RESUMO
During development, the heart grows by addition of progenitor cells to the poles of the primordial heart tube. In the zebrafish, Wilms tumor 1 transcription factor a (wt1a) and b (wt1b) genes are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a and wt1b expression is maintained in proepicardial cells, it is downregulated in pericardial cells that contributes cardiomyocytes to the developing heart. Sustained wt1b expression in cardiomyocytes reduced chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a- and wt1b-expressing cardiomyocytes changed their cell-adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1a and wt1b act as a break for cardiomyocyte differentiation, and ectopic wt1a and wt1b expression in cardiomyocytes can lead to their transdifferentiation into epicardial-like cells.
Assuntos
Miócitos Cardíacos , Peixe-Zebra , Animais , Regulação da Expressão Gênica no Desenvolvimento , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Pericárdio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The oxidative phosphorylation (OXPHOS) system is a dynamic system in which the respiratory complexes coexist with super-assembled quaternary structures called supercomplexes (SCs). The physiological role of SCs is still disputed. Here, we used zebrafish to study the relevance of respiratory SCs. We combined immunodetection analysis and deep data-independent proteomics to characterize these structures and found similar SCs to those described in mice, as well as novel SCs including III2 + IV2 , I + IV, and I + III2 + IV2 . To study the physiological role of SCs, we generated two null allele zebrafish lines for supercomplex assembly factor 1 (scaf1). scaf1-/- fish displayed altered OXPHOS activity due to the disrupted interaction of complexes III and IV. scaf1-/- fish were smaller in size and showed abnormal fat deposition and decreased female fertility. These physiological phenotypes were rescued by doubling the food supply, which correlated with improved bioenergetics and alterations in the metabolic gene expression program. These results reveal that SC assembly by Scaf1 modulates OXPHOS efficiency and allows the optimization of metabolic resources.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Fatores de Processamento de Serina-Arginina/metabolismo , Peixe-Zebra , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/genética , Feminino , Camundongos , Membranas Mitocondriais/metabolismo , Fosforilação Oxidativa , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
In the zebrafish (Danio rerio), regeneration and fibrosis after cardiac injury are not mutually exclusive responses. Upon cardiac cryoinjury, collagen and other extracellular matrix (ECM) proteins accumulate at the injury site. However, in contrast to the situation in mammals, fibrosis is transient in zebrafish and its regression is concomitant with regrowth of the myocardial wall. Little is known about the cells producing this fibrotic tissue or how it resolves. Using novel genetic tools to mark periostin b- and collagen 1alpha2 (col1a2)-expressing cells in combination with transcriptome analysis, we explored the sources of activated fibroblasts and traced their fate. We describe that during fibrosis regression, fibroblasts are not fully eliminated but become inactivated. Unexpectedly, limiting the fibrotic response by genetic ablation of col1a2-expressing cells impaired cardiomyocyte proliferation. We conclude that ECM-producing cells are key players in the regenerative process and suggest that antifibrotic therapies might be less efficient than strategies targeting fibroblast inactivation.
Assuntos
Fibroblastos/fisiologia , Coração/fisiologia , Regeneração/fisiologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , Moléculas de Adesão Celular/biossíntese , Linhagem da Célula , Temperatura Baixa/efeitos adversos , Colágeno Tipo XII/biossíntese , Colágeno Tipo XII/genética , Endocárdio/patologia , Matriz Extracelular/metabolismo , Fibrose , Regulação da Expressão Gênica , Genes Reporter , Traumatismos Cardíacos/genética , Traumatismos Cardíacos/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , RNA Mensageiro/biossíntese , Transcriptoma , Peixe-Zebra , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genéticaRESUMO
Myeloproliferative neoplasms (MPNs) are diseases caused by mutations in the haematopoietic stem cell (HSC) compartment. Most MPN patients have a common acquired mutation of Janus kinase 2 (JAK2) gene in HSCs that renders this kinase constitutively active, leading to uncontrolled cell expansion. The bone marrow microenvironment might contribute to the clinical outcomes of this common event. We previously showed that bone marrow nestin(+) mesenchymal stem cells (MSCs) innervated by sympathetic nerve fibres regulate normal HSCs. Here we demonstrate that abrogation of this regulatory circuit is essential for MPN pathogenesis. Sympathetic nerve fibres, supporting Schwann cells and nestin(+) MSCs are consistently reduced in the bone marrow of MPN patients and mice expressing the human JAK2(V617F) mutation in HSCs. Unexpectedly, MSC reduction is not due to differentiation but is caused by bone marrow neural damage and Schwann cell death triggered by interleukin-1ß produced by mutant HSCs. In turn, in vivo depletion of nestin(+) cells or their production of CXCL12 expanded mutant HSC number and accelerated MPN progression. In contrast, administration of neuroprotective or sympathomimetic drugs prevented mutant HSC expansion. Treatment with ß3-adrenergic agonists that restored the sympathetic regulation of nestin(+) MSCs prevented the loss of these cells and blocked MPN progression by indirectly reducing the number of leukaemic stem cells. Our results demonstrate that mutant-HSC-driven niche damage critically contributes to disease manifestation in MPN and identify niche-forming MSCs and their neural regulation as promising therapeutic targets.
