Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.     

Zebrafish Jak2a plays a crucial role in definitive hematopoiesis and blood vessel formation

We examined the role of zebrafish (Danio rerio) Jak2a, a homolog of mammalian Jak2, in the developing embryo by injecting in vitro synthesized Jak2a shRNA into zebrafish zygotes. Blood circulation was suppressed in Jak2a shRNA-injected embryos from 24 hours post fertilization (hpf) and all embryos died with enlarged pericardium, shortened body lengths, and defects in some vasculature within 8 days post fertilization. O-dianisidine staining of red blood cells revealed normal blood island formation with no circulating red blood cells. As in Jak2^−/− transgenic mice, expression of definitive Ba1 globin was significantly reduced in Jak2a knockdown embryos at 36 hpf, whereas expression of other hematopoietic markers, primitive be1 globin, gata-1, and scl, were unaffected. More importantly, blood vessel formation was disturbed in Jak2a knockdown embryos as revealed by alkaline phosphatase staining at 72 hpf. Thus, our data indicate that zebrafish Jak2a is important in both definitive hematopoiesis and blood vessel formation.     

Developmental expression of zebrafish emx1 during early embryogenesis

Emx family homeobox genes, Emx1 and Emx2, play an essential role in rostral brain development in mammalian embryos. Here we report a zebrafish emx family gene, emx1, which is more similar to the mouse Emx1 gene than the previously reported zebrafish emx1 gene; we propose to rename that gene emx3. The expression of emx1 is first detected around the 10-somite stage in the pineal gland (epiphysis) primodium in the developing anterior brain and in the pronephric primodium within the intermediate mesoderm. emx1 expression in the epiphysis has not been reported in other species. Expression in the epiphysis is suppressed at 23 h post-fertilization (hpf) in the floating head (flh) mutant, in which development of the epiphysis is impaired. Subsequently, emx1 is expressed in the telencephalon, as reported in mammals, and can be detected in the olfactory placode and in a small group of cells in the forebrain at 25 hpf. In the mesoderm, emx1 expression is gradually concentrated in the posterior pronephric duct during somitogenesis, and becomes expressed predominantly in the urogenital opening at 25 hpf. Thus, emx1 displays a unique expression pattern that is distinct from the patterns of emx2 and emx3.     

己烯雌酚抑制斑马鱼精子发生及其可能的分子机制

为研究内分泌干扰物己烯雌酚(DES)对鱼类精巢发育和配子发生的影响, 研究用DES(0.1、1和10 g/L, 暴露20d)对内分泌干扰研究的经典模式动物斑马鱼(Danio rerio)雄性成鱼进行了处理。组织学研究结果表明, DES严重影响斑马鱼精子发生。同时, 研究克隆了斑马鱼与生殖细胞发育和减数分裂相关的vasa、dmc1的部分cDNA, 对其组织和细胞表达模式进行了研究。结果表明, vasa仅表达于精巢的精原细胞、初级精母细胞和卵巢不同时期的生殖细胞; 而dmc1则表达于精巢精母细胞和卵巢卵母细胞发育早期。半定量PCR结果表明, DES处理后vasa的表达没有明显变化; 而dmc1的表达则被明显抑制, 且呈时间依赖性和剂量依赖性效应; 而转录因子dmrt1和雄激素合成关键酶基因P450 11的表达也被显著抑制。因此本研究推测, DES可能通过抑制dmrt1和P450 11的表达诱导了斑马鱼生殖细胞凋亡; 并通过抑制dmc1的表达阻碍了减数分裂。       

Inhibition of BMP activity by the FGF signal promotes posterior neural development in zebrafish

