RESUMO
Non-healing wounds are among the main causes of morbidity and mortality. We recently described a novel, serum-free ex vivo expansion system, the quantity and quality culture system (QQc), which uses peripheral blood mononuclear cells (PBMNCs) for effective and noninvasive regeneration of tissue and vasculature in murine and porcine models. In this prospective clinical study, we investigated the safety and efficacy of QQ-cultured peripheral blood mononuclear cell (MNC-QQ) therapy for chronic non-healing ischemic extremity wounds. Peripheral blood was collected from 9 patients with 10 chronic (>1 month) non-healing wounds (8 males, 1 female; 64-74 years) corresponding to ischemic extremity ulcers. PBMNCs were isolated and cultured using QQc. Within a 20-cm area surrounding the ulcer, 2 × 107 cells were injected under local anesthesia. Wound healing was monitored photometrically every 2 weeks. The primary endpoint was safety, whereas the secondary endpoint was efficacy at 12-week post-injection. All patients remained ambulant, and no deaths, other serious adverse events, or major amputations were observed for 12 weeks after cell transplantation. Six of the 10 cases showed complete wound closure with an average wound closure rate of 73.2% ± 40.1% at 12 weeks. MNC-QQ therapy increased vascular perfusion, skin perfusion pressure, and decreased pain intensity in all patients. These results indicate the feasibility and safety of MNC-QQ therapy in patients with chronic non-healing ischemic extremity wounds. As the therapy involves transplanting highly vasculogenic cells obtained from a small blood sample, it may be an effective and highly vasculogenic strategy for limb salvage.
Assuntos
Leucócitos Mononucleares , Úlcera , Feminino , Humanos , Masculino , Extremidades , Estudos de Viabilidade , Isquemia/terapia , Estudos ProspectivosAssuntos
Modelos Teóricos , Neovascularização Patológica , Animais , Células Cultivadas , Humanos , CamundongosRESUMO
The quality and quantity of endothelial progenitor cells (EPCs) are impaired in patients with diabetes mellitus patients, leading to reduced tissue repair during autologous EPC therapy. This study aimed to address the limitations of the previously described serum-free Quantity and Quality Control Culture System (QQc) using CD34+ cells by investigating the therapeutic potential of a novel mononuclear cell (MNC)-QQ. MNCs were isolated from 50 mL of peripheral blood of patients with diabetes mellitus and healthy volunteers (n = 13 each) and subjected to QQc for 7 days in serum-free expansion media with VEGF, Flt-3 ligand, TPO, IL-6, and SCF. The vascular regeneration capability of MNC-QQ cells pre- or post-QQc was evaluated with an EPC colony-forming assay, FACS, EPC culture, tube formation assay, and quantitative real time PCR. For in vivo assessment, 1 × 104 pre- and post-MNC-QQc cells from diabetic donors were injected into a murine wound-healing model using Balb/c nude mice. The percentage of wound closure and angio-vasculogenesis was then assessed. This study revealed vasculogenic, anti-inflammatory, and wound-healing effects of MNC-QQ therapy in both in vitro and in vivo models. This system addresses the low efficiency and efficacy of the current naïve MNC therapy for wound-healing in diabetic patients. As this technique requires a simple blood draw, isolation, and peripheral blood MNC suspension culture for only a week, it can be used as a simple and effective outpatient-based vascular and regenerative therapy for patients with diabetes mellitus.
