Mutations in the zebrafish hmgcs1 gene reveal a novel function for isoprenoids during red blood cell development.

Erythropoiesis is the process by which new red blood cells (RBCs) are formed and defects in this process can lead to anemia or thalassemia. The GATA1 transcription factor is an established mediator of RBC development. However, the upstream mechanisms that regulate the expression of GATA1 are not completely characterized. Cholesterol is 1 potential upstream mediator of GATA1 expression because previously published studies suggest that defects in cholesterol synthesis disrupt RBC differentiation. Here we characterize RBC development in a zebrafish harboring a single missense mutation in the hmgcs1 gene (Vu57 allele). hmgcs1 encodes the first enzyme in the cholesterol synthesis pathway and mutation of hmgcs1 inhibits cholesterol synthesis. We analyzed the number of RBCs in hmgcs1 mutants and their wild-type siblings. Mutation of hmgcs1 resulted in a decrease in the number of mature RBCs, which coincides with reduced gata1a expression. We combined these experiments with pharmacological inhibition and confirmed that cholesterol and isoprenoid synthesis are essential for RBC differentiation, but that gata1a expression is isoprenoid dependent. Collectively, our results reveal 2 novel upstream regulators of RBC development and suggest that appropriate cholesterol homeostasis is critical for primitive erythropoiesis.

Trypanosoma carassii infection in goldfish (Carassius auratus L.): changes in the expression of erythropoiesis and anemia regulatory genes.

Trypanosoma carassii is a flagellated bloodstream parasite of cyprinid fish with pathogenesis manifesting primarily as anemia in experimentally infected fish. This anemia is characterized by decreases in the number of circulating red blood cells (RBCs) during peak parasitemia. We examined changes in the key blood metrics and expression of genes known to be important in the regulation of erythropoiesis. Increasing parasitemia was strongly correlated with an overall decrease in the total number of circulating RBCs. Gene expression of key erythropoiesis regulators (EPO, EPOR, GATA1, Lmo2, and HIFα) and proinflammatory cytokines (IFNγ and TNFα) were measured and their expressions differed from those in fish made anemic by injections of phenylhydrazine (PHZ). Significant upregulation of pro-erythropoietic genes was observed in PHZ-induced anemia, but not during peak parasitic infection. Previously, we reported on functional characterization of goldfish erythropoietin (rgEPO) and its ability to induce survival and differentiation of erythroid progenitor cells in vitro. Treatment of goldfish during the infection with rgEPO reduced the severity of anemia but failed to fully prevent the onset of the anemic state in infected fish. Proinflammatory cytokines have been implicated in the suppression of erythropoiesis during trypanosomiasis, specifically the cytokines TNFα, IFNγ, and IL-1ß. Analysis of key proinflammatory cytokines revealed that mRNA levels of IFNγ and TNFα were upregulated in response to infection, but only TNFα increased in response to PHZ treatment. Synergistic activity of the proinflammatory cytokines may be required to sustain prolonged anemia. These findings provide insight into the relationship between T. carassii and host anemia and suggest that T. carassii may directly or indirectly suppress host erythropoiesis.

Ribonuclease inhibitor 1 regulates erythropoiesis by controlling GATA1 translation.

Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.

Epo reprograms the epigenome of erythroid cells.

The hormone erythropoietin (Epo) is required for erythropoiesis, yet its molecular mechanism of action remains poorly understood, particularly with respect to chromatin dynamics. To investigate how Epo modulates the erythroid epigenome, we performed epigenetic profiling using an ex vivo murine cell system that undergoes synchronous erythroid maturation in response to Epo stimulation. Our findings define the repertoire of Epo-modulated enhancers, illuminating a new facet of Epo signaling. First, a large number of enhancers rapidly responded to Epo stimulation, revealing a cis-regulatory network of Epo-responsive enhancers. In contrast, most of the other identified enhancers remained in an active acetylated state during Epo signaling, suggesting that most erythroid enhancers are established at an earlier precursor stage. Second, we identified several hundred super-enhancers that were linked to key erythroid genes, such as Tal1, Bcl11a, and Mir144/451. Third, experimental and computational validation revealed that many predicted enhancer regions were occupied by TAL1 and enriched with DNA-binding motifs for GATA1, KLF1, TAL1/E-box, and STAT5. Additionally, many of these cis-regulatory regions were conserved evolutionarily and displayed correlated enhancer:promoter acetylation. Together, these findings define a cis-regulatory enhancer network for Epo signaling during erythropoiesis, and provide the framework for future studies involving the interplay of epigenetics and Epo signaling.