The expression patterns of region-specific neuroectodermal genes and fate-map analyses in zebrafish gastrulae suggest that posterior neural development is initiated by nonaxial signals, distinct from organizer-derived secreted bone morphogenetic protein (BMP) antagonists. This notion is further supported by the misexpression of a constitutively active form of zebrafish BMP type IA receptor (CA-BRIA) in the zebrafish embryos. It effectively suppressed the anterior neural marker, otx2, but not the posterior marker, hoxb1b. Furthermore, we demonstrated that the cells in the presumptive posterior neural region lose their neural fate only when CA-BRIA and Xenopus dominant-negative fibroblast growth factor (FGF) receptors (XFD) are coexpressed. The indications are that FGF signaling is involved in the formation of the posterior neural region, counteracting the BMP signaling pathway within the target cells. We then examined the functions of Fgf3 in posterior neural development. Zebrafish fgf3 is expressed in the correct place (dorsolateral margin) and at the correct time (late blastula to early gastrula stages), the same point that the most precocious posterior neural marker, hoxb1b, is first activated. Unlike other members of the FGF family, Fgf3 had little mesoderm-inducing activity. When ectopically expressed, Fgf3 expands the neural region with suppression of anterior neural fate. However, this effect was mediated by Chordino (zebrafish Chordin), because Fgf3 induces chordino expression in the epiblast and Fgf3-induced neural expansion was substantially suppressed in dino mutants with mutated chordino genes. The results obtained in the present study reveal multiple actions of the FGF signal on neural development: it antagonizes BMP signaling within posterior neural cells, induces the expression of secreted BMP antagonists, and suppresses anterior neural fate.     

The chianti zebrafish mutant provides a model for erythroid-specific disruption of transferrin receptor 1

Iron is a crucial metal for normal development, being required for the production of heme, which is incorporated into cytochromes and hemoglobin. The zebrafish chianti (cia) mutant manifests a hypochromic, microcytic anemia after the onset of embryonic circulation, indicative of a perturbation in red blood cell hemoglobin production. We show that cia encodes tfr1a, which is specifically expressed in the developing blood and requisite only for iron uptake in erythroid precursors. In the process of isolating zebrafish tfr1, we discovered two tfr1-like genes (tfr1a and tfr1b) and a single tfr2 ortholog. Abrogation of tfr1b function using antisense morpholinos revealed that this paralog was dispensable for hemoglobin production in red cells. tfr1b morphants exhibited growth retardation and brain necrosis, similar to the central nervous system defects observed in the Tfr1 null mouse, indicating that tfr1b is probably used by non-erythroid tissues for iron acquisition. Overexpression of mouse Tfr1, mouse Tfr2, and zebrafish tfr1b partially rescued hypochromia in cia embryos, establishing that each of these transferrin receptors are capable of supporting iron uptake for hemoglobin production in vivo. Taken together, these data show that zebrafish tfr1a and tfr1b share biochemical function but have restricted domains of tissue expression, and establish a genetic model to study the specific function of Tfr1 in erythroid cells.     

Zebrafish Naked1 and Naked2 antagonize both canonical and non-canonical Wnt signaling

Wnt signaling controls a wide range of developmental processes and its aberrant regulation can lead to disease. To better understand the regulation of this pathway, we identified zebrafish homologues of Naked Cuticle (Nkd), Nkd1 and Nkd2, which have previously been shown to inhibit canonical Wnt/beta-catenin signaling. Zebrafish nkd1 expression increases substantially after the mid-blastula transition in a pattern mirroring that of activated canonical Wnt/beta-catenin signaling, being expressed in both the ventrolateral blastoderm margin and also in the axial mesendoderm. In contrast, zebrafish nkd2 is maternally and ubiquitously expressed. Overexpression of Nkd1 or Nkd2a suppressed canonical Wnt/beta-catenin signaling at multiple stages of early zebrafish development and also exacerbated the cyclopia and axial mesendoderm convergence and extension (C&E) defect in the non-canonical Wnt/PCP mutant silberblick (slb/wnt11). Thus, Nkds are sufficient to antagonize both canonical and non-canonical Wnt signaling. Reducing Nkd function using antisense morpholino oligonucleotides resulted in increased expression of canonical Wnt/beta-catenin target genes. Finally, reducing Nkd1 function in slb mutants suppressed the axial mesendoderm C&E defect. These data indicate that zebrafish Nkd1 and Nkd2 function to limit both canonical and non-canonical Wnt signaling.     

Vap (Vascular Associated Protein): a novel factor involved in erythropoiesis and angiogenesis

Both endothelial and erythroid cells are generated in the intermediate cell mass (ICM) during zebrafish embryogenesis, but the nature of the genes that contribute to the processes of erythrocyte maturation and blood vessel network formation is not fully understood. From our in situ-based screening, we have identified a novel factor, Vap (Vascular Associated Protein) that is predominantly expressed in the ICM, and subsequently enriched in endothelial cells. Vap expression in the ICM was drastically suppressed in the cloche mutant that has defects in both vasculogenesis and hematopoiesis, whereas Vap expression was not affected in the vlad tepes/gata1 mutant. Knockdown of Vap using anti-sense morpholinos (VAP-MO) not only resulted in decreased numbers of erythrocytes but also in the strong suppression of hemoglobin production. Further, we found that Vap knockdown caused the disorganization of the intersegmental vessels (ISVs), which show irregular branching. We propose that Vap plays an important role in the maturation of endothelial and erythroid cells in zebrafish.     

