Ventral Wall Of Dorsal Aorta Research Articles

Distinct regulatory networks control the development of macrophages of different origins in zebrafish

AbstractMacrophages are key components of the innate immune system and play pivotal roles in immune response, organ development, and tissue homeostasis. Studies in mice and zebrafish have shown that tissue-resident macrophages derived from different hematopoietic origins manifest distinct developmental kinetics and colonization potential, yet the genetic programs controlling the development of macrophages of different origins remain incompletely defined. In this study, we use zebrafish, where tissue-resident macrophages arise from the rostral blood island (RBI) and ventral wall of dorsal aorta (VDA), the zebrafish hematopoietic tissue equivalents to the mouse yolk sac and aorta-gonad-mesonephros for myelopoiesis, to address this issue. We show that RBI- and VDA-born macrophages are orchestrated by distinctive regulatory networks formed by the E-twenty-six (Ets) transcription factors Pu.1 and Spi-b, the zebrafish ortholog of mouse spleen focus forming virus proviral integration oncogene B (SPI-B), and the helix-turn-helix DNA-binding domain containing protein Irf8. Epistatic studies document that during RBI macrophage development, Pu.1 acts upstream of Spi-b, which, upon induction by Pu.1, partially compensates the function of Pu.1. In contrast, Pu.1 and Spi-b act in parallel and cooperatively to regulate the development of VDA-derived macrophages. Interestingly, these two distinct regulatory networks orchestrate the RBI- and VDA-born macrophage development largely by regulating a common downstream gene, Irf8. Our study indicates that macrophages derived from different origins are governed by distinct genetic networks formed by the same repertoire of myeloid-specific transcription factors.

Development of Multi-Lineage Hematopoiesis in Two Novel Zebrafish runx1 mutants

Long term hematopoietic stem cells are essential for the life-long maintenance of the hematopoietic system of an organism. The transcription factor RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during the embryo development. Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. However, we previously showed that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval "bloodless" phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish model, which requires verification in additional, independent models.In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we generated two new runx1 mutants, a deletion of 8 bp (runx1del8/del8) and a deletion of 25 bp (runx1del25/del25) within exon 4 of runx1, respectively, using the Transcription activator-like effector nucleases (TALENs) technology. These mutations cause frameshifts and premature terminations within the runt-homology domain, resulting in loss of function of runx1 (runx1-/-). Both runx1del8/del8 and runx1del25/del25 mutant embryos had normal primitive hematopoiesis but failed to develop definitive hematopoiesis. Time-lapse recordings with confocal microscopy revealed that, indeed, there was no emergence of HSCs from the ventral wall of dorsal aorta in the runx1-/- embryos. The runx1-/- larvae gradually lost circulating primitive blood cells and became bloodless between 8 and 14 days post fertilization (dpf). However they gradually regained circulating blood cells between 15 and 20 dpf. Eventually, about 40% of runx1del8/del8 and runx1del25/del25 mutants developed to fertile adults with circulating blood cells of multi-lineages. Taken together, our data is consistent with the previously described runx1W84X/W84X phenotype and supports the possibility of a runx1-independent mechanism for HSC formation and definitive hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Temporal-Spatial Resolution Fate Mapping Reveals Distinct Origins for Embryonic and Adult Microglia in Zebrafish

Microglia are CNS resident macrophages, and they play important roles in neural development and function. Recent studies have suggested that murine microglia arise from a single source, the yolk sac (YS), yet these studies lack spatial resolution to define the bona fide source(s) for microglia. Here, using light-induced high temporal-spatial resolution fate mapping, we challenge this single-source view by showing that microglia in zebrafish arise from multiple sources. The embryonic/larval microglia originate from the rostral blood island (RBI) region, the equivalent of mouse YS for myelopoiesis, whereas the adult microglia arise from the ventral wall of dorsal aorta (VDA) region, a tissue also producing definitive hematopoiesis in mouse. We further show that the VDA-region-derived microglia are Runx1 dependent, but cMyb independent, and developmentally regulated differently from the RBI region-derived microglia. Our study establishes a new paradigm for investigating the development and function of distinct microglia populations.

