RESUMO
The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancy (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 was shown to regulate inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to epigenetically repress two inflammatory signaling pathways in neutrophils: Toll-like receptor 4 (TLR4) and type I interferon (IFN) signaling. RUNX1 loss in GMPs augments neutrophils' inflammatory response to the TLR4 ligand lipopolysaccharide through increased expression of the TLR4 coreceptor CD14. RUNX1 binds Cd14 and other genes encoding proteins in the TLR4 and type I IFN signaling pathways whose chromatin accessibility increases when RUNX1 is deleted. Transcription factor footprints for the effectors of type I IFN signaling-the signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRFs)-were enriched in chromatin that gained accessibility in both GMPs and neutrophils when RUNX1 was lost. STAT1::STAT2 and IRF motifs were also enriched in the chromatin of retrotransposons that were derepressed in RUNX1-deficient GMPs and neutrophils. We conclude that a major direct effect of RUNX1 loss in GMPs is the derepression of type I IFN and TLR4 signaling, resulting in a state of fixed maladaptive innate immunity.
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Neutrófilos , Receptor 4 Toll-Like , Receptor 4 Toll-Like/metabolismo , Monócitos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Citocinas/metabolismo , Cromatina/metabolismo , Fator de Transcrição STAT1/metabolismoRESUMO
Germ line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous-mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.
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Subunidade alfa 2 de Fator de Ligação ao Core , Leucemia Mieloide Aguda , Animais , Humanos , Macaca mulatta , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/patologia , Trombopoese , FenótipoRESUMO
ABSTRACT: Deleterious germ line RUNX1 variants cause the autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM), characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematologic malignancies (HMs). We launched a FPDMM natural history study and, from January 2019 to December 2021, enrolled 214 participants, including 111 patients with 39 different RUNX1 variants from 45 unrelated families. Seventy of 77 patients had thrombocytopenia, 18 of 18 had abnormal platelet aggregometry, 16 of 35 had decreased platelet dense granules, and 28 of 55 had abnormal bleeding scores. Nonmalignant bone marrows showed increased numbers of megakaryocytes in 12 of 55 patients, dysmegakaryopoiesis in 42 of 55, and reduced cellularity for age in 30 of 55 adult and 17 of 21 pediatric cases. Of 111 patients, 19 were diagnosed with HMs, including myelodysplastic syndrome, acute myeloid leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, and smoldering myeloma. Of those 19, 18 were relapsed or refractory to upfront therapy and referred for stem cell transplantation. In addition, 28 of 45 families had at least 1 member with HM. Moreover, 42 of 45 patients had allergic symptoms, and 24 of 30 had gastrointestinal (GI) symptoms. Our results highlight the importance of a multidisciplinary approach, early malignancy detection, and wider awareness of inherited disorders. This actively accruing, longitudinal study will genotype and phenotype more patients with FPDMM, which may lead to a better understanding of the disease pathogenesis and clinical course, which may then inform preventive and therapeutic interventions. This trial was registered at www.clinicaltrials.gov as #NCT03854318.
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Neoplasias Hematológicas , Leucemia Mieloide Aguda , Transtornos Mieloproliferativos , Trombocitopenia , Adulto , Humanos , Criança , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Estudos Longitudinais , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/complicações , Trombocitopenia/genética , Transtornos Mieloproliferativos/complicações , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/complicaçõesRESUMO
BACKGROUND: Genome editing using CRISPR/Cas9 has become a powerful tool in zebrafish to generate targeted gene knockouts models. However, its use for targeted knock-in remains challenging due to inefficient homology directed repair (HDR) pathway in zebrafish, highlighting the need for efficient and cost-effective screening methods. RESULTS: Here, we present our fluorescent PCR and capillary electrophoresis based screening approach for knock-in using a single-stranded oligodeoxynucleotide donor (ssODN) as a repair template for the targeted insertion of epitope tags, or single nucleotide changes to recapitulate pathogenic human alleles. For the insertion of epitope tags, we took advantage of the expected change in size of the PCR product. For point mutations, we combined fluorescent PCR with restriction fragment length polymorphism (RFLP) analysis to distinguish the fish with the knock-in allele. As a proof-of-principle, we present our data on the generation of fish lines with insertion of a FLAG tag at the tcnba locus, an HA tag at the gata2b locus, and a point mutation observed in Gaucher disease patients in the gba gene. Despite the low number of germline transmitting founders (1-5%), combining our screening methods with prioritization of founder fish by fin biopsies allowed us to establish stable knock-in lines by screening 12 or less fish per gene. CONCLUSIONS: We have established a robust pipeline for the generation of zebrafish models with precise integration of small DNA sequences and point mutations at the desired sites in the genome. Our screening method is very efficient and easy to implement as it is PCR-based and only requires access to a capillary sequencer.