Downregulation of GATA1 drives impaired hematopoiesis in primary myelofibrosis.

Primary myelofibrosis (PMF) is a clonal hematologic malignancy characterized by BM fibrosis, extramedullary hematopoiesis, circulating CD34+ cells, splenomegaly, and a propensity to evolve to acute myeloid leukemia. Moreover, the spleen and BM of patients harbor atypical, clustered megakaryocytes, which contribute to the disease by secreting profibrotic cytokines. Here, we have revealed that megakaryocytes in PMF show impaired maturation that is associated with reduced GATA1 protein. In investigating the cause of GATA1 downregulation, our gene-expression study revealed the presence of the RPS14-deficient gene signature, which is associated with defective ribosomal protein function and linked to the erythroid lineage in 5q deletion myelodysplastic syndrome. Surprisingly, reduced GATA1 expression and impaired differentiation were limited to megakaryocytes, consistent with a proproliferative effect of a GATA1 deficiency on this lineage. Importantly, expression of GATA1 effectively rescued maturation of PMF megakaryocytes. Together, these results suggest that ribosomal deficiency contributes to impaired megakaryopoiesis in myeloproliferative neoplasms.

Reprogramming of human peripheral blood monocytes to erythroid lineage by blocking of the PU-1 gene expression.

In hematopoietic system development, PU.1 and GATA-1 as lineage-specific transcription factors (TF) are expressed in common myeloid progenitors. The cross antagonism between them ascertains gene expression programs of monocytic and erythroid cells, respectively. This concept in transdifferentiation approaches has not been well considered yet, especially in intralineage conversion systems. To demonstrate whether PU.1 suppression induces monocyte lineage conversion into red blood cells, a combination of three PU.1-specific siRNAs was implemented to knock down PU.1 gene expression and generate the balance in favor of GATA-1 expression to induce erythroid differentiation. For this purpose, monocytes were isolated from human peripheral blood and transfected by PU.1 siRNAs. In transfected monocytes, the rate of PU.1 expression in mRNA level was significantly decreased until 0.38 ± 0.118 when compared to untreated monocytes at 72 h (p value ≤0.05) which resulted in significant overexpression of GATA1 of 16.1 ± 0.343-fold compared to the untreated group (p value ≤0.01). Subsequently, overexpression of hemoglobin (α 13.26 ± 1.34-fold; p value≤0.0001) and ß-globin (37.55 ± 16.56-fold; p value≤0.0001) was observed when compared to control groups. The results of western immunoblotting confirm those findings too. While, reduced expression of monocyte, CD14 gene, was observed in qRT-PCR and flow cytometry results. Our results suggest that manipulating the ratio of the two TFs in bifurcation differentiation pathways via applying siRNA technology can possibly change the cells' fate as a safe way for therapeutics application.

A Child With Dyserythropoietic Anemia and Megakaryocyte Dysplasia Due to a Novel 5'UTR GATA1s Splice Mutation.

We describe a child with dyserythropoietic anemia, thrombocytosis, functional platelet defect, and megakaryocyte dysplasia. We show that (i) this constellation of hematopoietic abnormalities was due to a germline mutation within the 5' untranslated region (5'UTR) of globin transcription factor 1 (GATA1); (ii) the mutation impaired a 5'UTR GATA1 splicing site, with promoted production of the shortened GATA1 isoform lacking the N-terminus; and (iii) expression of the GATA1 N-terminus is restricted to erythroblasts and megakaryocytes in normal marrow, consistent with the patient's abnormal erythropoiesis and megakaryopoiesis. Our findings provide insights into the clinically relevant in vivo function of the N-terminal domain of GATA1 in human hematopoiesis.

