of the foetal male germ cell development pathway.
Asunto(s)
Células Germinativas/citología , Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Proteínas Proto-Oncogénicas c-ret/fisiología , Animales , Apoptosis , Ciclo Celular , Diferenciación Celular , Supervivencia Celular , Femenino , Células Germinativas/fisiología , Masculino , Ratones , Transducción de Señal , Testículo/citología , Testículo/fisiologíaRESUMEN
Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27(KIP1), p15(INK4B), activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility.
Asunto(s)
Feto/citología , Células Germinativas/citología , Mitosis , Teratoma/patología , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Susceptibilidad a Enfermedades/metabolismo , Susceptibilidad a Enfermedades/patología , Regulación hacia Abajo , Feto/metabolismo , Células Germinativas/metabolismo , Masculino , Ratones , Proteínas Nucleares/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción SOXB1/metabolismo , Testículo/citología , Testículo/metabolismo , Factores de TranscripciónRESUMEN
Mammalian germ cells are derived from the pluripotent epiblast and share features with pluripotent stem cells, including the expression of key genes that regulate developmental potency. The core genes Oct4, Sox2, and Nanog that regulate pluripotency in stem cells also perform important roles in regulating germ cell development and potentially in occurrence of germ line tumors in humans. Despite this, our understanding of the regulation of these genes during germ cell development remains limited. In this study we examine the regulation of pluripotency in the mouse fetal germ line. We show that male-specific methylation occurs in key functional elements of the Nanog and Sox2 promoters, and these genes are suppressed during early male germ cell differentiation. Furthermore, Oct4 translation is suppressed post-transcriptionally as germ cells differentiate down the male lineage and enter mitotic arrest. Combined, our data strongly support the conclusion that repression of the core machinery regulating pluripotency is a robust and early event involved in the differentiation of the male germ cell lineage. We hypothesize that active repression of pluripotency is required for fetal male germ cell differentiation and that failure of this mechanism may render germ cells susceptible to tumor formation.
Asunto(s)
Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Células Germinativas/citología , Células Madre Pluripotentes/citología , Animales , Linaje de la Célula/genética , Metilación de ADN , Proteínas de Homeodominio/genética , Masculino , Ratones , Proteína Homeótica Nanog , Regiones Promotoras Genéticas , Factores de Transcripción SOXB1/genéticaRESUMEN
During mouse embryonic development germ cells proliferate extensively until they commit to the male or female pathway and arrest in mitosis or meiosis respectively. Whilst the transition of female germ cells exiting the mitotic cell cycle and entering meiosis is well defined histologically, the essential cell cycle proteins involved in this process have remained unresolved. Using flow cytometry we have examined the entry of female germ cells into meiosis, their termination of DNA synthesis and entry into prophase I. Analysis of key G(2)/M cell cycle proteins revealed that entry into meiosis and cell cycle exit at G(2)/M involves repression of G(2)/M promoting Cyclin B1, coincident upregulation of G(2)/M repressing Cyclin B3 and robust establishment of the ATM/CHK2 pathway. By contrast we show that the ATR/CHK1 pathway is activated in male and female germ cells. This data indicates that an important G(2)/M surveillance mechanism operates during germ cell proliferation and that passage into meiotic G(2)/M involves the combined repression of G(2)/M through Cyclin B3 and activation of the key G(2)/M checkpoint regulatory network modulated through ATM and CHK2. This work shows that the core regulatory machinery that controls G(2)/M progression in mitotic cells is activated in female mouse germ cells as they enter meiosis.
Asunto(s)
Células Germinativas/metabolismo , Meiosis , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , División Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Ciclina B/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Fase G2 , Células Germinativas/citología , Masculino , Ratones , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Real-time PCR has become a popular method to analyze transcription of genes that are developmentally regulated during organogenesis of the testes and ovaries. However, the heterogenous cell populations and commitment to strikingly different developmental pathways of the germ and somatic cells in these organs complicate analysis of this process. The selection of suitable reference genes for quantifying gene expression in this system is essential, but to date it has not been sufficiently addressed. To rectify this problem, we have used fluorescence-activated cell sorting to purify germ cells from mouse fetal testes and ovaries and examined 16 common housekeeping genes for their suitability as reference genes. In pure populations of germ cells isolated from Embryonic Day 12.5 (E12.5) to E15.5 male and female gonads, Mapk1 and Sdha were identified as the most stable reference genes. Analysis of the heterogenous fraction of gonadal somatic cells revealed that Canx and Top1 were stable in both sexes, whereas a comparative analysis of germ and somatic cell populations identified Canx and Mapk1 as suitable reference genes through these developmental stages. Application of these reference genes to quantification of gene expression in developing gonads revealed that past assays, which employed nonverified reference genes, have in some cases provided misleading gene expression profiles. This study has identified suitable reference genes to directly compare expression profiles of genes expressed in germ and somatic cells of male and female fetal gonads. Application of these reference genes to expression analysis in fetal germ and somatic cells provides a more accurate system in which to profile gene expression in these tissues.
Asunto(s)
Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Gónadas/embriología , Organogénesis/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Animales , Calnexina/genética , Calnexina/metabolismo , Separación Celular , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Femenino , Citometría de Flujo , Gónadas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Embarazo , ARN/aislamiento & purificación , ARN/metabolismo , ARN/normas , Estabilidad del ARN , ARN sin Sentido/biosíntesis , ARN sin Sentido/metabolismo , Factores SexualesRESUMEN
During fetal mouse development, germ cells enter the developing gonad at embryonic day (E) 10-11. In response to signaling from the male or female gonad, the germ cells commit either to spermatogenesis at E12.5 and enter mitotic arrest or to oogenesis and enter meiotic arrest at E13.5. It is unclear whether male commitment of the germ line and mitotic arrest are directly associated or whether they are developmentally separate. In addition, the published data describing the timing of mitotic arrest are inconsistent, and the molecular processes underlying the control of the cell cycle during mitotic arrest also remain unknown. Using flow cytometric techniques, 5-bromo-2'-deoxyuridine labeling, and immunofluorescent analysis of cell proliferation, we have determined that germ cells in the embryonic mouse testis arrest in G0 during E12.5 and E14.5. This process is gradual and occurs in an unsynchronized manner. We have also purified germ cells and analyzed molecular changes in male germ cells as they exit the cell cycle. This has allowed us to identify a series of molecular events, including activation of p27(Kip1), p15(INK4b), and p16(INK4a); the dephosphorylation and degradation of retinoblastoma protein; and the suppression of CyclinE, which lead to mitotic arrest. For the first time, the data presented here accurately define the mitotic arrest of male germ cells by directly combining the analysis of cell cycle changes with the examination of functionally defined cell cycle regulators.