Primitive Myelopoiesis Research Articles

Ebf1 Is Involved in Primitive Myelopoiesis and Contributes in Leukemia Pathogenesis

Acute myeloid leukemia (AML) is caused by the abnormal proliferation and differentiation of a clonal population of myeloid progenitors. EBF1 is a key transcriptional factor in regulating hematopoiesis and has been linked to acute lymphoblastic leukemia (ALL). However, the relationship between AML and EBF1 mutation has not yet been studied. Recent research has shown that deleting EBF1 results in myeloid-biased progenitors and decreased HSC quiescence and repopulation capacity. This suggests that EBF1 is involved in earlier hematopoiesis and has additional functions beyond B-cell committed development. Our clinical center conducted whole-exome sequencing on 231 cases of acute myeloid leukemia and identified four patients with an Ebf1 mutation. This corresponds to a mutation frequency of 1.73% and the mutations were identified as p.Q477H (c.1434G>T), p.S513P (c.1537T>C), p.C534F (c.1601GT), and p.P570A (c.1708C>G). Data from the cBioPortal database showed that AML patients with Ebf1 deep deleted mutation had shortened survival. To better understand the role of Ebf1 in AML pathogenesis, we generated an ebf1a-deficient zebrafish mutant. Further research showed that the ebf1a deficiency caused neutrophilia during primitive hematopoiesis. BrdU staining in Tg(coronin1a: GFP) labeled myeloid progenitors was significantly higher in ebf1a-deficient larvae than in wild-type larvae, indicating that ebf1a deficiency led to increased proliferating myeloid progenitors which would take responsibility for the neutrophilia in early-developing larvae. We conducted experiments using four RNA variants (p.Q477H, p.S513P, p.C534F, and p.P570A) injected into ebf1a deficiency zebrafish larvae to verify their pathogenicity. Our findings showed that while EBF1 p.Q477H and p.S513P caused a decrease in neutrophils, p.C534F resulted in neutrophilia, indicating that it might be a loss-of-function mutation. To further understand the mechanism, we introduced EBF1 p.C534F to AML cell culture, and CCk8 and cell cycle analysis showed increased proliferation rates and disturbed cell cycle balance, leading to increased proliferation of AML cells of EBF1-C534F mutation. The cells bearing this mutation showed a higher rate of proliferation, likely due to a disturbance in the balance of the cell cycle. These findings provide valuable insight into the potential pathogenetic mechanism of EBF1 p.C534F. Our investigation into the cause of ebf1 deficiency disrupting normal hematopoiesis led us to perform bulk RNA-sequencing with sorted myeloid-progenitors labeled by Tg:(coronin1a-GFP) in 30hpf zebrafish larvae. We found that leukocyte differentiation and myeloid cell cycle-related genes such as lyz, lygl1, coro1a, lcp1, cebpa, spi1b, mpx, irf8, il6, il34, etc were upregulated in ebf1a deficiency zebrafish. Additionally, anxa1c, anxa1b, Cish, and nlrc3l, which have been reported to suppress lymphoid proliferation but increase myeloid expansion, were down-regulated. Go enrichment analysis showed that the significant genes were associated with hematopoietic disorders including AML and MDS. To address the issue of disturbed cell cycle balance caused by EBF1 deficiency and loss-of-function mutation, we sought to suppress the G1-S transition via CDK inhibitor. We treated EBF1-C534F, EBF1-WT, and control AML cell culture with CDK4 inhibitor: AMG925. We were surprised to find that the CDK4 inhibitor sufficiently suppressed the proliferation and reversed the prolonged S phase caused by EBF1 mutation. We investigated the myeloid-bias function of Ebf1 deficiency and the potential pathogenesis of EBF1 mutations of AML from the development view and were surprised to find CDK inhibitors as a potential drug for EBF1-deficient AML patients.

Cohesin Components Stag1 and Stag2 Differentially Influence Haematopoietic Mesoderm Development in Zebrafish Embryos

Cohesin is a multiprotein complex made up of core subunits Smc1, Smc3, and Rad21, and either Stag1 or Stag2. Normal haematopoietic development relies on crucial functions of cohesin in cell division and regulation of gene expression via three-dimensional chromatin organization. Cohesin subunit STAG2 is frequently mutated in myeloid malignancies, but the individual contributions of Stag variants to haematopoiesis or malignancy are not fully understood. Zebrafish have four Stag paralogues (Stag1a, Stag1b, Stag2a, and Stag2b), allowing detailed genetic dissection of the contribution of Stag1-cohesin and Stag2-cohesin to development. Here we characterize for the first time the expression patterns and functions of zebrafish stag genes during embryogenesis. Using loss-of-function CRISPR-Cas9 zebrafish mutants, we show that stag1a and stag2b contribute to primitive embryonic haematopoiesis. Both stag1a and stag2b mutants present with erythropenia by 24 h post-fertilization. Homozygous loss of either paralogue alters the number of haematopoietic/vascular progenitors in the lateral plate mesoderm. The lateral plate mesoderm zone of scl-positive cells is expanded in stag1a mutants with concomitant loss of kidney progenitors, and the number of spi1-positive cells are increased, consistent with skewing toward primitive myelopoiesis. In contrast, stag2b mutants have reduced haematopoietic/vascular mesoderm and downregulation of primitive erythropoiesis. Our results suggest that Stag1 and Stag2 proteins cooperate to balance the production of primitive haematopoietic/vascular progenitors from mesoderm.