Functions of IDH1 and its mutation in the regulation of developmental hematopoiesis in zebrafish

Isocitrate dehydrogenase 1 mutation (IDH1R132) was recently identified in AML by next generation sequencing, occurring in 10% of AML with normal cytogenetics. The cancer-associated mutation may result in novel enzymatic activity whereby a-ketoglutarate is converted to 2-hydroxyglutarate (2-HG). The latter may inhibit the function of a-ketoglutarate-dependent dioxygenases, e.g. TET2 and PHDs and result in DNA and histone hypermethylation and hence leukemia initiation. In this study, we examined the role of zebrafish idh1 (zidh1) and the phenotypes of human IDH1R132H expression in embryonic hematopoiesis. The amino acid sequence of zidh1 was remarkably similar to human IDH1. z1dh1 gene was expressed around the AGM region at 36 hpf and in the CHT at 48 hpf. Morpholino knockdown of zidh1 significantly increased gene expression associated with early myeloid progenitor (pu.1) and decreased those of late myelomonocytic differentiation (l-plastin, mpeg1 and mpo) at 18 hpf. Definitive hematopoiesis was also reduced, characterized by down-regulation of c-myb expression along the ventral wall of dorsal aorta at 36 hpf and CHT at 48 and 96 hpf and decreased rag1 expression at 96 hpf. To validate our observations, we constructed a TALEN pair targeting exon 1 of zidh1 and injected the TALEN mRNAs into 1-cell embryos, which showed high mutation frequency (more than 95%) of targeted indels by sequencing. These injected embryos also demonstrated similar hematopoietic phenotypes with that of the zidh1 morphants. Microinjection of cDNA encoding human IDH1R132H and zebrafish idh1R146H but not wild-type IDH1 at 1 cell-stage significantly increased pu.1, l-plastin, mpeg1 and mpo expression and 2-HG level at 24 hpf. The effects could be inhibited by AGI-5198, a specific inhibitor of human IDH1R132H. Overexpression of zebrafish tet2 mRNA could partially rescue the hematopoietic phenotypes of increased myeloid expression by human IDH1R132H. Co-overexpression of human IDH1R132H and FLT3-ITD on zebrafish significantly increased myelopoiesis in the embryos. The observations underscored a hitherto undescribed role of idh1 in zebrafish hematopoiesis and the potential of zebrafish in modeling human AML.

Zebrafish eaf1 suppresses foxo3b expression to modulate transcriptional activity of gata1 and spi1 in primitive hematopoiesis

Studies implicate a potential role for EAF1 in MLL–ELL induced leukemogenesis; however the biological function of EAF1 in this process remains unknown. In this study, we show that knockdown of zebrafish eaf1 by morpholinos caused serious defects in both primitive and definitive hematopoiesis. Using microarray analysis, we identified foxo3b as a target gene suppressed by eaf1. Ectopic expression of foxo3b in embryos mimicked the phenotypes exhibited in eaf1 morphants, except that foxo3b had no effect on runx1 and c-myb expression while eaf1 morphants did not express these markers in the ventral wall of dorsal aorta. Subsequent experiments showed that a dominant negative form of Foxo3b (dn-foxo3b) partially restored primitive hematopoietic defects in eaf1 morphants, suggesting that foxo3b might serve as a key factor for mediating eaf1 function in primitive hematopoiesis. Furthermore, we observed that foxo3b inhibited the transcriptional activity of gata1 and spi1 through protein–protein interaction. Our findings not only suggest a function of eaf1 on hematopoiesis in vivo, but also reveal a novel regulatory pathway, eaf1–foxo3b–gata1/spi1, that may shed light on the role of EAF1 in MLL–ELL induced leukemogenesis.

Thrombin Receptor Regulates Hematopoiesis and Endothelial-to-Hematopoietic Transition

Hematopoietic development and vascular development are closely related physiological processes during vertebrate embryogenesis. Recently, endothelial-to-hematopoietic transition (EHT) was demonstrated to be critical for hematopoietic stem and progenitor cell induction, but its underlying regulatory mechanisms remain poorly understood. Here we show that thrombin receptor (F2r), a protease-activated G protein-coupled receptor required for vascular development, functions as a negative regulator during hematopoietic development. F2r is significantly upregulated during hematopoietic differentiation of mouse embryonic stem cells (mESCs) and zebrafish hematopoietic development. Pharmacological or genetic inhibition of F2r promotes hematopoietic differentiation, whereas F2r overexpression shows opposite effects. Further mechanistic studies reveal that F2r-RhoA/ROCK pathway inhibits EHT in vitro and negatively regulates zebrafish EHT and hematopoietic stem cell induction in vivo. Taken together, this study demonstrates a fundamental role of F2r-RhoA/ROCK pathway in vertebrate hematopoiesis and EHT, as well as an important molecular mechanism coordinating hematopoietic and vascular development.