Assuntos
Diabetes Mellitus , Leucócitos Mononucleares , Cicatrização , Animais , Meios de Cultura Livres de Soro , Humanos , Leucócitos Mononucleares/transplante , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização FisiológicaRESUMO
BACKGROUND: Fat graft ischemia impedes us from having satisfying long-term results. The quality and quantity culture is a 1-week cell culture that increases the vasculogenic potential of peripheral blood mononuclear cells (PBMNC). This in vivo murine model investigates whether enrichment with quality and quantity-cultured human mononuclear cells (MNC-QQ) improves the vascularization in the human fat graft and whether this decreases the tissue loss. METHODS: Human adipose tissue, PBMNC, MNC-QQ, and stromal vascular fraction were prepared. First, PBMNC, MNC-QQ, and stromal vascular fraction were compared in vitro for vasculogenic potential by endothelial progenitor cell colony-forming and culture assays. Second, 0.25-g fat grafts were created with 1 × 106 PBMNC (n = 16), 1 × 106 MNC-QQ (n = 16), 1 × 106 stromal vascular fraction (n = 16), or phosphate-buffered saline as control (n = 16) before grafting in BALB/c nude mice. Grafts were analyzed for weight persistence, vessel formation by CD31 immunohistochemistry, and angiogenic markers by quantitative polymerase chain reaction. RESULTS: MNC-QQ develop more definitive endothelial progenitor cell colonies and more functional endothelial progenitor cells compared to PBMNC and stromal vascular fraction. Weight persistence after 7 weeks was significantly higher in grafts with MNC-QQ (89.8 ± 3.5 percent) or stromal vascular fraction (90.1 ± 4.2 percent) compared with control (70.4 ± 6.3 percent; p < 0.05). MNC-QQ-enriched grafts had the highest vessel density (96.6 ± 6.5 vessels/mm2; control, 70.4 ± 5.6 vessels/mm2; p < 0.05). MNC-QQ exerted a direct vasculogenic effect through vascular integration and a potential paracrine vascular endothelial growth factor-mediated effect. CONCLUSION: Quality and quantity-cultured human mononuclear cells containing endothelial progenitor cells stimulate fat graft vascularization and enhance graft survival in a rodent recipient.
Assuntos
Tecido Adiposo/transplante , Sobrevivência de Enxerto/fisiologia , Leucócitos Mononucleares/transplante , Neovascularização Fisiológica , Adipócitos/fisiologia , Tecido Adiposo/citologia , Animais , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Modelos Animais de Doenças , Células Progenitoras Endoteliais/fisiologia , Feminino , Humanos , Leucócitos Mononucleares/fisiologia , Masculino , Camundongos , Pessoa de Meia-Idade , Cultura Primária de Células , Células Estromais/transplanteRESUMO
The transplantation of endothelial progenitor cells (EPCs) is used to promote wound angiogenesis. In patients with chronic wounds and accompanying morbidities, EPCs are often compromised in number and function. To overcome these limitations, we previously developed a quality and quantity controlled (QQ) culture system to enrich peripheral blood mononuclear cells (PBMNCs) in EPCs. To evaluate the wound healing efficacy of mononuclear cells (MNCs) harvested after QQ culture (QQMNCs), preclinical studies were performed on large animals. MNCs harvested from the blood of healthy human subjects were cultured in the presence of angiogenic cytokines and growth factors in a serum-free medium for 7 days. A total of 5 × 106 QQMNCs per full-thickness skin defect or control saline was injected into wounds induced in cyclosporine-immunosuppressed pigs. EPC colony-forming assays revealed a significantly higher number of definitive (partially differentiated) EPC colony-forming units in QQMNCs. Flow cytometry evaluation of QQMNC surface markers showed enrichment of CD34+ and CD133+ stem cell populations, significant reduction in CCR2+ cell percentages, and a greater than 10-fold increase in the percentage of anti-inflammatory M2-type macrophages (CD206+ cells) compared with PBMNCs. Wounds treated with QQMNCs had a significantly higher closure rate. Wounds were harvested, frozen, and sectioned at day 21 postoperatively. Hematoxylin and eosin staining revealed that the epithelization of QQMNC-treated wounds was more advanced than in controls. Treated wounds developed granulation tissue with more mature collagen and larger capillary networks. CD31 and human mitochondrial co-staining confirmed the presence of differentiated human cells within newly formed vessels. Real-time polymerase chain reaction (PCR) showed upregulation of interleukin 6 (IL-6), IL-10, and IL-4 in the wound bed, suggesting paracrine activity of the transplanted QQMNCs. Our data demonstrate for the first time that QQ culture of MNCs obtained from a small amount of peripheral blood yields vasculogenic and therapeutic cells effective in wound healing.