Assuntos
Células-Tronco Hematopoéticas/patologia , Transtornos Mieloproliferativos/patologia , Neoplasias/patologia , Fibras Nervosas/patologia , Nicho de Células-Tronco , Sistema Nervoso Simpático/patologia , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Agonistas de Receptores Adrenérgicos beta 3/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Progressão da Doença , Feminino , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Interleucina-1beta/metabolismo , Janus Quinase 2/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/patologia , Camundongos , Transtornos Mieloproliferativos/tratamento farmacológico , Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fibras Nervosas/efeitos dos fármacos , Nestina/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Receptores Adrenérgicos beta 3/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/patologia , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiopatologiaRESUMO
Metals and metalloids are integral to biological processes and play key roles in physiology and metabolism. Nonetheless, overexposure to some metals or lack of others can lead to serious health consequences. In this study, eight zebrafish facilities collaborated to generate a multielement analysis of their centralized recirculating water systems. We report a first set of average concentrations for 46 elements detected in zebrafish facilities. Our results help to establish an initial baseline for trouble-shooting purposes, and in general for safe ranges of metal concentrations in recirculating water systems, supporting reproducible scientific research outcomes with zebrafish.
Assuntos
Metaloides , Poluentes Químicos da Água , Animais , Metaloides/análise , Metaloides/metabolismo , Água , Poluentes Químicos da Água/análise , Peixe-Zebra/metabolismoRESUMO
Bumped kinase inhibitors (BKIs) are effective against a variety of apicomplexan parasites. Fifteen BKIs with promising in vitro efficacy against Neospora caninum tachyzoites, low cytotoxicity in mammalian cells, and no toxic effects in non-pregnant BALB/c mice were assessed in pregnant mice. Drugs were emulsified in corn oil and were applied by gavage for 5 days. Five BKIs did not affect pregnancy, five BKIs exhibited ~15-35% neonatal mortality and five compounds caused strong effects (infertility, abortion, stillbirth and pup mortality). Additionally, the impact of these compounds on zebrafish (Danio rerio) embryo development was assessed by exposing freshly fertilised eggs to 0.2-50 µM of BKIs and microscopic monitoring of embryo development in a blinded manner for 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows the calculation of an impact score (Si) indicating at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for nine compounds no clear correlation between Si and pregnancy outcome was observed. However, the three BKIs affecting zebrafish embryos only at high concentrations (≥40 µM) did not impair mouse pregnancy at all, and the three compounds that inhibited zebrafish embryo development already at 0.2 µM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has limited predictive value to foresee pregnancy outcome in BKI-treated mice. We conclude that maternal health-related factors such as cardiovascular, pharmacokinetic and/or bioavailability properties also contribute to BKI-pregnancy effects.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Naftalenos/toxicidade , Neospora/efeitos dos fármacos , Piperidinas/toxicidade , Pirazóis/toxicidade , Pirimidinas/toxicidade , Quinolinas/toxicidade , Toxoplasma/efeitos dos fármacos , Animais , Linhagem Celular , Coccidiose/tratamento farmacológico , Feminino , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Naftalenos/farmacocinética , Naftalenos/farmacologia , Neospora/crescimento & desenvolvimento , Piperidinas/farmacocinética , Piperidinas/farmacologia , Gravidez , Complicações na Gravidez/induzido quimicamente , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Pirazóis/farmacocinética , Pirazóis/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Quinolinas/farmacocinética , Quinolinas/farmacologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/tratamento farmacológico , Peixe-Zebra/embriologiaRESUMO
During heart regeneration in the zebrafish, fibrotic tissue is replaced by newly formed cardiomyocytes derived from preexisting ones. It is unclear whether the heart is composed of several cardiomyocyte populations bearing different capacity to replace lost myocardium. Here, using sox10 genetic fate mapping, we identify a subset of preexistent cardiomyocytes in the adult zebrafish heart with a distinct gene expression profile that expanded after cryoinjury. Genetic ablation of sox10+ cardiomyocytes impairs cardiac regeneration, revealing that these cells play a role in heart regeneration.