Thalidomide can promote erythropoiesis by induction of STAT5 and repression of external pathway of apoptosis resulting in increased expression of GATA-1 transcription factor.

BACKGROUND: Thalidomide was shown to stimulate erythropoiesis and increase hemoglobin level in multiple myeloma patients, but way of such activity remains unclear. The aim of the study was to investigate the mechanisms of thalidomide stimulating effect on erythroid differentiation. METHODS: Hematopoietic stem cells were isolated from bone marrow aspirates taken from myeloma patients and cultured with or without thalidomide. Then the generation of erythroid cells and the expression of STAT5, GATA-1, GATA-2, selected caspases and Bcl-2 family proteins in erythroid cells were assessed using flow cytometry and real-time PCR. RESULTS: The generation of erythroblasts was higher in thalidomide than in control cultures (63.9% vs. 55.8%, p < 0.001). The expression of caspase 3 (cytometry 947.3 vs. 1021.0, p = 0.025; PCR 12.9 vs. 16.3, p = 0.025) and caspase 8 (cytometry 1050.8 vs. 1168.5, p = 0.033; PCR 16.2 vs. 17.8, p = 0.004) was significantly lower in thalidomide than in control cultures. The expression of STAT5 (cytometry 331.5 vs. 276.1, p = 0.015; PCR 24.3 vs. 21.1, p = 0.003) and GATA-1 (cytometry 259.7 vs. 232.0, p = 0.027; PCR 18.9 vs. 16.5, p = 0.003) was higher in thalidomide than in control cultures. CONCLUSION: Our results suggest that thalidomide enhances expression of STAT5 in response of erythroid cells to erythropoietin and as a result of caspase 3 suppression. Moreover it may exert inhibitory effect on an external pathway of caspases activation with consequent decreased degradation of GATA-1 transcription factor by downstream caspases.

The Transcription Factor Ehf Is Involved in TGF-ß-Induced Suppression of FcεRI and c-Kit Expression and FcεRI-Mediated Activation in Mast Cells.

FcεRI, which is composed of α, ß, and γ subunits, plays an important role in IgE-mediated allergic responses. TGF-ß1 has been reported to suppress FcεRI and stem cell factor receptor c-Kit expression on mast cell surfaces and to suppress mast cell activation induced by cross-linking of FcεRI. However, the molecular mechanism by which these expressions and activation are suppressed by TGF-ß1 remains unclear. In this study, we found that the expression of Ets homologous factor (Ehf), a member of the Ets family transcriptional factors, is upregulated by TGF-ß/Smad signaling in mouse bone marrow-derived mast cells (BMMCs). Forced expression of Ehf in BMMCs repressed the transcription of genes encoding FcεRIα, FcεRIß, and c-Kit, resulting in a reduction in cell surface FcεRI and c-Kit expression. Additionally, forced expression of Ehf suppressed FcεRI-mediated degranulation and cytokine production. Ehf inhibited the promoter activity of genes encoding FcεRIα, FcεRIß, and c-Kit by binding to these gene promoters. Furthermore, the mRNA levels of Gata1, Gata2, and Stat5b were lower in BMMCs stably expressing Ehf compared with control cells. Because GATA-1 and GATA-2 are positive regulators of FcεRI and c-Kit expression, decreased expression of GATAs may be also involved in the reduction of FcεRI and c-Kit expression. Decreased expression of Stat5 may contribute to the suppression of cytokine production by BMMCs. In part, mast cell response to TGF-ß1 was mimicked by forced expression of Ehf, suggesting that TGF-ß1 suppresses FcεRI and c-Kit expression and suppresses FcεRI-mediated activation through upregulation of Ehf.

Polarity Protein Scrib Facilitates Endothelial Inflammatory Signaling.