Assuntos
Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/transplante , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/transplante , Neovascularização Fisiológica , Cicatrização , Adulto , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Humanos , Masculino , Controle de Qualidade , SuínosRESUMO
Autologous endothelial progenitor cell (EPC) therapy is commonly used to stimulate angiogenesis in ischemic repair and wound healing. However, low total numbers and functional deficits of EPCs make autologous EPC therapy ineffective in diabetes. Currently, no known ex vivo culture techniques can expand and/or ameliorate the functional deficits of EPCs for clinical usage. Recently, we showed that a quality-quantity culture (QQc) system restores the vasculogenic and wound-healing efficacy of murine diabetic EPCs. To validate these results and elucidate the mechanism in a translational study, we evaluated the efficacy of this QQc system to restore the vasculogenic potential of diabetic human peripheral blood (PB) CD34+ cells. CD34+ cells purified from PB of diabetic and healthy patients were subjected to QQc. Gene expression, vascular regeneration, and expression of cytokines and paracrine mediators were analyzed. Pre- or post-QQc diabetic human PB-CD34+ cells were transplanted into wounded BALB/c nude mice and streptozotocin-induced diabetic mice to assess functional efficacy. Post-QQc diabetic human PB-CD34+ cell therapy significantly accelerated wound closure, re-epithelialization, and angiogenesis. The higher therapeutic efficacy of post-QQc diabetic human PB-CD34+ cells was attributed to increased differentiation ability of diabetic CD34+ cells, direct vasculogenesis, and enhanced expression of angiogenic factors and wound-healing genes. Thus, QQc can significantly enhance the therapeutic efficacy of human PB-CD34+ cells in diabetic wounds, overcoming the inherent limitation of autologous cell therapy in diabetic patients, and could be useful for treatment of not only wounds but also other ischemic diseases. Stem Cells Translational Medicine 2018;7:428-438.
Assuntos
Antígenos CD34/metabolismo , Células Sanguíneas/fisiologia , Neovascularização Fisiológica/fisiologia , Cicatrização/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Sanguíneas/metabolismo , Diferenciação Celular/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Progenitoras Endoteliais , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Adulto JovemRESUMO
Koshu is indigenous to Japan and considered the most important wine grape in Japan. Koshu grape berry possesses characteristics that make it unique from European V. vinifera as wine grape. However, the physiological characteristics of Koshu leaf and internode remain unknown. An understanding of those characteristics would contribute to improvements in Koshu cultivation, thereby enhancing grape berry and wine quality. To identify the genes responsible for the physiological characteristics of Koshu, we comprehensively analyzed leaf and internode differences at the transcriptome level between Koshu and Pinot Noir by RNA sequencing. A total of 248 and 131 differentially expressed genes (DEGs) were detected in leaves and internodes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of these DEGs revealed that "flavonoid biosynthesis" and "glutathione metabolism" pathways were significantly enriched in Koshu leaves. On the other hand, when internodes were compared, "flavonoid"-related GO terms were specifically detected in Koshu. KEGG pathway enrichment analysis suggested that the expression of such genes as leucoanthocyanidin reductase and flavonol synthase in the flavonoid biosynthesis pathway was higher in Koshu than Pinot Noir. Measurement of the relative expression levels of these genes by RT-qPCR validated the results obtained by RNA sequencing. The characteristics of Koshu leaf and internode, which are expected to produce flavonoids with antibacterial activity and UV protection function, would suit Japanese climate as a survival strategy.