Assuntos
Miócitos Cardíacos/metabolismo , Regeneração , Fatores de Transcrição SOXE/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Coração/fisiologia , Miócitos Cardíacos/fisiologia , Fatores de Transcrição SOXE/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Organ regeneration is preceded by the recruitment of innate immune cells, which play an active role during repair and regrowth. Here, we studied macrophage subtypes during organ regeneration in the zebrafish, an animal model with a high regenerative capacity. We identified a macrophage subpopulation expressing Wilms tumor 1b (wt1b), which accumulates within regenerating tissues. This wt1b+ macrophage population exhibited an overall pro-regenerative gene expression profile and different migratory behavior compared to the remainder of the macrophages. Functional studies showed that wt1b regulates macrophage migration and retention at the injury area. Furthermore, wt1b-null mutant zebrafish presented signs of impaired macrophage differentiation, delayed fin growth upon caudal fin amputation, and reduced cardiomyocyte proliferation following cardiac injury that correlated with altered macrophage recruitment to the regenerating areas. We describe a pro-regenerative macrophage subtype in the zebrafish and a role for wt1b in organ regeneration.
Assuntos
Nadadeiras de Animais/fisiologia , Coração/fisiologia , Macrófagos/metabolismo , Regeneração , Proteínas WT1/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Macrófagos/citologia , Proteínas WT1/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The H2.0-like homeobox transcription factor (HLX) regulates hematopoietic differentiation and is overexpressed in Acute Myeloid Leukemia (AML), but the mechanisms underlying these functions remain unclear. We demonstrate here that HLX overexpression leads to a myeloid differentiation block both in zebrafish and human hematopoietic stem and progenitor cells (HSPCs). We show that HLX overexpression leads to downregulation of genes encoding electron transport chain (ETC) components and upregulation of PPARδ gene expression in zebrafish and human HSPCs. HLX overexpression also results in AMPK activation. Pharmacological modulation of PPARδ signaling relieves the HLX-induced myeloid differentiation block and rescues HSPC loss upon HLX knockdown but it has no effect on AML cell lines. In contrast, AMPK inhibition results in reduced viability of AML cell lines, but minimally affects myeloid progenitors. This newly described role of HLX in regulating the metabolic state of hematopoietic cells may have important therapeutic implications.
Assuntos
Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/fisiologia , Leucemia Mieloide Aguda/metabolismo , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Autofagia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Regulação Leucêmica da Expressão Gênica , Hematopoese , Proteínas de Homeodomínio/genética , Humanos , Células K562 , Leucemia Mieloide Aguda/genética , Potencial da Membrana Mitocondrial , PPAR gama/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Estrogens are potent regulators of mature hematopoietic cells; however, their effects on primitive and malignant hematopoietic cells remain unclear. Using genetic and pharmacological approaches, we observed differential expression and function of estrogen receptors (ERs) in hematopoietic stem cell (HSC) and progenitor subsets. ERα activation with the selective ER modulator (SERM) tamoxifen induced apoptosis in short-term HSCs and multipotent progenitors. In contrast, tamoxifen induced proliferation of quiescent long-term HSCs, altered the expression of self-renewal genes, and compromised hematopoietic reconstitution after myelotoxic stress, which was reversible. In mice, tamoxifen treatment blocked development of JAK2(V617F)-induced myeloproliferative neoplasm in vivo, induced apoptosis of human JAK2(V617F+) HSPCs in a xenograft model, and sensitized MLL-AF9(+) leukemias to chemotherapy. Apoptosis was selectively observed in mutant cells, and tamoxifen treatment only had a minor impact on steady-state hematopoiesis in disease-free animals. Together, these results uncover specific regulation of hematopoietic progenitors by estrogens and potential antileukemic properties of SERMs.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Janus Quinase 2/metabolismo , Leucemia/metabolismo , Células Progenitoras Mieloides/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/administração & dosagem , Tamoxifeno/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Receptor alfa de Estrogênio/genética , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Humanos , Janus Quinase 2/genética , Leucemia/tratamento farmacológico , Leucemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Células Progenitoras Mieloides/fisiologia , Proteínas de Fusão Oncogênica/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine patient management. A discovery exome sequencing screen (n = 17), followed by a prevalence screen (n = 60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2 and BPTF), cell division (STAG2, SMC1A and SMC1B) and DNA repair (ATM, ERCC2 and FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated or lost in UBC, mainly in tumors of low stage or grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally stable tumors, and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a new UBC tumor suppressor acting through mechanisms that are different from its role in preventing aneuploidy.