ADTRP regulates TFPI expression via transcription factor POU1F1 involved in coronary artery disease.

Genomic variants in both ADTRP and TFPI genes are associated with risk of coronary artery disease (CAD). ADTRP regulates TFPI expression and endothelial cell functions involved in the initiation of atherosclerotic CAD. ADTRP also specifies primitive myelopoiesis and definitive hematopoiesis by upregulating TFPI expression. However, the underlying molecular mechanism is unknown. Here we show that transcription factor POU1F1 is the key by which ADTRP regulates TFPI expression. Luciferase reporter assays, chromatin-immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) in combination with analysis of large and small deletions of the TFPI promoter/regulatory region were used to identify the molecular mechanism by which ADTRP regulates TFPI expression. Genetic association was assessed using case-control association analysis and phenome-wide association analysis (PhenGWA). ADTRP regulates TFPI expression at the transcription level in a dose-dependent manner. The ADTRP-response element was localized to a 50bp region between -806bp and -756bp upstream of TFPI transcription start site, which contains a binding site for POU1F1. Deletion of POU1F1-binding site or knockdown of POU1F1 expression abolished ADTRP-mediated transcription of TFPI. ChIP and EMSA demonstrated that POU1F1 binds to the ADTRP response element. Genetic analysis identified significant association between POU1F1 variants and risk of CAD. PhenGWA identified other phenotypic traits associated with the ADTRP-POU1F1-TFPI axis such as lymphocyte count (ADTRP), waist circumference (TFPI), and standing height (POU1F1). These data identify POU1F1 as a transcription factor that regulates TFPI transcription in response to ADTRP, and link POU1F1 variants to risk of CAD for the first time.

Zebrafish microRNA miR-210-5p inhibits primitive myelopoiesis by silencing foxj1b and slc3a2a mRNAs downstream of gata4/5/6 transcription factor genes

Zebrafish gata4/5/6 genes encode transcription factors that lie on the apex of the regulatory hierarchy in primitive myelopoiesis. However, little is known about the roles of microRNAs in gata4/5/6-regulated processes. Performing RNA-Seq deep sequencing analysis of the expression changes of microRNAs in gata4/5/6-knockdown embryos, we identified miR-210-5p as a regulator of zebrafish primitive myelopoiesis. Knocking down gata4/5/6 (generating gata5/6 morphants) significantly increased miR-210-5p expression, whereas gata4/5/6 overexpression greatly reduced its expression. Consistent with inhibited primitive myelopoiesis in the gata5/6 morphants, miR-210-5p overexpression repressed primitive myelopoiesis, indicated by reduced numbers of granulocytes and macrophages. Moreover, knocking out miR-210 partially rescued the defective primitive myelopoiesis in zebrafish gata4/5/6-knockdown embryos. Furthermore, we show that the restrictive role of miR-210-5p in zebrafish primitive myelopoiesis is due to impaired differentiation of hemangioblast into myeloid progenitor cells. By comparing the set of genes with reduced expression levels in the gata5/6 morphants to the predicted target genes of miR-210-5p, we found that foxj1b and slc3a2a, encoding a forkhead box transcription factor and a solute carrier family 3 protein, respectively, are two direct downstream targets of miR-210-5p that mediate its inhibitory roles in zebrafish primitive myelopoiesis. In summary, our results reveal that miR-210-5p has an important role in the genetic network controlling zebrafish primitive myelopoiesis.

Zebrafish miR-462-731 regulates hematopoietic specification and pu.1-dependent primitive myelopoiesis

MicroRNAs (miRNAs) play significant roles in both embryonic hematopoiesis and hematological malignancy. Zebrafish miR-462-731 cluster is orthologous of miR-191-425 in human which regulates proliferation and tumorigenesis. In our previous work, miR-462-731 was found highly and ubiquitously expressed during early embryogenesis. In this study, by loss-of-function analysis (morpholino knockdown combined with CRISRP/Cas9 knockout) and mRNA profiling, we suggest that miR-462-731 is required for normal embryonic development by regulating cell survival. We found that loss of miR-462/miR-731 caused a remarkable decrease in the number of erythroid cells as well as an ectopic myeloid cell expansion at 48 hpf, suggesting a skewing of myeloid-erythroid lineage differentiation. Mechanistically, miR-462-731 provides an instructive input for pu.1-dependent primitive myelopoiesis through regulating etsrp/scl signaling combined with a novel pu.1/miR-462-731 feedback loop. On the other hand, morpholino (MO) knockdown of miR-462/miR-731 resulted in an expansion of posterior blood islands at 24 hpf, which is a mild ventralization phenotype resulted from elevation of BMP signaling. Rescue experiments with both BMP type I receptor inhibitor dorsomorphin and alk8 MO indicate that miR-462-731 acts upstream of alk8 within the BMP/Smad signaling pathway and functions as a novel endogenous BMP antagonist. Besides, an impairment of angiogenesis was observed in miR-462/miR-731 morphants. The specification of arteries and veins was also perturbed, as characterized by the irregular patterning of efnb2a and flt4 expression. Our study unveils a previously unrecognized role of miR-462-731 in BMP/Smad signaling mediated hematopoietic specification of mesodermal progenitors and demonstrates a miR-462-731 mediated regulatory mechanism driving primitive myelopoiesis in the ALPM. We also show a requirement for miR-462-731 in regulating arterial-venous specification and definitive hematopoietic stem cell (HSC) production. The current findings might provide further insights into the molecular mechanistic basis of miRNA regulation of embryonic hematopoiesis and hematological malignancy.