Assuntos
Análise de Sequência de RNA/métodos , Vitis/anatomia & histologia , Vitis/genética , Frutas/anatomia & histologia , Frutas/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Japão , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Transcriptoma , VinhoRESUMO
Vitis vinifera cv. Koshu is an indigenous grape cultivar that has been cultivated for more than a thousand years in Japan and one of the most important cultivars in white winemaking. To improve Koshu wine quality, it is necessary to identify the metabolites in Koshu berry. We conducted a comprehensive and comparative lipidome analysis of Koshu and Pinot Noir berries cultivated in the same location in Japan using GC-MS/MS for fatty acids and LC-MS for glycerolipids and glycerophospholipids. Koshu skins and juices contained 22 and 19 fatty acids, respectively, whereas 23 and 20 fatty acids were detected in Pinot Noir skins and juices. C22:6n3 and C24:0 contents in Koshu skins were two and three times higher than those in Pinot Noir skins. C24:0 content in Koshu juices was also higher than that in Pinot Noir juices. Forty-nine lipid components (six digalactosyldiacylglycerols, one monogalactosyldiacylglycerol, 10 phosphatidylcholines, 12 phosphatidylethanolamines, and 20 triglycerides) were detected in Pinot Noir and Koshu skins. Strong peaks were observed for MGDG 36:6, DGDG 36:6, PC 34:2, PC 36:5, TG 54:6, TG 54:7, and TG 54:8 in Koshu skins. The contents of 36 of the 49 lipid components were significantly higher in Pinot Noir skins than Koshu skins. Pinot Noir skins contained more lipids whose alkyl chains have more than 18 carbons than Koshu skins. Further analysis of both lipid profiles revealed that the number of double bonds in a fatty acid molecule in Pinot Noir skins and juices was significantly larger than that in Koshu skins and juices. A strong relationship exists between the heat requirement of grapevine cultivars and the level of fatty acid desaturation. C18-fatty acids were the major components in Koshu and Pinot Noir berries. The expression levels of C18-fatty acid desaturases regulated the accumulation of C18-unsaturated fatty acids in berry skins. The loss of C18:3 in Koshu berries at the end of ripening was observed. Koshu might effectively convert C18:3 into (Z)-hex-3-enal for the production of C6-aroma compounds. These findings by the lipidome analysis are expected to contribute to the improvement of Koshu wine aroma and breeding strategies of cold-tolerant Koshu grapevines.
Assuntos
Metabolismo dos Lipídeos , Vitis/metabolismo , Ácidos Graxos/análise , Cromatografia Gasosa-Espectrometria de Massas , Reprodutibilidade dos Testes , Especificidade da Espécie , Espectrometria de Massas em TandemRESUMO
We investigated the effect of vanillylacetone (VA) on anthocyanin accumulation with aim of improving grape berry coloration. Spraying Vitis vinifera cv. Muscat Bailey A berries with VA at veraison increased sugar/acid ratio, an indicator of maturation and total anthocyanin accumulation. To elucidate the molecular mechanism underlying the effect of VA on anthocyanin accumulation, in vitro VA treatment of a grapevine cell culture was carried out. Endogenous abscisic acid (ABA) content was higher in the VA-treated cell cultures than in control at 3h after treatment. Consistent with this, the relative expression levels of anthocyanin-synthesis-related genes, including DFR, LDOX, MybA1 and UFGT, in VA-treated cell cultures were much higher than those in control, and high total anthocyanin accumulation was noted in the VA-treated cell cultures as well. These results suggest that VA up-regulates the expression of genes leading to anthocyanin accumulation by inducing endogenous ABA. In addition, VA increased total anthocyanin content in a dose-dependent manner. Although VA treatment in combination with exogenous ABA did not exhibit any synergistic effect, treatment with VA alone showed an equivalent effect to that with exogenous ABA alone on total anthocyanin accumulation. These findings point to the possibility of using VA for improving grape berry coloration.