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Mutação Puntual , Peixe-Zebra , Humanos , Animais , Peixe-Zebra/genética , Epitopos , PesquisaRESUMO
ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3' splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. Studies of ZRSR2-depleted cell lines and ZRSR2-mutated patient samples revealed its essential role in the U12-dependent minor spliceosome. However, the role of ZRSR2 during embryonic development is not clear, as its function is compensated for by Zrsr1 in mice. Here, we utilized the zebrafish model to investigate the role of zrsr2 during embryonic development. Using CRISPR/Cas9 technology, we generated a zrsr2-knockout zebrafish line, termed zrsr2hg129/hg129 (p.Trp167Argfs*9) and examined embryo development in the homozygous mutant embryos. zrsr2hg129/hg129 embryos displayed multiple developmental defects starting at 4 days post fertilization (dpf) and died after 8 dpf, suggesting that proper Zrsr2 function is required during embryonic development. The global transcriptome analysis of 3 dpf zrsr2hg129/hg129 embryos revealed that the loss of Zrsr2 results in the downregulation of essential metabolic pathways and the aberrant retention of minor introns in about one-third of all minor intron-containing genes in zebrafish. Overall, our study has demonstrated that the role of Zrsr2 as a component of the minor spliceosome is conserved and critical for proper embryonic development in zebrafish.
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Splicing de RNA , Ribonucleoproteínas , Peixe-Zebra , Animais , Camundongos , Desenvolvimento Embrionário , Íntrons/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Sítios de Splice de RNA , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Spliceossomos/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The transcription factor RUNX1, a pivotal regulator of HSCs and haematopoiesis, is a frequent target of chromosomal translocations, point mutations or altered gene/protein dosage. These modifications lead or contribute to the development of myelodysplasia, leukaemia or platelet disorders. A better understanding of how regulatory elements contribute to fine-tune the RUNX1 expression in haematopoietic tissues could improve our knowledge of the mechanisms responsible for normal haematopoiesis and malignancy insurgence. The cohesin RAD21 was reported to be a regulator of RUNX1 expression in the human myeloid HL60 cell line and during primitive haematopoiesis in zebrafish. In our study, we demonstrate that another cohesin, NIPBL, exerts positive regulation of RUNX1 in three different contexts in which RUNX1 displays important functions: in megakaryocytes derived from healthy donors, in bone marrow samples obtained from adult patients with acute myeloid leukaemia and during zebrafish haematopoiesis. In this model, we demonstrate that alterations in the zebrafish orthologue nipblb reduce runx1 expression with consequent defects in its erythroid and myeloid targets such as gata1a and spi1b in an opposite way to rad21. Thus, also in the absence of RUNX1 translocation or mutations, additional factors such as defects in the expression of NIPBL might induce haematological diseases.
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Proteínas de Ciclo Celular/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação Leucêmica da Expressão Gênica , Hematopoese/genética , Proteínas de Peixe-Zebra/genética , Adulto , Idoso , Animais , Células da Medula Óssea/metabolismo , Proteínas de Ciclo Celular/metabolismo , Criança , Estudos de Coortes , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação para Baixo/genética , Humanos , Leucemia Mieloide Aguda/genética , Megacariócitos/metabolismo , Pessoa de Meia-Idade , Doadores de Tecidos , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The nucleophosmin 1 gene (NPM1) is the most frequently mutated gene in acute myeloid leukemia. Notably, NPM1 mutations are always accompanied by additional mutations such as those in cohesin genes RAD21, SMC1A, SMC3, and STAG2 but not in the cohesin regulator, nipped B-like (NIPBL). In this work, we analyzed a cohort of adult patients with acute myeloid leukemia and NPM1 mutation and observed a specific reduction in the expression of NIPBL but not in other cohesin genes. In our zebrafish model, overexpression of the mutated form of NPM1 also induced downregulation of nipblb, the zebrafish ortholog of human NIPBL To investigate the hematopoietic phenotype and the interaction between mutated NPM1 and nipblb, we generated a zebrafish model with nipblb downregulation which showed an increased number of myeloid progenitors. This phenotype was due to hyper-activation of the canonical Wnt pathway: myeloid cells blocked in an undifferentiated state could be rescued when the Wnt pathway was inhibited by dkk1b mRNA injection or indomethacin administration. Our results reveal, for the first time, a role for NIPBL during zebrafish hematopoiesis and suggest that an interplay between NIPBL/NPM1 may regulate myeloid differentiation in zebrafish and humans through the canonical Wnt pathway and that dysregulation of these interactions may drive leukemic transformation.