Identification of a new adtrp1-tfpi regulatory axis for the specification of primitive myelopoiesis and definitive hematopoiesis.

A genomic variant in the human ADTRP [androgen-dependent tissue factor (TF) pathway inhibitor (TFPI) regulating protein] gene increases the risk of coronary artery disease, the leading cause of death worldwide. TFPI is the TF pathway inhibitor that is involved in coagulation. Here, we report that adtrp and tfpi form a regulatory axis that specifies primitive myelopoiesis and definitive hematopoiesis, but not primitive erythropoiesis or vasculogenesis. In zebrafish, there are 2 paralogues for adtrp (i.e., adtrp1 and adtrp2). Knockdown of adtrp1 expression inhibits the specification of hemangioblasts, as shown by decreased expression of the hemangioblast markers, etsrp, fli1a, and scl; blocks primitive hematopoiesis, as shown by decreased expression of pu.1, mpo, and l-plastin; and disrupts the specification of hematopoietic stem cells (definitive hematopoiesis), as shown by decreased expression of runx1 and c-myb However, adtrp1 knockdown does not affect erythropoiesis during primitive hematopoiesis (no effect on gata1 or h-bae1) or vasculogenesis (no effect on kdrl, ephb2a, notch3, dab2, or flt4). Knockdown of adtrp2 expression does not have apparent effects on all markers tested. Knockdown of adtrp1 reduced the expression of tfpi, and hematopoietic defects in adtrp1 morphants were rescued by tfpi overexpression. These data suggest that the regulation of tfpi expression is one potential mechanism by which adtrp1 regulates primitive myelopoiesis and definitive hematopoiesis.-Wang, L., Wang, X., Wang, L., Yousaf, M., Li, J., Zuo, M., Yang, Z., Gou, D., Bao, B., Li, L., Xiang, N., Jia, H., Xu, C., Chen, Q., Wang, Q. K. Identification of a new adtrp1-tfpi regulatory axis for the specification of primitive myelopoiesis and definitive hematopoiesis.

Transgenic Zebrafish Expressing the Human TEL-AML1 Oncogene Have Aberrant Developmental Myelopoiesis

The t(12;21)(p13;q22) chromosomal translocation resulting in the TEL-AML1 fusion gene is the most common translocation in childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) and is found in approximately 25% of cases. The translocation arises in utero, and can be found in approximately 1% of newborn infants. However, less than 1% of these children develop leukaemia and this translocation is not observed in adult ALL. This suggests that the cell of origin resulting in the eventual transformation to BCP-ALL is present and disrupted early during development but does not represent a potent oncoprotein. Further evidence for this is provided by animal models show that TEL-AML1 alone is necessary but not sufficient to trigger overt ALL.To further delineate the effects of TEL-AML1 during developmental haematopoiesis we utilized a zebrafish model, Tg(ZβA:hTEL-AML1-EGFP), that expresses human TEL-AML1 (hTEL-AML1) under the control of zebrafish β-actin gene promoter. Previous studies have shown that approximately 2% of these fish develop B-cell leukemia with a long latency.Primitive erythropoiesis was studied using whole mount in situ hybridisation (WISH) in Tg(ZβA:EGFP-hTEL-AML1+/-) and siblings. The expression pattern of ikarosand gata1a showed that primitive erythroid cells develop normally in Tg(ZβA:EGFP-hTEL-AML1+/-). The development of erythroid cells at later stages was studied using o-dianisidine staining, to assess the haemoglobinization of erythrocytes. Our results suggest normal differentiation of erythroblasts into erythrocytes in Tg(ZβA:EGFP-hTEL-AML1+/-) embryos and normal haemoglobinization of erythrocytes.We next examined myelopoiesis in Tg(ZβA:EGFP-hTEL-AML1)using WISH for cebpα and lcp1, and Sudan Black (SB) staining to label mature granulocytes. Primitive myelopoiesis was unperturbed by hTEL-AML1 at 18hpf, however we observed an increased number of SB positive cells commencing at 2 days post fertilization (dpf). SB positive cells continued to increase in number until 4dpf when the difference became less pronounced and was gone by 7dpf (Figure 1). SB positive myeloid cells at these stages are most likely to be derived from the earliest definitive hematopoietic precursors, such as the transient bi-potent erythro-myeloid progenitor (EMP) because primitive myeloid cells are predominantly macrophage-like cells that do not express SB. By 4dpf when definitive hematopoietic stem and progenitor cell (HSPC) derived myelopoiesis is dominant, the observed difference in mature myeloid cell numbers has resolved.The earliest lymphoid cells in zebrafish embryos are thymocytes. Thymocytes were visualized using WISH for gata3 (GATA binding protein 3), lck (T-cell specific tyrosine kinase) and rag1 (recombination activating gene 1). Expression of all three markers was unpertubed indicating that early T cell development is not affected by hTEL-AML1 fusion protein expression.B lymphoid cells have not been convincingly observed in zebrafish until 3 weeks post fertilization despite evidence of VDJ rearrangements in whole embryos from 4dpf. We attempted to identify evidence of B lymphoid cells using WISH for pax5 and cd79b, however we were not able to identify any cells before 10dpf. This supports the observation that B cells do not arise until later in development, or are too low in expression level or few in number to identify by WISH.In order to more closely recapitulate aspects of TEL-AML1 leukemia in humans, where one the remaining endogenouscopy of TEL is frequently lost at diagnosis of BCP-ALL, we have generated a knockout of zebrafish etv6 using CRISPR/Cas9. 4 Etv6 guides targeting zebrafish etv6 were more than 90% efficient at cleaving their DNA target. F0 animals with this mutational burden survived to adulthood with no increase in mortality. By contrast F0 Tg(ZβA:EGFP-hTEL-AML1+/-) injected with etv6 crispr had an increased mortality and showed a variety of developmental abnormalities. Assessment of their haematopoietic compartment is ongoing.In summary we show that early myelopoiesis is disrupted during development in a zebrafish transgenic model expressing human TELAML1, and that this occurs prior to the stage at which definitive pluripotent HSPC-derived myelopoiesis is dominant. This supports the hypothesis that effects of TELAML1 during developmental haematopoiesis underpin its oncogenic potential during childhood. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Chemical Modifier Screen in Zebrafish Mutants for the Spliceosomal Component Sf3b1reveals Genotype-Selective Vulnerabilities

