TET2 Plays an Essential Role in Erythropoiesis by Regulating Lineage-Specific Genes via DNA Oxidative Demethylation in a Zebrafish Model

Although epigenetic modulation is critical for a variety of cellular activities, its role in erythropoiesis remains poorly understood. Ten-eleven translocation (TET) molecules participate in methylcytosine (5mC) hydroxylation, which results in DNA demethylation in several biological processes. In this research, the role of TETs in erythropoiesis was investigated by using the zebrafish model, where three TET homologs were identified. These homologs share conserved structural domains with their mammalian counterparts. Zebrafish TETs mediate the conversion of 5mC to hydroxymethylcytosine (5hmC) in zebrafish embryos, and the deletion of TET2 inhibits erythropoiesis by suppressing the expression of the scl, gata-1, and cmyb genes. TET2-upregulated lineage-specific genes and erythropoiesis are closely associated with the occurrence of 5hmC and demethylation in the intermediate CpG promoters (ICPs) of scl, gata-1, cmyb, which frequently occur at specific regions or CpG sites of these ICPs. Moreover, TET2 regulates the formation and differentiation of erythroid progenitors, and deletion of TET2 leads to erythrocyte dysplasia and anemia. Here, we preliminarily proved that TET2 plays an essential role in erythrocyte development by regulating lineage-specific genes via DNA oxidative demethylation. This report is anticipated to broaden current information on hematopoiesis and pathogenesis of hematopoiesis-related diseases.     

How does blood cell concentration modulate cardiovascular parameters in developing zebrafish (Danio rerio)?

Blood flow and shear forces are considered to be important parameters possibly stimulating angiogenesis or cardiovascular remodeling. The main objective of this study was to test the hypothesis that a significant reduction in shear forces as a consequence of a significant isovolemic anemia induced by microsurgical techniques during early larval development of the zebrafish might induce a compensatory stimulation of erythropoiesis and/or induce a modification of cardiac activity or even the formation of the heart and may influence the shaping of the vascular bed. Blood from 2 day old zebrafish larvae was withdrawn and replaced by zebrafish Ringer's solution, so that the blood cell concentration was reduced by at least 75%. At 5 days post fertilization (dpf) a partial recovery in blood cell concentration was observed and reached a value of 814.55+/-85.42 cells/nL, while in control animals blood cell concentration amounted to 1856.00+/-131.59 cells/nL. At 7 dpf the value of blood cell concentration was 1023.89+/-95.75 cells/nL versus 1701.54+/-146.03 cells/nL in control animals. Compared to control animals, heart rate and cardiac output were significantly reduced in anemic animals and alterations in the formation of the vascular bed were also observed. A significant decrease in the end-diastolic volume suggested that ventricular volume was reduced. Thus, within a few days zebrafish larvae were nearly able to compensate for an isovolemic anemia by an enhanced erythropoiesis. However, several changes in cardiovascular system indicated that phenotypic plasticity is established even at an early developmental stage.     

Behavioral screening for neuroactive drugs in zebrafish

The larval zebrafish has emerged asa vertebrate model system amenable to small molecule screens for probing diverse biological pathways. Two large-scale small molecule screens examined the effects of thousands of drugs on larval zebrafish sleep/wake and photomotor response behaviors. Both screens identified hundreds of molecules that altered zebrafish behavior in distinct ways. The behavioral profiles induced by these small molecules enabled the clustering of compounds according to shared phenotypes. This approach identified regulators of sleep/wake behavior and revealed the biological targets for poorly characterized compounds. Behavioral screening for neuroactive small molecules in zebrafish is an attractive complement to in vitro screening efforts, because the complex interactions in the vertebrate brain can only be revealed in vivo.     

Small molecule screen for compounds that affect vascular development in the zebrafish retina

Blood vessel formation in the vertebrate eye is a precisely regulated process. In the human retina, both an excess and a deficiency of blood vessels may lead to a loss of vision. To gain insight into the molecular basis of vessel formation in the vertebrate retina and to develop pharmacological means of manipulating this process in a living organism, we further characterized the embryonic zebrafish eye vasculature, and performed a small molecule screen for compounds that affect blood vessel morphogenesis. The screening of approximately 2000 compounds revealed four small molecules that at specific concentrations affect retinal vessel morphology but do not produce obvious changes in trunk vessels, or in the neuronal architecture of the retina. Of these, two induce a pronounced widening of vessel diameter without a substantial loss of vessel number, one compound produces a loss of retinal blood vessels accompanied by a mild increase of their diameter, and finally one other generates a severe loss of retinal vessels. This work demonstrates the utility of zebrafish as a screening tool for small molecules that affect eye vasculature and presents several compounds of potential therapeutic importance.     