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Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proteínas Cromossômicas não Histona/metabolismo , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/patologia , Mutação , Proteínas Nucleares/genética , Adulto , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Hematopoese , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Nucleofosmina , Fenótipo , Via de Sinalização Wnt , Peixe-Zebra , CoesinasRESUMO
CBFß and RUNX1 form a DNA-binding heterodimer and are both required for hematopoietic stem cell (HSC) generation in mice. However, the exact role of CBFß in the production of HSCs remains unclear. Here, we generated and characterized 2 zebrafish cbfb null mutants. The cbfb(-/-) embryos underwent primitive hematopoiesis and developed transient erythromyeloid progenitors, but they lacked definitive hematopoiesis. Unlike runx1 mutants, in which HSCs are not formed, nascent, runx1(+)/c-myb(+) HSCs were formed in cbfb(-/-) embryos. However, the nascent HSCs were not released from the aorta-gonad-mesonephros (AGM) region, as evidenced by the accumulation of runx1(+) cells in the AGM that could not enter circulation. Moreover, wild-type embryos treated with an inhibitor of RUNX1-CBFß interaction, Ro5-3335, phenocopied the hematopoietic defects in cbfb(-/-) mutants, rather than those in runx1(-/-) mutants. Finally, we found that cbfb was downstream of the Notch pathway during HSC development. Our data suggest that runx1 and cbfb are required at 2 different steps during early HSC development. CBFß is not required for nascent HSC emergence but is required for the release of HSCs from AGM into circulation. Our results also indicate that RUNX1 can drive the emergence of nascent HSCs in the AGM without its heterodimeric partner CBFß.
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Fator de Ligação a CCAAT/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Peixe-Zebra/genética , Animais , Fator de Ligação a CCAAT/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Técnicas de Inativação de Genes , Hibridização In Situ , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
OBJECTIVE: Lymphangiogenesis is regulated by transcription factors and by growth factor pathways, but their interplay has not been extensively studied so far. We addressed this issue in zebrafish. APPROACH AND RESULTS: Mutations in the transcription factor-coding gene SOX18 and in VEGFR3 cause lymphedema, and the VEGFR3/Flt4 ligand VEGFC plays an evolutionarily conserved role in lymphangiogenesis. Here, we report a strong genetic interaction between Sox18 and VegfC in the early phases of lymphatic development in zebrafish. Knockdown of sox18 selectively impaired lymphatic sprouting from the cardinal vein and resulted in defective lymphatic thoracic duct formation. Sox18 and the related protein Sox7 play redundant roles in arteriovenous differentiation. We used a novel transgenic line that enables inducible expression of a dominant-negative mutant form of mouse Sox18 protein. Our data led us to conclude that Sox18 is crucially involved in lymphangiogenesis after arteriovenous differentiation. Combined partial knockdown of sox18 and vegfc, using subcritical doses of specific morpholinos, revealed a synergistic interaction in both venous and lymphatic sprouting from the cardinal vein and greatly impaired thoracic duct formation. CONCLUSIONS: This interaction suggests a previously unappreciated crosstalk between the growth factor and transcription factor pathways that regulate lymphangiogenesis in development and disease.