OBJECTIVE: The polarity protein Scrib is highly expressed in endothelial cells and is required for planar cell polarity. Scrib also facilitates recycling of integrin α5 to the plasma membrane. Because integrin α5 signals the presence of the inflammatory matrix protein fibronectin, we hypothesized that Scrib contributes to endothelial inflammatory signaling. APPROACH AND RESULTS: Cytokine treatment of human umbilical vein endothelial cells induced an inflammatory response as evident by the induction of vascular cell adhesion molecule-1 (VCAM-1). Downregulation of Scrib greatly attenuated this effect. In endothelial-specific conditional Scrib knockout mice, in vivo lipopolysaccharide treatment resulted in an impaired VCAM-1 induction. These effects were functionally relevant because Scrib small interfering RNAs in human umbilical vein endothelial cells attenuated the VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α. In vivo, tamoxifen-induced endothelial-specific deletion of Scrib resulted in a reduced VCAM-1-mediated leukocyte adhesion in response to tumor necrosis factor-α in the mouse cremaster model. This effect was specific for Scrib and not mediated by other polarity proteins. Moreover, it did not involve integrin α5 or classic pathways supporting inflammatory signaling, such as nuclear factor κ light chain enhancer of activated B-cells or MAP kinases. Co-immunoprecipitation/mass spectrometry identified the zinc finger transcription factor GATA-like protein-1 as a novel Scrib interacting protein. Small interfering RNA depletion of GATA-like protein-1 decreased the tumor necrosis factor-α-stimulated VCAM-1 induction to a similar extent as loss of Scrib did. Silencing of Scrib reduced GATA-like protein-1 protein, but not mRNA abundance. CONCLUSIONS: Scrib is a novel proinflammatory regulator in endothelial cells, which maintains the protein expression of GATA-like protein-1.

Exploring erythropoiesis of common carp (Cyprinus carpio) using an in vitro colony assay in the presence of recombinant carp kit ligand A and erythropoietin.

The use of in vitro colony assays in mammals has contributed to identification of erythroid progenitor cells such as burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors, and serves to examine functions of erythropoietic growth factors like Erythropoietin (Epo) and Kit ligand. Here, we established an in vitro colony-forming assay capable of investigating erythropoiesis in carp (Cyprinus carpio), cloned and functionally characterized recombinant homologous molecules Epo and Kit ligand A (Kitla), and identified three distinct erythroid progenitor cells in carp. Recombinant carp Epo induced the formation of CFU-E-like and BFU-E-like erythroid colonies, expressing erythroid marker genes, ß-globin, epor and gata1. Recombinant carp Kitla alone induced limited colony formation, whereas a combination of Kitla and Epo dramatically enhanced erythroid colony formation and colony cell growth, as well as stimulated the formation of thrombocytic/erythroid colonies expressing not only erythroid markers but also thrombocytic markers, cd41 and c-mpl. Utilizing this colony assay to examine the distribution of distinct erythroid progenitor cells in carp, we demonstrated that carp head and trunk kidney play a primary role in erythropoiesis, while the spleen plays a secondary. Furthermore, we showed that presumably bi-potent thrombocytic/erythroid progenitor cells localize principally in the trunk kidney. Our results indicate that teleost fish possess mechanisms of Epo- and Kitla-dependent erythropoiesis similar to those in other vertebrates, and also help to demonstrate the diversity of erythropoietic sites among vertebrates.

Embryonic stem cells as sources of donor-independent platelets.

The creation of a donor-independent source of platelets has been challenging; however, recent advances show growing promise for alternative platelet sources. Pluripotent stem cells have the capacity to differentiate into mature megakaryocytes with the ability to produce functional platelets. In this issue of JCI, Noh et al. provide a proof-of-principle demonstration that embryonic stem cells can be used to produce platelets on a clinical scale by controlling the level of the transcription factor GATA1. This study emphasizes the importance of precise regulation of gene expression for regenerative medicine applications.

Inducible Gata1 suppression expands megakaryocyte-erythroid progenitors from embryonic stem cells.