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.     

Phosphorylation of ELAVL1 (Ser219/Ser316) mediated by PKC is required for erythropoiesis

Elavl1 (also known as HuR), an RNA binding protein highly conserved between zebrafish and human, regulates gene expression by stabilizing target mRNA. Our previous studies have uncovered that the predominant isoform elavl1a is required for zebrafish embryonic erythropoiesis. However, the exact mechanism of how elav11 spatiotemporally stabilizes target mRNAs to regulate specific erythropoiesis is not yet understood. Here we show that phosphorylation of elavl1a at Ser219 and Ser316 by PKC is necessarily required for cytosolic shuttling from the nucleus to stabilize gata1 mRNA and thus promotes erythropoiesis. Knockdown of elavl1a resulted in the hindrance of erythropoiesis and Hemin-induced erythroid differentiation of human myeloid leukemia K562 cells. Interestingly, inhibition of PKC reproduced the phenotype seen during zebrafish embryogenesis and erythroid differentiation of myeloid leukemia. Mechanistically, Hemin induced elavl1a export from nuclear to cytoplasmic space in K562 cells in a manner dependent on phosphorylation on Ser219 and Ser316, as overexpression of elavl1a with mutations on Ser219 and Ser316 resulted in erythropoiesis failure. Additionally, co-administration of low doses of elavl1a morpholino (MO) and three PKC inhibitors showed a combined effect in zebrafish embryonic erythropoiesis dysplasia. In conclusion, our study reveals that PKC-mediated phosphorylation of elavl1a at Ser219 and Ser316 sites controls its nucleo-cytoplasmic translocation in zebrafish, thereby regulating embryonic erythropoiesis.     

Macrocytic Anemia and Mitochondriopathy Resulting from a Defect in Sideroflexin 4

We used exome sequencing to identify mutations in sideroflexin 4 (SFXN4) in two children with mitochondrial disease (the more severe case also presented with macrocytic anemia). SFXN4 is an uncharacterized mitochondrial protein that localizes to the mitochondrial inner membrane. sfxn4 knockdown in zebrafish recapitulated the mitochondrial respiratory defect observed in both individuals and the macrocytic anemia with megaloblastic features of the more severe case. In vitro and in vivo complementation studies with fibroblasts from the affected individuals and zebrafish demonstrated the requirement of SFXN4 for mitochondrial respiratory homeostasis and erythropoiesis. Our findings establish mutations in SFXN4 as a cause of mitochondriopathy and macrocytic anemia.     

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.     

Small molecule screening identifies targetable zebrafish pigmentation pathways

Small molecules complement genetic mutants and can be used to probe pigment cell biology by inhibiting specific proteins or pathways. Here, we present the results of a screen of active compounds for those that affect the processes of melanocyte and iridophore development in zebrafish and investigate the effects of a few of these compounds in further detail. We identified and confirmed 57 compounds that altered pigment cell patterning, number, survival, or differentiation. Additional tissue targets and toxicity of small molecules are also discussed. Given that the majority of cell types, including pigment cells, are conserved between zebrafish and other vertebrates, we present these chemicals as molecular tools to study developmental processes of pigment cells in living animals and emphasize the value of zebrafish as an in vivo system for testing the on- and off-target activities of clinically active drugs.     

Identification of Small Molecule Activators of BMP Signaling

Bone Morphogenetic Proteins (BMPs) are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A) with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ∼5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad), Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.     

Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis

Hepcidin, a key regulator of iron metabolism, decreases intestinal absorption of iron and its release from macrophages. Iron, anemia, hypoxia, and inflammation were reported to influence hepcidin expression. To investigate regulation of the expression of hepcidin and other iron-related genes, we manipulated erythropoietic activity in mice. Erythropoiesis was inhibited by irradiation or posttransfusion polycythemia and stimulated by phenylhydrazine administration and erythropoietin. Gene expression of hepcidin and other iron-related genes (hemojuvelin, DMT1, ferroportin, transferrin receptors, ferritin) in the liver was measured by the real-time polymerase chain reaction. Hepcidin expression increased despite severe anemia when hematopoiesis was inhibited by irradiation. Suppression of erythropoiesis by posttransfusion polycythemia or irradiation also increased hepcidin mRNA levels. Compensated hemolysis induced by repeated phenylhydrazine administration did not change hepcidin expression. The decrease caused by exogenous erythropoeitin was blocked by postirradiation bone marrow suppression. The hemolysis and anemia decrease hepcidin expression only when erythropoiesis is functional; on the other hand, if erythropoiesis is blocked, even severe anemia does not lead to a decrease of hepcidin expression, which is indeed increased. We propose that hepcidin is exclusively sensitive to iron utilization for erythropoiesis and hepatocyte iron balance, and these changes are not sensed by other genes involved in the control of iron metabolism in the liver.     

A Phenotypic Screen in Zebrafish Identifies a Novel Small-Molecule Inducer of Ectopic Tail Formation Suggestive of Alterations in Non-Canonical Wnt/PCP Signaling

Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators of the BMP and Wnt signaling pathways. One of the bioactive plant extracts identified in this screen – Jasminum gilgianum, an Oleaceae species native to Papua New Guinea – induced ectopic tails during zebrafish embryonic development. As ectopic tail formation occurs when BMP or non-canonical Wnt signaling is inhibited during the tail protrusion process, we suspected a constituent of this extract to act as a modulator of these pathways. A bioassay-guided isolation was carried out on the basis of this zebrafish phenotype, identifying para-coumaric acid methyl ester (pCAME) as the active compound. We then performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos, including a tissue-specific marker analysis of the secondary tails. We found pCAME to synergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails, and causing convergence-extension defects in compound-treated embryos. These results indicate that pCAME may interfere with non-canonical Wnt signaling. Inhibition of Jnk, a downstream target of Wnt/PCP signaling (via morpholino antisense knockdown and pharmacological inhibition with the kinase inhibitor SP600125) phenocopied pCAME-treated embryos. However, immunoblotting experiments revealed pCAME to not directly inhibit Jnk-mediated phosphorylation of c-Jun, suggesting additional targets of SP600125, and/or other pathways, as possibly being involved in the ectopic tail formation activity of pCAME. Further investigation of pCAME’s mechanism of action will help determine this compound’s pharmacological utility.     

Kupffer cells support extramedullary erythropoiesis induced by nitrogen-containing bisphosphonate in splenectomized mice

Our previous study indicated that injecting nitrogen-containing bisphosphonate (NBP) induced the site of erythropoiesis to shift from the bone marrow (BM) to the spleen. This was due to the depletion of BM-resident macrophages, which support erythropoiesis. In this study, we examined NBP treatment-induced extramedullary hematopoiesis in splenectomized mice, focusing on hepatic hematopoiesis. NBP-treated mice did not display anemia or significant change in erythropoietin production, while megakaryopoiesis and erythropoiesis were constantly observed in the liver. Erythroblastic islands were detected in the sinusoidal lumen. Kupffer cells expressed VCAM-1 following NBP treatment, which is an important factor for erythroblast differentiation. Cl₂MBP-liposome treatment depleted the erythroblastic islands, and decreased the number of hematopoietic cells in the liver, as determined by colony forming assays. Together, these results indicate that Kupffer cells support erythropoiesis, acting as stromal cells in the liver, and that they might act as a niche for hematopoietic precursor cells in an emergency.     

Understanding dioxin developmental toxicity using the zebrafish model

Zebrafish (Danio rerio) have advantages over mammals as an animal model for investigating developmental toxicity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin, TCDD), a persistent global contaminant, is the most comprehensively studied developmental toxicant in zebrafish. The hallmark responses of TCDD developmental toxicity manifested in zebrafish larvae include edema, anemia, hemorrhage, and ischemia associated with arrested growth and development. Heart and vasculature development and function are severely impaired, and jaw malformations occur secondary to inhibited chondrogenesis. The swim bladder fails to inflate, and the switch from embryonic to adult erythropoiesis is blocked. This profile of developmental toxicity responses, commonly referred to as "blue sac syndrome" because the edematous yolk sac appears blue, is observed in the larval form of all freshwater fish species exposed to TCDD at the embryonic stage of development. Components of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AHR/ARNT) signaling pathway in zebrafish have been identified and functionally characterized. Their role in mediating TCDD toxicity has been determined using morpholinos to specifically knockdown the translation of zfAHR1, zfAHR2, zfARNT1, and zfARNT2 mRNAs, respectively, and a line of zfARNT2 null mutant zebrafish has provided further insight. These studies have shown that zfAHR2 and zfARNT1 mediate TCDD developmental toxicity. In addition, the growing use of molecular and genomic tools for research on zebrafish have led to advances in our understanding of the mechanism of TCDD developmental toxicity at the molecular level, including the recent finding that toxicity is not mediated by increased cytochrome P4501A (zfCYP1A) expression.     