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Regulação da Expressão Gênica no Desenvolvimento , Linfangiogênese/genética , Fatores de Transcrição SOXF/genética , Transdução de Sinais/genética , Fator C de Crescimento do Endotélio Vascular/genética , Animais , Animais Geneticamente Modificados , Vasos Sanguíneos/embriologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos , Modelos Animais , Domínios e Motivos de Interação entre Proteínas/genética , Fatores de Transcrição SOXF/metabolismo , Sensibilidade e Especificidade , Fator C de Crescimento do Endotélio Vascular/metabolismo , Peixe-ZebraRESUMO
ABSTRACT: Familial platelet disorder with associated myeloid malignancies (FPDMM) is caused by germline RUNX1 mutations and characterized by thrombocytopenia and increased risk of hematologic malignancies. We recently launched a longitudinal natural history study for patients with FPDMM. Among 27 families with research genomic data by the end of 2021, 26 different germline RUNX1 variants were detected. Besides missense mutations enriched in Runt homology domain and loss-of-function mutations distributed throughout the gene, splice-region mutations and large deletions were detected in 6 and 7 families, respectively. In 25 of 51 (49%) patients without hematologic malignancy, somatic mutations were detected in at least 1 of the clonal hematopoiesis of indeterminate potential (CHIP) genes or acute myeloid leukemia (AML) driver genes. BCOR was the most frequently mutated gene (in 9 patients), and multiple BCOR mutations were identified in 4 patients. Mutations in 6 other CHIP- or AML-driver genes (TET2, DNMT3A, KRAS, LRP1B, IDH1, and KMT2C) were also found in ≥2 patients without hematologic malignancy. Moreover, 3 unrelated patients (1 with myeloid malignancy) carried somatic mutations in NFE2, which regulates erythroid and megakaryocytic differentiation. Sequential sequencing data from 19 patients demonstrated dynamic changes of somatic mutations over time, and stable clones were more frequently found in older adult patients. In summary, there are diverse types of germline RUNX1 mutations and high frequency of somatic mutations related to clonal hematopoiesis in patients with FPDMM. Monitoring changes in somatic mutations and clinical manifestations prospectively may reveal mechanisms for malignant progression and inform clinical management. This trial was registered at www.clinicaltrials.gov as #NCT03854318.
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Transtornos Herdados da Coagulação Sanguínea , Transtornos Plaquetários , Neoplasias Hematológicas , Leucemia Mieloide Aguda , Transtornos Mieloproliferativos , Humanos , Idoso , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Transtornos Mieloproliferativos/genética , Neoplasias Hematológicas/genética , Genômica , Células Germinativas/patologiaRESUMO
Germline RUNX1 mutations lead to familial platelet disorder with associated myeloid malignancies (FPDMM), which is characterized by thrombocytopenia and a life-long risk (35-45%) of hematological malignancies. We recently launched a longitudinal natural history study for patients with FPDMM at the NIH Clinical Center. Among 29 families with research genomic data, 28 different germline RUNX1 variants were detected. Besides missense mutations enriched in Runt homology domain and loss-of-function mutations distributed throughout the gene, splice-region mutations and large deletions were detected in 6 and 7 families, respectively. In 24 of 54 (44.4%) non-malignant patients, somatic mutations were detected in at least one of the clonal hematopoiesis of indeterminate potential (CHIP) genes or acute myeloid leukemia (AML) driver genes. BCOR was the most frequently mutated gene (in 9 patients), and multiple BCOR mutations were identified in 4 patients. Mutations in 7 other CHIP or AML driver genes ( DNMT3A, TET2, NRAS, SETBP1, SF3B1, KMT2C , and LRP1B ) were also found in more than one non-malignant patient. Moreover, three unrelated patients (one with myeloid malignancy) carried somatic mutations in NFE2 , which regulates erythroid and megakaryocytic differentiation. Sequential sequencing data from 19 patients demonstrated dynamic changes of somatic mutations over time, and stable clones were more frequently found in elderly patients. In summary, there are diverse types of germline RUNX1 mutations and high frequency of somatic mutations related to clonal hematopoiesis in patients with FPDMM. Monitoring dynamic changes of somatic mutations prospectively will benefit patientsâ™ clinical management and reveal mechanisms for progression to myeloid malignancies. Key Points: Comprehensive genomic profile of patients with FPDMM with germline RUNX1 mutations. Rising clonal hematopoiesis related secondary mutations that may lead to myeloid malignancies.
