RESUMEN
The closely related C. elegans MEG-1 and MEG-2 proteins localize to P granules during a brief period of embryogenesis when the germ lineage is being separated from the soma. Embryonic primordial germ cells still develop in the absence of MEG activity, but major defects emerge during larval stages when germ cells fail to proliferate or differentiate normally, resulting in sterility. To investigate meg-1 function, we conducted a targeted RNAi screen for enhancers and suppressors of meg-1 sterility. Here, we show that meg-1 interacts with multiple pathways that promote germ cell proliferation and survival. Surprisingly, we found that two nanos family members had opposing effects on the meg-1 phenotype. Loss of nos-3 suppressed the meg-1 phenotype, restoring fertility, while loss of nos-2 enhanced the meg-1 phenotype, abolishing proliferation and causing early and pronounced germ cell degeneration. Together, our analyses suggest that, under circumstances that favor proliferation, MEG function is not essential for germ cells to proliferate, although it is important for optimal proliferation. Additionally, MEG activity is likely more directly involved in germ cell survival than previously thought. genesis 49:380-391, 2011.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proliferación Celular , Células Germinativas/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/embriología , Supervivencia Celular/genética , Trastornos del Desarrollo Sexual/genética , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Epistasis Genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/citología , Proteínas Luminiscentes/genética , Masculino , Interferencia de ARN , Proteínas de Unión al ARN/genética , Factores de Tiempo , Proteína Fluorescente RojaRESUMEN
Erythropoietin (EPO's) actions on erythroblasts are ascribed largely to survival effects. Certain studies, however, point to EPO-regulated proliferation. To investigate this problem in a primary system, Kit(pos)CD71(high) erythroblasts were prepared from murine bone marrow, and were first used in the array-based discovery of EPO-modulated cell-cycle regulators. Five cell-cycle progression factors were rapidly up-modulated: nuclear protein 1 (Nupr1), G1 to S phase transition 1 (Gspt1), early growth response 1 (Egr1), Ngfi-A binding protein 2 (Nab2), and cyclin D2. In contrast, inhibitory cyclin G2, p27/Cdkn1b, and B-cell leukemia/lymphoma 6 (Bcl6) were sharply down-modulated. For CYCLIN G2, ectopic expression also proved to selectively attenuate EPO-dependent UT7epo cell-cycle progression at S-phase. As analyzed in primary erythroblasts expressing minimal EPO receptor alleles, EPO repression of cyclin G2 and Bcl6, and induction of cyclin D2, were determined to depend on PY343 (and Stat5) signals. Furthermore, erythroblasts expressing a on PY-null EPOR-HM allele were abnormally distributed in G0/G1. During differentiation divisions, EPOR-HM Ter119(pos) erythroblasts conversely accumulated in S-phase and faltered in an apparent EPO-directed transition to G0/G1. EPO/EPOR signals therefore control the expression of select cell-cycle regulatory genes that are proposed to modulate stage-specific decisions for erythroblast cell-cycle progression.
Asunto(s)
Médula Ósea/metabolismo , Proteínas de Ciclo Celular/metabolismo , División Celular , Eritroblastos/citología , Eritroblastos/metabolismo , Eritropoyetina/farmacología , Alelos , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Regulación de la Expresión Génica , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismoRESUMEN
Epo's erythropoietic capacity is ascribed largely to its antiapoptotic actions. In part via gene profiling of bone marrow erythroblasts, Epo is now shown to selectively down-modulate the adhesion/migration factors chemokine receptor-4 (Cxcr4) and integrin alpha-4 (Itga4) and to up-modulate growth differentiation factor-3 (Gdf3), oncostatin-M (OncoM), and podocalyxin like-1 (PODXL). For PODXL, Epo dose-dependent expression of this CD34-related sialomucin was discovered in Kit(+)CD71(high) proerythroblasts and was sustained at subsequent Kit(-)CD71(high) and Ter119(+) stages. In vivo, Epo markedly induced PODXL expression in these progenitors and in marrow-resident reticulocytes. This was further associated with a rapid release of PODXL(+) reticulocytes to blood. As studied in erythroblasts expressing minimal Epo receptor (EpoR) alleles, efficient PODXL induction proved dependence on an EpoR-PY343 Stat5 binding site. Moreover, in mice expressing an EpoR-HM F343 allele, compromised Epo-induced PODXL expression correlated with abnormal anucleated red cell representation in marrow. By modulating this select set of cell-surface adhesion molecules and chemokines, Epo is proposed to mobilize erythroblasts from a hypothesized stromal niche and possibly promote reticulocyte egress to blood.
Asunto(s)
Eritroblastos/fisiología , Eritropoyetina/farmacología , Receptores de Eritropoyetina/genética , Sialoglicoproteínas/genética , Animales , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Núcleo Celular/fisiología , Eritroblastos/efectos de los fármacos , Citometría de Flujo , Perfilación de la Expresión Génica , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sialoglicoproteínas/efectos de los fármacos , Sialomucinas/efectos de los fármacos , Sialomucinas/genéticaRESUMEN
Committed erythroid progenitor cells require exposure to erythropoietin (Epo) for their survival and for their quantitatively regulated transition to red blood cells. With regard to Epo signal transduction mechanisms, much has been learned from analyses in cell line models, fetal liver or spleen-derived primary erythroblasts and human CD34pos progenitor cells from cord blood or mobilized bone marrow. Presently, we have developed an ex vivo system that efficiently supports the expansion and development of murine adult bone-marrow-derived erythroid progenitor cells. This system is outlined together with its demonstrated utility in studying (for the first time) the signaling capacities of two knocked-in phosphotyrosine-deficient Epo receptor alleles (EpoR-H and EpoR-HM). Ways in which these studies advance an understanding of core Epo signal transduction events are outlined. Also introduced are two new putative negative regulators of Epo-dependent erythropoiesis, DYRK3 and DAPK2 kinases.