Immuno- and erythropoiesis in mice with graft vs host disease against a background of immunodeficiency

In BALB/c mice immunodeficiency was induced by the transfer of lymphocytes immune to alloantigen. This model is one of experimental models of AIDS. The work was aimed at the study of disturbances in the immuno--and erythropoiesis in immunodeficient mice. The state of erythropoiesis was evaluated by the level of level of hemoglobin, hematocrit and the content of reticulocytes in peripheral blood, by the number of erythroid bursitis-forming units and the percentage of erythrokaryocytes in the marrow, as well as by the number of colony-forming units in the spleen by days 5 and 8. The study revealed that in BALB/c mice hypoplastic anemia, accompanied by the decreased phagocytic activity of macrophages and the reduced production of interleukin 1 and tumor necrosis factor, developed on months 5-6 of the disease. Macrophagal dysfunction was supposed to be one of the causes of hypoplastic anemia in immunodeficient mice.     

In Vivo Regulation of Erythropoiesis by Chemically Inducible Dimerization of the Erythropoietin Receptor Intracellular Domain

Erythropoietin (Epo) and its receptor (EpoR) are required for the regulation of erythropoiesis. Epo binds to the EpoR homodimer on the surface of erythroid progenitors and erythroblasts, and positions the intracellular domains of the homodimer to be in close proximity with each other. This conformational change is sufficient for the initiation of Epo-EpoR signal transduction. Here, we established a system of chemically regulated erythropoiesis in transgenic mice expressing a modified EpoR intracellular domain (amino acids 247–406) in which dimerization is induced using a specific compound (chemical inducer of dimerization, CID). Erythropoiesis is reversibly induced by oral administration of the CID to the transgenic mice. Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects. Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo. We propose that the CID-dependent dimerization system combined with the EpoR intracellular domain and the Gata1 gene regulatory region generates a novel peroral strategy for the treatment of anemia.     

Identification of genetic and chemical modulators of zebrafish mechanosensory hair cell death

Inner ear sensory hair cell death is observed in the majority of hearing and balance disorders, affecting the health of more than 600 million people worldwide. While normal aging is the single greatest contributor, exposure to environmental toxins and therapeutic drugs such as aminoglycoside antibiotics and antineoplastic agents are significant contributors. Genetic variation contributes markedly to differences in normal disease progression during aging and in susceptibility to ototoxic agents. Using the lateral line system of larval zebrafish, we developed an in vivo drug toxicity interaction screen to uncover genetic modulators of antibiotic-induced hair cell death and to identify compounds that confer protection. We have identified 5 mutations that modulate aminoglycoside susceptibility. Further characterization and identification of one protective mutant, sentinel (snl), revealed a novel conserved vertebrate gene. A similar screen identified a new class of drug-like small molecules, benzothiophene carboxamides, that prevent aminoglycoside-induced hair cell death in zebrafish and in mammals. Testing for interaction with the sentinel mutation suggests that the gene and compounds may operate in different pathways. The combination of chemical screening with traditional genetic approaches is a new strategy for identifying drugs and drug targets to attenuate hearing and balance disorders.     

Studies on the erythropoietic effect of plasma from anemic toads both with and without testis.

Injection of plasma from experimentally induced anemic toad with intact testis increases erythropoiesis in starved toads evidenced by the increase of red blood cell, hemoglobin and hematocrit, whereas the plasma of castrated and phenylhydrazine-HCl treated anemic toad failed to do so. It can be suggested that the erythropoiesis stimulating factor (ESF) is produced from the testis of toad and the production of this factor (ESF) was found to increase during anemia in an attempt to correct the anemic condition.