Pituitary corticotroph ontogeny and regulation in transgenic zebrafish

We characterized zebrafish proopiomelanocortin (POMC) gene promoter, and sequence analysis revealed that the promoter contains regulatory elements conserved among vertebrate species. To monitor the ontogeny of the pituitary POMC lineage in living vertebrates, we generated transgenic zebrafish expressing green fluorescent protein (GFP) driven by the POMC promoter. Zebrafish POMC-GFP is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18-20 h post fertilization (hpf). POMC-GFP-positive cells then fuse into a single-cell mass within the pituitary anlage after 24 hpf and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Immunohistochemical studies with ACTH and alphaMSH antisera showed that POMC-GFP was mainly targeted to both anterior and posterior pituitary corticotrophs, whereas posterior pituitary region melanotrophs did not express GFP. To determine in vivo zebrafish corticotroph responses, dexamethasone (10(-5) m) was added to live embryos, which selectively suppressed POMC-GFP expression in the anterior group of corticotrophs, suggesting a distinct domain that is responsive to glucocorticoid feedback. Transgenic zebrafish with specific POMC-GFP expression in pituitary corticotrophs offers a powerful genetic system for in vivo study of vertebrate corticotroph lineage development.     

Molecular characterization and expression pattern of taurine transporter in zebrafish during embryogenesis

Taurine and its transporter (TauT) are expressed in preimplantation embryos, but their role in embryogenesis is not known. To investigate the role of TauT during embryonic development, we cloned and functionally characterized the zebrafish TauT. The zebrafish TauT cDNA codes for a protein of 625 amino acids which is highly homologous to mammalian TauT. When expressed in mammalian cells, zebrafish TauT mediates taurine uptake in a Na(+)/Cl(-)-dependent manner with a Na(+):Cl(-):taurine stoichiometry of 2:1:1. In the zebrafish embryo, taurine and TauT mRNA are present during early cleavage stages, indicating that both the transporter and its substrate are maternally derived. During embryogenesis, zygotic expression of TauT mRNA is evident in the retina, brain, heart, kidney, and blood vessels. Knockdown of TauT by antisense morpholino oligonucleotides leads to cell death in the central nervous system and increased mortality. These findings suggest a specific role for TauT during development in vertebrates.     

The Impact of Tumor Nitric Oxide Production on VEGFA Expression and Tumor Growth in a Zebrafish Rat Glioma Xenograft Model

To investigate the effect of nitric oxide on tumor development, we established a rat tumor xenograft model in zebrafish embryos. The injected tumor cells formed masses in which nitric oxide production could be detected by the use of the cell-permeant DAF-FM-DA (diaminofluorophore 4-amino-5-methylamino-2’-7’-difluorofluorescein diacetate) and DAR-4M-AM (diaminorhodamine-4M). This method revealed that nitric oxide production could be co-localized with the tumor xenograft in 46% of the embryos. In 85% of these embryos, tumors were vascularized and blood vessels were observed on day 4 post injection. Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA. In the xenografted embryos we also found increased zebrafish vegfa expression. Glioma and zebrafish derived Vegfa and tumor Cyclin D1 expression could be down regulated by the nitric oxide scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or CPTIO. We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma.     

A crucial interaction between embryonic red blood cell progenitors and paraxial mesoderm revealed in spadetail embryos

Zebrafish embryonic red blood cells (RBCs) develop in trunk intermediate mesoderm (IM), and early macrophages develop in the head, suggesting that local microenvironmental cues regulate differentiation of these two blood lineages. spadetail (spt) mutant embryos, which lack trunk paraxial mesoderm (PM) due to a cell-autonomous defect in tbx16, fail to produce embryonic RBCs but retain head macrophage development. In spt mutants, initial hematopoietic gene expression is absent in trunk IM, although endothelial and pronephric expression is retained, suggesting that early blood progenitor development is specifically disrupted. Using cell transplantation, we reveal that spt is required cell autonomously for early hematopoietic gene expression in trunk IM. Further, we uncover an interaction between embryonic trunk PM and blood progenitors that is essential for RBC development. Importantly, our data identify a hematopoietic microenvironment that allows embryonic RBC production in the zebrafish.