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The current view of hematopoiesis considers leukocytes on a continuum with distinct developmental origins, and which exert non-overlapping functions. However, there is less known about the function and phenotype of ontogenetically distinct neutrophil populations. In this work, using a photoconvertible transgenic zebrafish line; Tg(mpx:Dendra2), we selectively label rostral blood island-derived and caudal hematopoietic tissue-derived neutrophils in vivo during steady state or upon injury. By comparing the migratory properties and single-cell expression profiles of both neutrophil populations at steady state we show that rostral neutrophils show higher csf3b expression and migration capacity than caudal neutrophils. Upon injury, both populations share a core transcriptional profile as well as subset-specific transcriptional signatures. Accordingly, both rostral and caudal neutrophils are recruited to the wound independently of their distance to the injury. While rostral neutrophils respond uniformly, caudal neutrophils respond heterogeneously. Collectively, our results reveal that co-existing neutrophils populations with ontogenically distinct origin display functional differences.
Assuntos
Neutrófilos , Peixe-Zebra , Animais , Peixe-Zebra/genética , Neutrófilos/metabolismo , Animais Geneticamente Modificados , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , HematopoeseRESUMO
The microenvironment is an important regulator of intertumoral trafficking and activity of immune cells. Understanding how the immune system can be tailored to maintain anti-tumor killing responses in metastatic disease remains an important goal. Thus, immune mediated eradication of metastasis requires the consideration of organ specific microenvironmental cues. Using a xenograft model of melanoma metastasis in adult zebrafish, we perturbed the dynamic balance between the infiltrating immune cells in the metastatic setting using a suite of different transgenic zebrafish. We employed intravital imaging coupled with metabolism imaging (FLIM) to visualize and map the organ specific metabolism with near simultaneity in multiple metastatic lesions. Of all the MHC complexes examined for brain and skeletal metastases, we determined that there is an organ specific expression of mhc1uba (human ortholog, MR1) for both the melanoma cells and the resident and infiltrating immune cells. Specifically, immune clusters did not express mhc1uba in brain metastatic lesions in immune competent fish. Finally, the differential immune response drove organ specific metabolism where tumor glycolysis was increased in brain metastases compared to skeletal and parental lines as measured using fluorescence lifetime imaging microscopy (FLIM). As MR1 belongs to the MHC class I molecules and is a target of immunotherapeutic drugs, we believe that our data presents an opportunity to understand the relationship between organ specific tumor metabolism and drug efficacy in the metastatic setting.
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The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancies (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 regulates inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to restrict the inflammatory response of neutrophils to toll-like receptor 4 (TLR4) signaling. Loss of RUNX1 in GMPs increased the TLR4 coreceptor CD14 on neutrophils, which contributed to neutrophilsâ™ increased inflammatory cytokine production in response to the TLR4 ligand lipopolysaccharide. RUNX1 loss increased the chromatin accessibility of retrotransposons in GMPs and neutrophils and induced a type I interferon signature characterized by enriched footprints for signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRF) in opened chromatin, and increased expression of interferon-stimulated genes. The overproduction of inflammatory cytokines by neutrophils was reversed by inhibitors of type I IFN signaling. We conclude that RUNX1 restrains the chromatin accessibility of retrotransposons in GMPs and neutrophils, and that loss of RUNX1 increases proinflammatory cytokine production by elevating tonic type I interferon signaling.
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Throughout their lifetime, fish maintain a high capacity for regenerating complex tissues after injury. We utilized a larval tail regeneration assay in the zebrafish Danio rerio, which serves as an ideal model of appendage regeneration due to its easy manipulation, relatively simple mixture of cell types, and superior imaging properties. Regeneration of the embryonic zebrafish tail requires development of a blastema, a mass of dedifferentiated cells capable of replacing lost tissue, a crucial step in all known examples of appendage regeneration. Using this model, we show that tail amputation triggers an obligate metabolic shift to promote glucose metabolism during early regeneration similar to the Warburg effect observed in tumor forming cells. Inhibition of glucose metabolism did not affect the overall health of the embryo but completely blocked the tail from regenerating after amputation due to the failure to form a functional blastema. We performed a time series of single-cell RNA sequencing on regenerating tails with and without inhibition of glucose metabolism. We demonstrated that metabolic reprogramming is required for sustained TGF-ß signaling and blocking glucose metabolism largely mimicked inhibition of TGF-ß receptors, both resulting in an aberrant blastema. Finally, we showed using genetic ablation of three possible metabolic pathways for glucose, that metabolic reprogramming is required to provide glucose specifically to the hexosamine biosynthetic pathway while neither glycolysis nor the pentose phosphate pathway were necessary for regeneration.