Assuntos
Ácido Abscísico/metabolismo , Antocianinas/metabolismo , Regulação da Expressão Gênica de Plantas , Guaiacol/análogos & derivados , Proteínas de Plantas/genética , Regulação para Cima , Vitis/genética , Técnicas de Cultura de Células , Frutas/metabolismo , Guaiacol/metabolismo , Proteínas de Plantas/metabolismo , Vitis/metabolismoRESUMO
Essential thrombocythemia (ET) is a subtype of myeloproliferative neoplasms. Approximately half of the patients with ET harbor a gain-of-function mutation in the JAK2 gene (JAK2-V617F), a small percentage have mutations in codon 515 of MPL (thrombopoietin receptor) gene, and the rest have neither mutation. Pregnancy is a rare complication of ET, and it has been reported that the number of blood platelets falls with pregnancy in ET patients and the number of blood platelets increases again after a delivery and this phenomenon is observed in JAK2-V617F-positive and JAK2-V617F-negative patients. We report the first case of an ET patient with MPL mutations, whose platelet count improved with the onset of menopause, not pregnancy, and the MPL mutation also simultaneously disappeared.
Assuntos
Menopausa , Mutação , Receptores de Trombopoetina/genética , Trombocitemia Essencial/genética , Feminino , Humanos , Pessoa de Meia-Idade , Avaliação de Resultados da Assistência ao Paciente , Prognóstico , Trombocitemia Essencial/diagnósticoRESUMO
BACKGROUND: Basic fibroblast growth factors (bFGFs) play a crucial role in wound healing by promoting fibroblast proliferation and neovascularization. However, drawback of bFGF is short half-life in free form. Gelatin has a capability of sustaining growth factors, which are gradually released while degradation. The purpose of this study is to see whether bFGF-impregnated gelatin sheet is effective in a murine model and whether it could also be available for patients in a safe manner. METHODS: Full-thickness skin defect was created on C57BL/6J mice and covered with bFGF with gelatin sheet (group A), bFGF without gelatin sheet (group B), phosphate buffer saline (PBS) with gelatin sheet (group C), and only PBS (group D). Wound healing was evaluated in terms of percent wound closure, granulation thickness, wound maturity, and vascular density. Clinical trial was conducted for patients who received either acute or chronic ulcers. The sheets were put onto the wounds and covered by hydrocolloid dressing, which was changed weekly. RESULTS: Groups A and B exhibited better wound healing than groups C and D in all aspects. Moreover, group A showed better results than group B at day 7 in terms of wound closure, collagen maturity, and vascularity. Efficacy without any adverse events was found in the clinical series. CONCLUSIONS: These findings suggest that control-released bFGF using gelatin sheet is effective for promoting wound healing. Such therapeutic strategy was considered to offer several clinical advantages including rapid healing and reduction of the dressing change with less patient discomfort.
Assuntos
Quelantes de Ferro/uso terapêutico , Sobrecarga de Ferro/tratamento farmacológico , Sobrecarga de Ferro/etiologia , Síndromes Mielodisplásicas/complicações , Síndromes Mielodisplásicas/diagnóstico , Reação Transfusional , Medula Óssea/metabolismo , Medula Óssea/patologia , Humanos , Ferro/metabolismo , Sobrecarga de Ferro/diagnóstico , Fígado/metabolismo , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/terapia , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
Nanos (Nos) is an evolutionarily conserved protein essential for the maintenance of primordial germ cells (PGCs). In Drosophila, the PGCs or pole cells express head involution defective (hid), which is required for caspase activation, but its translation is repressed by maternal Nos. In the absence of Nos activity, translation of hid mRNA into protein induces apoptosis in pole cells. However, it remains unclear how hid mRNA is regulated in pole cells. Here, we report that hid expression requires eiger (egr), a tumor necrosis factor ligand (TNF) homologue, which is induced in pole cells by decapentaplegic (dpp). In addition, we demonstrate that p53 and loki (lok), a damage-activated kinase known to be required for p53 phosphorylation, are both required for hid expression in pole cells. Since maternal lok mRNA is enriched in pole cells, it is possible that ubiquitously distributed p53 is activated in pole cells by maternal Lok. We propose that hid expression is activated in a pole cell-specific manner by loki/p53 and dpp/egr during embryogenesis.