Methionine aminopeptidase 2 is required for HSC initiation and proliferation

AbstractIn a chemical screening, we tested the antiangiogenic effects of fumagillin derivatives and identified fumagillin as an inhibitor of definitive hematopoiesis in zebrafish embryos. Fumagillin is known to target methionine aminopeptidase II (MetAP2), an enzyme whose function in hematopoiesis is unknown. We investigated the role of MetAP2 in hematopoiesis by using zebrafish embryo and human umbilical cord blood models. Zebrafish metap2 was expressed ubiquitously during early embryogenesis and later in the somitic region, the caudal hematopoietic tissue, and pronephric duct. metap2 was inhibited by morpholino and fumagillin treatment, resulting in increased mpo expression at 18 hours postfertilization and reduced c-myb expression along the ventral wall of dorsal aorta at 36 hours postfertilization. It also disrupted intersegmental vessels in Tg(fli1:gfp) embryos without affecting development of major axial vasculatures. Inhibition of MetAP2 in CB CD34+ cells by fumagillin had no effect on overall clonogenic activity but significantly reduced their engraftment into immunodeficient nonobese diabetes/severe combined immunodeficiency mice. metap2 knock-down in zebrafish and inhibition by fumagillin in zebrafish and human CB CD34+ cells inhibited Calmodulin Kinase II activity and induced ERK phosphorylation. This study demonstrated a hithertoundescribed role of MetAP2 in definitive hematopoiesis and a possible link to noncanonical Wnt and ERK signaling.

Definitive hematopoietic stem/progenitor cells manifest distinct differentiation output in the zebrafish VDA and PBI

One unique feature of vertebrate definitive hematopoiesis is the ontogenic switching of hematopoietic stem cells from one anatomical compartment or niche to another. In mice, hematopoietic stem cells are believed to originate in the aorta-gonad-mesonephros (AGM), subsequently migrate to the fetal liver (FL) and finally colonize the bone marrow (BM). Yet, the differentiation potential of hematopoietic stem cells within early niches such as the AGM and FL remains incompletely defined. Here, we present in vivo analysis to delineate the differentiation potential of definitive hematopoietic stem/progenitor cells (HSPCs) in the zebrafish AGM and FL analogies, namely the ventral wall of dorsal aorta (VDA) and the posterior blood island (PBI), respectively. Cell fate mapping and analysis of zebrafish runx1(w84x) and vlad tepes (vlt(m651)) mutants revealed that HSPCs in the PBI gave rise to both erythroid and myeloid lineages. However, we surprisingly found that HSPCs in the VDA were not quiescent but were uniquely adapted to generate myeloid but not erythroid lineage cells. We further showed that such distinct differentiation output of HSPCs was, at least in part, ascribed to the different micro-environments present in these two niches. Our results highlight the importance of niche in shaping the differentiation output of developing HSPCs.

Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development

AbstractThe development of vertebrate definitive hematopoiesis is featured by temporally and spatially dynamic distribution of hematopoietic stem/progenitor cells (HSPCs). It is proposed that the migration of definitive HSPCs, at least in part, accounts for this unique characteristic; however, compelling in vivo lineage evidence is still lacking. Here we present an in vivo analysis to delineate the migration route of definitive HSPCs in the early zebrafish embryo. Cell-marking analysis was able to first map definitive HSPCs to the ventral wall of dorsal aorta (DA). These cells were subsequently found to migrate to a previously unappreciated organ, posterior blood island (PBI), located between the caudal artery and caudal vein, and finally populate the kidney, the adult hematopoietic organ. These findings demonstrate that the PBI acts as an intermediate hematopoietic organ in a manner analogous to the mammalian fetal liver to sustain definitive hematopoiesis before adult kidney hematopoiesis occurs. Thus our study unambiguously documents the in vivo trafficking of definitive HSPCs among developmentally successive hematopoietic compartments and underscores the ontogenic conservation of definitive hematopoiesis between zebrafish and mammals.