Myelodysplastic syndrome (MDS) is a disorder arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis. Mutations in the spliceosomal component SF3B1 (Splicing Factor 3B, subunit 1) are prevalent in MDS, but how this leads to aberrant hematopoiesis is unclear. We elucidated the in vivo consequences of sf3b1 misregulation on hematopoiesis and splicing using a zebrafish sf3b1 (sf3b1hi3394) loss-of-function mutant. Primitive erythropoiesis initiates normally in sf3b1 mutants, as evidenced by normal expression of scl (stem cell leukemia), gata1 and beta-globin, but maturation is defective as shown by decreased hemoglobinization and an increase in immature erythroblasts at 48 hours post fertilization (hpf). Similarly, primitive myelopoiesis initiates normally as early expression of scl and pu.1 are normal, but expression of the differentiation markers l-plastin and myeloperoxidase are diminished in mutants at 24 and 28 hpf. Hemogenic endothelial and HSPC markers runx1 and gata2b are also diminished, while the pan-endothelial marker kdrl (kinase insert domain receptor like) and aorta-specific markers notch1b and notch3 are normally expressed at 24 hpf, indicating hematopoietic induction is more sensitive to sf3b1 loss than vascular formation. RNA-seq analysis of sf3b1 mutants revealed large-scale splicing defects and alterations, particularly affecting introns with suboptimal splice sites. Comparison of RNA-seq data from zebrafish sf3b1 mutants and MDS patients harboring SF3B1 mutations revealed a significant overlap in misspliced genes and in the corresponding affected pathways, including many pathways implicated in MDS pathology such as TGFβ (Transforming Growth Factor-Beta) and immune signaling. Together, these results show conservation between zebrafish mutants and human cells with SF3B1 mutations. We next took a chemical genetic approach and discovered sf3b1 mutants were more sensitive than wild type siblings to treatment with E7107, a small molecule inhibitor of the spliceosome, indicating a potential therapeutic window for spliceosome modulators in MDS. Motivated by this finding, we performed a large-scale chemical modifier screen and identified many new lead compounds (6 out of 562 screened) that also show synthetic lethality in sf3b1 mutants. These small molecules are enriched in those with anti-inflammatory properties, suggesting aberrant immune signaling could underlie some of the defects in sf3b1 mutants. Together, our data demonstrate the molecular and cellular consequences of sf3b1 loss in vivo and establish sf3b1 zebrafish mutants as a novel model for discovery of therapeutics for MDS. DisclosuresSmith:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment.