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RUNX1 is essential for the generation of hematopoietic stem cells (HSCs). Runx1-null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, although zebrafish embryos with a runx1 W84X mutation have defects in early definitive hematopoiesis, some runx1W84X/W84X embryos can develop to fertile adults with blood cells of multilineages, raising the possibility that HSCs can emerge without RUNX1. Here, using 3 new zebrafish runx1-/- lines, we uncovered the compensatory mechanism for runx1-independent hematopoiesis. We show that, in the absence of a functional runx1, a cd41-green fluorescent protein (GFP)+ population of hematopoietic precursors still emerge from the hemogenic endothelium and can colonize the hematopoietic tissues of the mutant embryos. Single-cell RNA sequencing of the cd41-GFP+ cells identified a set of runx1-/--specific signature genes during hematopoiesis. Significantly, gata2b, which normally acts upstream of runx1 for the generation of HSCs, was increased in the cd41-GFP+ cells in runx1-/- embryos. Interestingly, genetic inactivation of both gata2b and its paralog gata2a did not affect hematopoiesis. However, knocking out runx1 and any 3 of the 4 alleles of gata2a and gata2b abolished definitive hematopoiesis. Gata2 expression was also upregulated in hematopoietic cells in Runx1-/- mice, suggesting the compensatory mechanism is conserved. Our findings indicate that RUNX1 and GATA2 serve redundant roles for HSC production, acting as each other's safeguard.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Fator de Transcrição GATA2/metabolismo , Hemangioblastos , Proteínas de Peixe-Zebra/metabolismo , Animais , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Fator de Transcrição GATA2/genética , Hematopoese/genética , Células-Tronco Hematopoéticas , Camundongos , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Prox1, the vertebrate homolog of prospero in Drosophila melanogaster, is a divergent homeogene that regulates cell proliferation, fate determination and differentiation during vertebrate embryonic development. RESULTS: Here we report that, in zebrafish, prox1 is widely expressed in several districts of the Central Nervous System (CNS). Specifically, we evidenced prox1 expression in a group of neurons, already positive for otp1, located in the hypothalamus at the level of the posterior tuberculum (PT). Prox1 knock-down determines the severe loss of hypothalamic catecholaminergic (CA) neurons, identified by tyrosine hydroxylase (TH) expression, and the synergistic prox1/otp1 overexpression induces the appearance of hypothalamic supernumerary TH-positive neurons and ectopic TH-positive cells on the yolk epitelium. CONCLUSION: Our findings indicate that prox1 activity is crucial for the proper development of the otp1-positive hypothalamic neuronal precursors to their terminal CA phenotype.
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Catecolaminas/genética , Proteínas de Homeodomínio/genética , Hipotálamo/embriologia , Neurônios/citologia , Proteínas Supressoras de Tumor/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Animais , Contagem de Células , Diferenciação Celular , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Hipotálamo/citologia , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peixe-Zebra/genéticaRESUMO
Zebrafish (Danio rerio) has emerged as a powerful animal model to study developmental processes and human diseases. The introduction of CRISPR/Cas9 as a genome editing tool allowed the generation of genetic mutants with high-throughput (Varshney et al., 2015) and has opened the possibility to understand gene function not only during embryonic stages but also in larval stages. Therefore, there is an increasing need to optimize methods for embryo and larvae dissociation that allow the generation of single cell suspension for fluorescence-activated cell sorting (FACS), RNA extraction and single cell RNA-sequencing.
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During embryonic development, cell type-specific transcription factors promote cell identities, while epigenetic modifications are thought to contribute to maintain these cell fates. Our understanding of how genetic and epigenetic modes of regulation work together to establish and maintain cellular identity is still limited, however. Here, we show that DNA methyltransferase 3bb.1 (dnmt3bb.1) is essential for maintenance of hematopoietic stem and progenitor cell (HSPC) fate as part of an early Notch-runx1-cmyb HSPC specification pathway in the zebrafish. Dnmt3bb.1 is expressed in HSPC downstream from Notch1 and runx1, and loss of Dnmt3bb.1 activity leads to reduced cmyb locus methylation, reduced cmyb expression, and gradual reduction in HSPCs. Ectopic overexpression of dnmt3bb.1 in non-hematopoietic cells is sufficient to methylate the cmyb locus, promote cmyb expression, and promote hematopoietic development. Our results reveal an epigenetic mechanism supporting the maintenance of hematopoietic cell fate via DNA methylation-mediated perdurance of a key transcription factor in HSPCs.