Transfusion of donor-derived platelets is commonly used for thrombocytopenia, which results from a variety of clinical conditions and relies on a constant donor supply due to the limited shelf life of these cells. Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies; however, the majority of current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. In both mice and humans, mutations in the gene-encoding transcription factor GATA1 cause an accumulation of proliferating, developmentally arrested megakaryocytes, suggesting that GATA1 suppression in ES and iPS cell-derived hematopoietic progenitors may enhance megakaryocyte production. Here, we engineered ES cells from WT mice to express a doxycycline-regulated (dox-regulated) shRNA that targets Gata1 transcripts for degradation. Differentiation of these cells in the presence of dox and thrombopoietin (TPO) resulted in an exponential (at least 10¹³-fold) expansion of immature hematopoietic progenitors. Dox withdrawal in combination with multilineage cytokines restored GATA1 expression, resulting in differentiation into erythroblasts and megakaryocytes. Following transfusion into recipient animals, these dox-deprived mature megakaryocytes generated functional platelets. Our findings provide a readily reproducible strategy to exponentially expand ES cell-derived megakaryocyte-erythroid progenitors that have the capacity to differentiate into functional platelet-producing megakaryocytes.

KLF1 stabilizes GATA-1 and TAL1 occupancy in the human ß-globin locus.

KLF1 is an erythroid specific transcription factor that binds to regulatory regions of erythroid genes. Binding sites of KLF1 are often found near binding sites of GATA-1 and TAL1. In the ß-globin locus, KLF1 is required for forming active chromatin structure, although its role is unclear. To explore the role of KLF1 in transcribing the human γ-globin genes, we stably reduced the expression of KLF1 in erythroid K562 cells, compromising its association in the ß-globin locus. The γ-globin transcription was reduced with disappearance of active chromatin structure of the locus in the KLF1 knockdown cells. Interestingly, GATA-1 and TAL1 binding was reduced in the ß-globin locus, even though their expressions were not affected by KLF1 knockdown. The KLF1-dependent GATA-1 and TAL1 binding was observed in the adult locus transcribing the ß-globin gene and in several erythroid genes, where GATA-1 occupancy is independent from TAL1. These results indicate that KLF1 plays a role in facilitating and/or stabilizing GATA-1 and TAL1 occupancy in the erythroid genes, contributing to the generation of active chromatin structure such as histone acetylation and chromatin looping.

Distinct regulation of the anterior and posterior myeloperoxidase expression by Etv2 and Gata1 during primitive Granulopoiesis in zebrafish.

Neutrophilic granulocytes are the most abundant type of myeloid cells and form an essential part of the innate immune system. In vertebrates the first neutrophils are thought to originate during primitive hematopoiesis, which precedes hematopoietic stem cell formation. In zebrafish embryos, it has been suggested that primitive neutrophils may originate in two distinct sites, the anterior (ALPM) and posterior lateral plate mesoderm (PLPM). An ETS-family transcription factor Etsrp/Etv2/ER71 has been implicated in vasculogenesis and hematopoiesis in multiple vertebrates. However, its role during neutrophil development is not well understood. Here we demonstrate using zebrafish embryos that Etv2 has a specific cell-autonomous function during primitive neutropoiesis in the anterior lateral plate mesoderm (ALPM) but has little effect on erythropoiesis or the posterior lateral plate mesoderm (PLPM) expression of neutrophil marker myeloperoxidase mpo/mpx. Our results argue that ALPM-derived neutrophils originate from etv2-expressing cells which downregulate etv2 during neutropoiesis. We further show that Scl functions downstream of Etv2 in anterior neutropoiesis. Additionally, we demonstrate that mpx expression within the PLPM overlaps with gata1 expression, potentially marking the cells with a dual myelo-erythroid potential. Intriguingly, initiation of mpx expression in the PLPM is dependent on gata1 but not etv2 function. Our results demonstrate that mpx expression is controlled differently in the ALPM and PLPM regions and describe novel roles for etv2 and gata1 during primitive neutropoiesis.

Ddx46 is required for multi-lineage differentiation of hematopoietic stem cells in zebrafish.

Balanced and precisely controlled processes between self-renewal and differentiation of hematopoietic stem cells (HSCs) into all blood lineages are critical for vertebrate definitive hematopoiesis. However, the molecular mechanisms underlying the maintenance and differentiation of HSCs have not been fully elucidated. Here, we show that zebrafish Ddx46, encoding a DEAD-box RNA helicase, is expressed in HSCs of the caudal hematopoietic tissue (CHT). The number of HSCs expressing the molecular markers cmyb or T-cell acute lymphocytic leukemia 1 (tal1) was markedly reduced in Ddx46 mutants. However, massive cell death of HSCs was not detected, and proliferation of HSCs was normal in the CHT of the mutants at 48 h postfertilization. We found that myelopoiesis occurred, but erythropoiesis and lymphopoiesis were suppressed, in Ddx46 mutants. Consistent with these results, the expression of spi1, encoding a regulator of myeloid development, was maintained, but the expression of gata1a, encoding a regulator of erythrocyte development, was downregulated in the mutants. Taken together, our results provide the first genetic evidence that zebrafish Ddx46 is required for the multilineage differentiation of HSCs during development, through the regulation of specific gene expressions.