The Spliceosomal Component Sf3b1 Is Essential for Hematopoietic Differentiation

Myelodysplastic syndrome (MDS) is a disorder arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis. A multitude of recurrent somatic mutations in spliceosomal components were recently identified in MDS that likely contribute to the pathogenesis of the disease. The lack of in vivo models to study cell-type specific effects of spliceosomal mutations limits our understanding of why such mutations lead to hematopoietic abnormalities. Using a zebrafish with a loss-of-function mutation in sf3b1 (sf3b1hi3394), an essential member of the spliceosome, we demonstrate hematopoietic cell differentiation and hematopoietic stem and progenitor cell (HSPC) specification are processes sensitive to spliceosomal malfunction. Primitive erythropoiesis initiates normally in sf3b1 mutants as evidenced by expression of scl in the posterior lateral plate mesoderm at 14 hours post fertilization (hpf) as well as gata1 and beta-globin at 24 hpf. Flow cytometry quantification of gata1:gfp positive erythrocytes showed sf3b1 mutants have 25% more cells at 24 hpf, but greater than 3-fold fewer cells at 36 and 48 hpf, time points when wild type erythroblasts are expanding and differentiating. At 48 hpf, we also observed decreased levels of o-dianisidine positive erythrocytes, low numbers of morphologically mature erythroblasts, and higher levels of immature erythroblasts in sf3b1 mutants. Similarly, we observed normal initiation of primitive myelopoiesis marked by stem cell leukemia (scl) expression in the anterior lateral plate mesoderm at 14 hpf, but diminished expression of more differentiated markers, l-plastin and myeloperoxidase at 24 and 28 hpf in sf3b1 mutants. Quantification of lysozyme C:dsred positivemyeloid cells using flow cytometry also showed 24-fold fewer mature myeloid cells in sf3b1 mutants at 36 hpf. Our data on primitive erythropoiesis and myelopoiesis indicate sf3b1 is required for hematopoietic cell differentiation. Additionally, sf3b1 mutants have diminished expression of the definitive HSPC marker runx1 within the aorta at 28 hpf. In contrast, we observed normal expression of the pan-endothelial marker kinase insert domain receptor-like (kdrl) and aorta-specific markers notch1b and notch3 at 24 hpf. Flow cytometry quantification of kdrl:gfp endothelial cells at 24 hpf shows no difference in the frequency of endothelial cells in sf3b1 mutants. Moreover, we observed fewer cmyb:gfp; kdrl:dsred double positive HSPCs along the dorsal aorta. The data suggest that decreased HSPC formation in sf3b1 mutants is due to a failure in hemogenic induction. From these studies, we show sf3b1 is required at specific stages of hematopoietic cell development. These results provide novel insight into the role of splicing in blood cell development, and can afford a deeper understanding of the mechanism of splicing regulation on the origins of MDS. DisclosuresNo relevant conflicts of interest to declare.

Multiple Roles for the Zebrafish Homologue of the Murine Evi1 Gene during Primitive Myelopoiesis and HSC Development

The Evi1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Several reports associate Evi1 expression with aggressiveness in myeloid leukemia. Since developmental pathways often reactivate in cancer, we hypothesized that Evi1 also plays critical roles during developmental hematopoiesis. Here, we employ the zebrafish model to study how evi1 modulates early blood development. We find that indeed zebrafish evi1 co-localizes with the hematopoietic markers scl, gata1, pu.1 and gata2 in the posterior lateral mesoderm and the rostral blood islands, indicating involvement in primitive hematopoiesis. Knockdown of evi1 via three independent Morpholino Oligonucleotides impairs embryonic myelopoiesis as shown by reduced pu.1, mpo and l-plastin staining, while not affecting hemangioblast formation and primitive erythropoiesis. Additionally, we observe reduced levels of cd41 expression upon evi1 knockdown, indicating that megakaryopoiesis is also impaired.Notably, at later time-points, evi1 is also expressed in the AGM region and evi1 morphants show strong reduction of runx1/c-myb expression in this region, demonstrating an additional role in hematopoietic stem cell (HSC) formation. Consistently, evi1 morphants lack ikaros+ lymphocyte precursor cells and rag1+ T-lymphocytes, and less circulating globin+, lyz+ and cd41+ cells are detected in transgenic fish analyzed by flow cytometry at 5 days post fertilization. To dissect the mechanisms by which evi1 regulates HSC formation, we furthermore analyzed genes specifically expressed in the dorsal aorta, where HSCs emerge from the hemogenic endothelium. We hypothesize that evi1 is indeed regulating HSC specification from hemogenic endothelial cells, since detailed analyses show defective aortic expression of efnb2a and dlc. How this effect is mediated is currently being investigated. Furthermore, TUNEL, anti-activated Caspase-3, anti-phospho Histone H3 and BrdU assays show both increased apoptosis and decreased proliferation in the AGM region of evi1 morphants as compared to control injected fish, suggesting important additional effects of evi1 on HSC biology beyond the specification step. Similar effects are seen in the rostral blood islands and are possibly responsible for the observed reduction of primitive myeloid progenitors.We also examined potential downstream targets of evi1. Previous reports in adult murine hematopoietic cells suggest that Evi1 affects HSC proliferation through regulation of Gata2. Indeed, gata2 co-injection enhances hematopoietic cell viability also in zebrafish embryos, and co-injection of gata2 mRNA is able to fully rescue both primitive myelopoiesis and HSC formation. We are currently investigating whether gata2 expression can compensate evi1 requirement during HSC specification or, alternatively, might rescue HSC numbers by amplifying residual HSCs that escape evi1 inhibition.Taken together, our data demonstrate that evi1 plays multiple roles during hematopoietic development, critically regulating the biology of primitive myeloid progenitors and pre-formed HSCs, as well as HSC specification from the hemogenic endothelium. Currently, we analyze the molecular mechanisms that mediate evi1 effects during these different regulatory steps and use the CRISPR/Cas9 system to generate evi1 mutant fish for further validation of our findings. DisclosuresNo relevant conflicts of interest to declare.