Assuntos
Apoptose/fisiologia , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Proteínas de Membrana/fisiologia , Neuropeptídeos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Quinase do Ponto de Checagem 2 , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero , Hibridização In Situ , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
In early Drosophila embryos, germ plasm is localized to the posterior pole region and is partitioned into the germline progenitors, known as pole cells. Germ plasm contains the maternal factors required for germline development. It has been proposed that germline-specific gene expression is initiated by the function of maternal factors that are enriched in the germ plasm. However, such factors have remained elusive. Here, we describe a genome-wide survey of maternal transcripts that encode for transcription factors and are enriched in the germ plasm. We isolated pole cells from blastodermal embryos by fluorescence-activated cell sorting (FACS) and then used these isolated cells in a microarray analysis. Among the 835 genes in the Gene Ontology (GO) category transcription regulator activity listed in FlyBase, 68 were found to be predominantly expressed in pole cells as compared to whole embryos. As the early pole cells are known to be transcriptionally quiescent, the listed transcripts are predicted to be maternal in origin. Our in situ hybridization analysis revealed that 27 of the 68 transcripts were enriched in the germ plasm. Among the 27 transcripts, six were found to be required for germline-specific gene expression of vasa and/or nanos by knockdown experiments using RNA interference (RNAi). The identified transcripts encode a transcriptional activator (ovo), components of the transcriptional initiation complexes (Trf2, bip2 and Tif-IA), and the Ccr4-Not complex (CG31716 and l(2)NC136). Our study demonstrates that germ plasm contains maternal transcripts encoding transcriptional regulators for germline-specific gene expression in pole cells.
Assuntos
Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , RNA/genética , Fatores de Transcrição/genética , Animais , Drosophila/genética , Drosophila/metabolismo , Embrião não Mamífero , Células Germinativas/citologia , Hibridização In Situ , Análise de Sequência com Séries de Oligonucleotídeos , RNA/metabolismo , Interferência de RNA , Fatores de Transcrição/metabolismoRESUMO
Drosophila germline stem cells are regulated by the somatic microenvironment, or "niche," which ensures that the stem cells can both self-renew and produce functional gametes throughout adult life. However, despite its prime importance, little is known about how niche formation is regulated during gonadal development. Here, we demonstrate that a receptor tyrosine kinase, Sevenless (Sev), is required to ensure that the niche develops in the anterior region of the male embryonic gonads. Sev is expressed in somatic cells within the posterior region of the gonads. Sev is activated by a ligand, Bride of sevenless (Boss), which is expressed by the germline, to prevent ectopic niche differentiation in the posterior gonadal somatic cells. Thus, we propose that signal transduction from germline to soma restricts expansion of the germline-stem-cell niche in the gonads.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Proteínas do Olho/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Peptídeos/metabolismo , Células-Tronco/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular/fisiologia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/fisiologia , Masculino , Glicoproteínas de Membrana/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Peptídeos/genética , Transdução de Sinais/fisiologia , Testículo/citologia , Testículo/embriologia , Testículo/metabolismoRESUMO
A hallmark of germline cells throughout the animal kingdom is their ability to execute meiosis. However, despite its prime importance, little is known about how germline progenitors acquire this ability. In Drosophila, the primordial germ cells (PGCs) are characterized by the inheritance of germ plasm, which contains maternal factors that have sufficient ability to direct germline development. Here, we show that a novel maternal factor, MAMO, is autonomously required in PGCs to produce functional gametes. MAMO protein which contains both a BTB/POZ (Broad Complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) domain and C(2)H(2) zinc finger motifs is enriched in PGCs during embryogenesis. The PGCs with reduced maternal MAMO activity are able to undergo oogenesis, but fail to execute meiosis properly. In the resulting oocytes, meiosis-specific chromosomal configurations are impaired. We additionally show that the decondensation of fertilized sperm nuclei is also affected in the eggs. We propose that maternal MAMO activates downstream genes to promote specialized morphological changes of both female meiotic chromosomes and the sperm nucleus, which are critical in zygote formation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Células Germinativas/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação a DNA/genética , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/genética , Feminino , Meiose/genética , Meiose/fisiologia , Dados de Sequência Molecular , Oogênese , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