Ncor1 and Ncor2 play essential but distinct roles in zebrafish primitive myelopoiesis.

Although Ncor1 and Ncor2, the co-repressors that can actively repress gene transcription through binding nuclear receptors in the absence of ligands, are crucial to vertebrate embryogenesis, their roles in its primitive myelopoiesis remain unknown. We investigated the function of ncor1 or ncor2 in zebrafish embryos by antisense morpholino knocking down technologies. Development of both mfap4(+) macrophages and mpx(+) neutrophils was abolished in ncor2 morphants, whereas development of mpx(+) neutrophils was depleted in ncor1 morphants. ncor2 was essential to the development of spi1b(+) myeloid precursors but not anterior hemangioblasts whereas ncor1 was dispensable to the specification of spi1b(+) myeloid precursors and anterior hemangioblasts. Overexpressing spi1b could partially rescue expressions of mfap4 and mpx in ncor2 morphants. Furthermore, overexpressing tal1/lmo2 could well rescue the defective myelopoiesis in both ncor1 and ncor2 morphants. Ncor1 and Ncor2 play essential but distinct roles in zebrafish primitive myelopoiesis. ncor2 could parallel with tal1/lmo2 and acted upstream of spi1b to produce mature macrophages and neutrophils during primitive myelopoiesis. The role of ncor1 in zebrafish myelopoiesis could be substituted by excessive Tal1/Lmo2.

Zebrafish Ortholog of Human DOT1L Regulates Primitive and Transient Definitive Hematopoiesis and Controls hoxa9 and meis1 Expression

AbstractAbstract 849 Objective:DOT1L is a H3K79 methyltransferase implicated in multiple biological processes including embryonic development, cell proliferation, DNA damage repair and hematopoiesis. Recently, it was reported that DOT1L interacts with various transcription factor MLL partner proteins, and that aberrant DOT1L methyltransferase activity is essential for the form of leukemogenesis mediated by MLL fusion oncoproteins. These findings led to current efforts in therapeutic targeting of DOT1L for MLL-rearranged leukemias. However, the role of DOT1L in hematopoiesis is incompletely understood, largely because all prior studies were conducted using mice where Dot1L inactivation leads to early embryonic lethality due in part to the failure of primitive hematopoiesis, precluding analysis Dot1L during the transition from primitive to definitive hematopoiesis. In this study we took advantage of a unique attribute of zebrafish to survive for several days without blood cells, which offers new possibilities to study effects of loss of function of genes that cause embryonic lethality in mice from hematopoietic defects. Here, we report a functional characterization of the zebrafish dot1l gene during development, with particular emphasis on its role in hematopoietic regulation. Methods and Results:We identified a single ortholog of human DOT1L on zebrafish chromosome 22 using the NCBI HomoloGene resource, with a predicted protein that is 66% identical to human DOT1L overall and has greatest similarity (96%) restricted to its methyltransferase domain. Highly conserved syntenic genes surrounding zebrafish dot1l and genes in chromosomal regions containing human DOT1L (chr. 19) and mouse Dot1L (chr. 10) suggested functional similarity and formed the basis to further investigate the zebrafish ortholog. By whole-mount in situ hybridization (WISH) dot1l was found as early as the 2-cell stage before zygotic gene expression starts, indicating maternally supplied transcripts in the embryo. Zygotic dot1l expression was detected at 20 hpf in the posterior intermediate cell mass (ICM) where primitive erythropoiesis occurs in zebrafish. Two different splice blocking morpholinos were used to inhibit dot1l pre-mRNA splicing in either wild type or fluorescent reporter lines to determine the consequences of zebrafish dot1l depletion. The morphants showed impaired growth, defective angiogenesis and cardiac dilatation, consistent with developmental defects in Dot1L−/−mice. Although Dot1L−/−murine embryos are anemic, o-dianisidine staining of the morphants at 48 hpf showed reduced circulating red cells during definitive hematopoiesis beyond when Dot1L−/− mice are viable. Reduced in vivo fluorescence in Tg(gata1:dsRed) dot1l morphants at 48 hpf also suggested an erythroid defect. WISH analysis of 24 hpf dot1l morphants revealed significant up-regulation of the myeloid marker pu.1 in the anterior lateral plate mesoderm (ALPM) where primitive myelopoiesis occurs in zebrafish, persistent and ectopic pu.1 expression in the ICM and reduced expression of the early progenitor marker gata2 and the erythroid marker gata1. Additionally at 20 hpf, Tg(pu.1:gfp) dot1l morphants suggested increased pu.1 expressing cells in the ALPM, yolk and ICM. These results indicate that dot1l plays essential roles in primitive erythropoiesis and primitive myelopoiesis and in the erythromyeloid cell fate decision during transient definitive hematopoiesis. Monitoring of hematopoietic and developmental marker genes by qRT-PCR in 24 hpf embryos confirmed the expansion of myelopoiesis and impairment of erythropoiesis. Interestingly, qRT-PCR analysis also revealed an entirely new finding that two key target genes in MLL-fusion-mediated leukemogenesis, hoxa9 and meis1, were downregulated in dot1l morphants. Conclusion:This work demonstrates a critical role of dot1l in zebrafish primitive erythropoiesis, in agreement with previous observations in Dot1L−/− mice, but reveals a new role of dot1l in erythromyeloid progenitor differentiation in transient definitive hematopoiesis. Furthermore, based on reduced hoxa9 and meis1 expression with dot1l depletion, we report for the first time that dot1l is a key regulator of hoxa9a and meis1 gene expression, human orthologs of both of which are key upregulated target genes in MLL leukemogenesis. These discoveries also have important implications for DOT1L directed therapies. Disclosures:No relevant conflicts of interest to declare.

Maternal pak4 expression is required for primitive myelopoiesis in zebrafish

Transcripts of pak4, the zebrafish ortholog of p21-activated kinase 4 (PAK4), are most abundant in the egg and fall to low levels by the end of gastrulation, after which expression is essentially ubiquitous. Translation of maternal mRNA into pak4 protein is first detectable at high stage (3.3hpf). Splice-blocking morpholino oligonucleotides (MOs) were used to prevent zygotic pak4 expression. This had no discernable effect on development through larval stages. In contrast, a translation-blocking MO, alone or in combination with the splice MOs, resulted in a complex lethal phenotype. In addition to disrupted somite development and other morphogenetic abnormalities, the knockdown of maternal pak4 expression led to alterations in regulatory gene expression in the primitive hematopoietic domains, leading to deficiencies in granulocyte and leukocyte lineages. At least some of the effects of pak4 knockdown on gene expression could be mimicked by treatment with actin depolymerization agents, suggesting a mechanistic link between regulation of microfilament dynamics by pak4 and regulation of gene expression in primitive myeloid cell differentiation.

Retinoic Acid Signaling Plays a Restrictive Role in Zebrafish Primitive Myelopoiesis

Retinoic acid (RA) is known to regulate definitive myelopoiesis but its role in vertebrate primitive myelopoiesis remains unclear. Here we report that zebrafish primitive myelopoiesis is restricted by RA in a dose dependent manner mainly before 11 hpf (hours post fertilization) when anterior hemangioblasts are initiated to form. RA treatment significantly reduces expressions of anterior hemangioblast markers scl, lmo2, gata2 and etsrp in the rostral end of ALPM (anterior lateral plate mesoderm) of the embryos. The result indicates that RA restricts primitive myelopoiesis by suppressing formation of anterior hemangioblasts. Analyses of ALPM formation suggest that the defective primitive myelopoiesis resulting from RA treatment before late gastrulation may be secondary to global loss of cells for ALPM fate whereas the developmental defect resulting from RA treatment during 10–11 hpf should be due to ALPM patterning shift. Overexpressions of scl and lmo2 partially rescue the block of primitive myelopoiesis in the embryos treated with 250 nM RA during 10–11 hpf, suggesting RA acts upstream of scl to control primitive myelopoiesis. However, the RA treatment blocks the increased primitive myelopoiesis caused by overexpressing gata4/6 whereas the abolished primitive myelopoiesis in gata4/5/6 depleted embryos is well rescued by 4-diethylamino-benzaldehyde, a retinal dehydrogenase inhibitor, or partially rescued by knocking down aldh1a2, the major retinal dehydrogenase gene that is responsible for RA synthesis during early development. Consistently, overexpressing gata4/6 inhibits aldh1a2 expression whereas depleting gata4/5/6 increases aldh1a2 expression. The results reveal that RA signaling acts downstream of gata4/5/6 to control primitive myelopoiesis. But, 4-diethylamino-benzaldehyde fails to rescue the defective primitive myelopoiesis in either cloche embryos or lycat morphants. Taken together, our results demonstrate that RA signaling restricts zebrafish primitive myelopoiesis through acting downstream of gata4/5/6, upstream of, or parallel to, cloche, and upstream of scl.

Irf8 regulates macrophage versus neutrophil fate during zebrafish primitive myelopoiesis

AbstractIn vertebrates, myeloid cells comprise polymorphonuclear and mononuclear lineages that arise from 2 successive waves of development: a transitory primitive wave giving rise to limited myeloid cells during embryonic stage and a definitive wave capable of producing myeloid cells throughout the fetal and adult life. One key unresolved question is what factors dictate polymorphonuclear versus mononuclear lineage fates during myelopoiesis. Here we show that during zebrafish embryogenesis interferon regulatory factor-8 (irf8) is expressed specifically in macrophages but not neutrophils. Suppression of Irf8 function in zebrafish causes a depletion of macrophages and an enhanced output of neutrophils but does not affect the overall number, proliferation, and survival of primitive myeloid cells. These data indicate that the skewed myeloid lineage development in Irf8 knockdown embryos results from a cell-fate switching. Such a conclusion is further supported by the observation showing that overexpression of Irf8 promotes macrophage formation at the expense of neutrophil development. Genetic epistasis analysis reveals that Irf8 acts downstream of Pu.1 but is insufficient to promote macrophage development in the absence of Pu.1. Our findings demonstrate that Irf8 is a critical determinant for neutrophil versus macrophage fate choice during zebrafish primitive myelopoiesis.