Nanos (Nos) is an evolutionarily conserved protein essential for the survival of primordial germ cells. In Drosophila, maternal Nos partitions into pole cells and suppresses apoptosis to permit proper germ-line development. However, how this critical event is regulated by Nos has remained elusive. Here, we report that Nos represses apoptosis of pole cells by suppressing translation of head involution defective (hid), a member of the RHG gene family that is required for Caspase activation. In addition, we demonstrate that hid acts in concert with another RHG gene, sickle (skl), to induce apoptosis. Expression of skl is induced in pole cells by maternal tao-1, a ste20-like serine/threonine kinase. Tao-1-dependent skl expression is required to potentiate hid activity. However, skl expression is largely suppressed in normal pole cells. Once the pole cells lack maternal Nos, Tao-1-dependent skl expression is fully activated, suggesting that skl expression is also restricted by Nos. These findings provide the first evidence that the germ line is maintained through the regulated expression of RHG genes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Neuropeptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Mães , Neuropeptídeos/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Elementos de RespostaRESUMO
Primordial germ cells (PGC) are the earliest identifiable germ cells in the embryo. To understand the molecular basis of germline development, isolation of pure PGC is required. We report here the use of fluorescence-activated cell sorting (FACS) to isolate pure populations of Drosophila pole cells, which are the presumptive primordial germ cells in flies. In order to fluorescently mark pole cells, we used an EGFP-vasa transgenic line that expresses green fluorescent protein (GFP) specifically and continuously in the germ line throughout the life cycle. The purity of FACS-sorted pole cells from embryos was confirmed by microscopic inspection and quantitative polymerase chain reaction. Moreover, by optimizing the sample preparation and the sorting protocol, embryonic gonads could also be isolated. This technique opens the way for genome-wide transcriptome analysis of germline cells. In a pilot experiment, we generated a cDNA library from purified embryonic gonad and identified a novel germline-specific gene, RpL22-like.
Assuntos
Linhagem Celular/citologia , Drosophila/embriologia , Citometria de Fluxo/métodos , Gametogênese/genética , Células Germinativas/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Linhagem Celular/fisiologia , RNA Helicases DEAD-box , DNA Complementar/genética , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Embrião não Mamífero/citologia , Etiquetas de Sequências Expressas , Feminino , Células Germinativas/química , Células Germinativas/citologia , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Masculino , RNA Helicases/análise , RNA Helicases/genética , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genéticaRESUMO
Meiosis is one of the fundamental characteristics of germ cells. In Drosophila, genetic screens have identified many genes required for meiotic division. However, it remains elusive as to when and how these meiotic genes are activated during germline development. To obtain insights into their regulatory mechanisms, we examined the expression of 38 meiotic genes in the germline progenitors, pole cells, during embryogenesis. We found that the transcripts of 12 meiotic genes were enriched in pole cells within the embryonic gonads. Among them, bag of marbles (bam), benign gonial cell neoplasia (bgcn), deadhead (dhd), matotopetli (topi) and twine (twe) were activated only in pole cells within the gonads, whereas the transcripts from grapes (grp), Kinesin-like protein at 3A (Klp3A), pavarotti (pav), lesswright (lwr), mei-P26, Topoisomerase 2 (Top2) and out at first (oaf) were distributed ubiquitously in early embryos and then became restricted to pole cells and to a subset of somatic tissues at later embryonic stages. The remaining meiotic genes were either expressed ubiquitously in the embryos (15 genes) or were undetectable in pole cells within the gonads (11 genes). These observations suggest that pole cells have already acquired the potential to express several meiotic genes. Our data will thus provide a useful basis for analyzing how the germline acquires a potential to execute meiosis.