Discovery and Characterization of Novel Vascular and Hematopoietic Genes Downstream of Etsrp in Zebrafish

The transcription factor Etsrp is required for vasculogenesis and primitive myelopoiesis in zebrafish. When ectopically expressed, etsrp is sufficient to induce the expression of many vascular and myeloid genes in zebrafish. The mammalian homolog of etsrp, ER71/Etv2, is also essential for vascular and hematopoietic development. To identify genes downstream of etsrp, gain-of-function experiments were performed for etsrp in zebrafish embryos followed by transcription profile analysis by microarray. Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel. Regulation of these genes by etsrp was confirmed by ectopic induction in etsrp overexpressing embryos and decreased expression in etsrp deficient embryos. Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development. The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development.

Eena Promotes Myeloid Proliferation through Stimulating ERK1/2 Phosphorylation in Zebrafish

The EEN (extra eleven nineteen) gene is one of the fusion partners of mixed-lineage leukemia, located on chromosome 19p13. Here we cloned two een genes (designated as eena and eenb) in zebrafish, which are assigned to linkage groups 8 and 2, respectively. Whole-mount in situ hybridization assay showed that eena and eenb have overlapping but distinct expression patterns during embryogenesis. Ubiquitous or targeted overexpression of eena, but not eenb, into wild-type or transgenic embryos (green fluorescent protein-labeled myeloid progenitors) induced a significant proliferation and ectopic distribution of myeloid progenitors in the yolk sac. Using a morpholino antisense gene knockdown approach, we showed that the number of myeloid progenitors and their downstream mature myelomonocytic cells was significantly decreased in the eena- deficient embryos. Mechanistically, overexpression of eena selectively stimulated ERK phosphorylation and increased the level of transcription factor c-Fos in vitro and in vivo, whereas eena lacking the Src homology 3 domain completely abolished these effects. Furthermore, a MAPK/ERK kinase (MEK) inhibitor, PD98059, blocked the eena-induced cell proliferation and activation of ERK signaling. The results suggest that eena plays an important role in the development of the myeloid cell through activation of the ERK pathway and may provide a valuable reference for future studies of the role of EEN in leukemogenesis.

A role for the transcription intermediary factor 2 in zebrafish myelopoiesis

TIF2 is fused with MOZ in the inv(8)(p11q13) acute myeloid leukemia. TIF2, member of the p160 family, is a histone acetyl transferase (HAT). Deletion of p160 genes were performed in mice. Some observations suggest that p160 family members may perform overlapping functions in mice. Therefore, we decided to choose the zebrafish model to study TIF2. The aim of this study was to characterize the role of this HAT during embryonic development. We use antisense, morpholino-modified oligomers to transiently knockdown tif2 gene, thus determining whether TIF2 plays a role in zebrafish early development. We show that tif2 is involved in embryogenesis and in primitive hematopoiesis. tif2-knockdown zebrafish embryos are smaller than controls, they demonstrate shorter tails, they display notochord deformation and they exhibit U-shaped tail somites. A synthetic RNA encoding human TIF2 rescues the tif2-knockdown phenotype. Analysis of fli1 expression by whole-mount in situ hybridization indicates normal angioblast specification, but altered localization of intersomitic vessels. The posterior intermediate cell mass, in which a part of primitive hematopoiesis occurs, is altered in tif2 morphants and whole-mount in situ hybridization analyses of l-plastin and mpx expression suggest a specific inhibition of granulocytic and macrophagic differentiation at late stages. These data indicate an important role for TIF2 in zebrafish primitive myelopoiesis.

Differential Regulation of Primitive Myelopoiesis in the Zebrafish by Spi-1/Pu.1 and C/ebp1

The zebrafish has become a powerful tool for analysis of vertebrate hematopoiesis. Zebrafish, unlike mammals, have a robust primitive myeloid pathway that generates both granulocytes and macrophages. It is not clear how this unique primitive myeloid pathway relates to mammalian definitive hematopoiesis. In this study, we show that the two myeloid subsets can be distinguished using RNA in situ hybridization. Using a morpholino-antisense gene knockdown approach, we have characterized the hematopoietic defects resulting from knockdown of the myeloid transcription factor gene pu.1 and the unique zebrafish gene c/ebp1. Severe reduction of pu.1 resulted in complete loss of primitive macrophage development, with effects on granulocyte development only with maximal knockdown. Reduction of c/ebp1 did not ablate initial macrophage or granulocyte development, but resulted in loss of expression of the secondary granule gene lys C. These data reveal strong functional conservation of pu.1 between zebrafish primitive myelopoiesis and mammalian definitive myelopoiesis. Further, these results are consistent with a conserved role between c/ebp1 and mammalian C/EBPE, whose ortholog in zebrafish has not been identified. These studies validate the examination of zebrafish primitive myeloid development as a model for human myelopoiesis, and form a framework for identification and analysis of myeloid mutants.