Expression of GATA1 in the ovine conceptus and endometrium during the peri-attachment period.

The transcription factor GATA1 is known to play an essential role in hematopoiesis, but its other roles have not been well characterized. The purpose of this study was to determine relationships between GATA1 and GATA2 and/or GATA3, and to identify their possible functions in ovine development. GATA1 mRNA was found in ovine conceptuses and endometrial epithelial regions of Day 15 (Day 0=day of estrus) cyclic and Days 15, 17, and 21 pregnant ovine uteri. GATA1 mRNA was strongly expressed in conceptuses on Day 21, when trophoblast attachment to the maternal endometrium progressed. Similarly, GATA1 protein expression was relatively high on Day 21. To localize GATA1 mRNA, ovine conceptuses and pregnant uteri were subjected to in situ hybridization on Days 15, 17, and 21, confirming that GATA1 mRNA was expressed in trophoblasts and uterine endometrial epithelial cells in these gestation days. The presence of GATA1 protein was further confirmed by immunohistochemistry. Because high GATA1 expression appeared to coincide with reduced GATA2/3 expression, a potential role of GATA1 was examined through transfection of a mouse Gata1 expression plasmid into bovine trophoblast F3 cells. This over-expression resulted in the down-regulation of endogenous GATA2 transcripts. These observations indicate that GATA1 exists in the ovine conceptus and uterus during the peri-attachment period, and suggest that GATA1 is integral to conceptus and endometrial development through the regulation of GATA2 and possibly other developmentally important genes.

Subacute thyroiditis and dyserythropoesis after influenza vaccination suggesting immune dysregulation.

Subacute thyroiditis (SAT) is an extremely rare complication of influenza vaccination. Several infectious agents have been related with SAT. It is also well known the association between HLA-B35 and the development of SAT. We describe a case of subacute thyroiditis and dyserythropoesis occurring shortly after administration of an influenza vaccine in a 55-year-old man with history of diabetes and psoriasis, family history of autoimmunity without clinical evidence of acute viral infection prior to the onset of symptoms. We propose that, the events occurring in the patient may be explained as result of complex interactions between the individual genetic background and environmental exposure to infectious agents that generated a pro-inflammatory status, where the vaccine was the trigger for the subsequent alterations in thyroid and bone marrow. These findings highlight the importance of immunogenetic factors involved in response to vaccination that is the central theme in the growing field of 'vaccinomics'.

Zebrafish enhancer trap line recapitulates embryonic aquaporin 1a expression pattern in vascular endothelial cells.

Aquaporin 1 (Aqp1) is a water channel protein, expressed widely in microvascular endothelial cells and implicated in mammalian tumor angiogenesis. However, its developmental expression has not yet been characterized in great detail. An enhancer trap screen was performed using a Tol2-derived GFP reporter in zebrafish embryos. An insertional Et(GBT-B1)tpl1 line was identified that has reporter insertion in the vicinity of the aqp1a gene. We further characterized the embryonic expression pattern of this GFP reporter line, as well as that of endogenous aqp1a. Both endogenous aqp1a and reporter GFP expression were restricted to the vascular endothelial cells within the dorsal aorta, cranial, intersegmental and other secondary vessels, but were absent in the axial venous vasculature. In addition, endogenous aqp1a expression was observed in both primitive and definitive hematopoietic erythroid progenitors, as well as in the otic vesicle, swim bladder, pneumatic duct, intestine and a subset of neurons within the retina and the midbrain-hindbrain region. We further show that gata1 and etsrp/etv2 function is required for hematopoietic and endothelial aqp1a expression, respectively. Aqp1a expression is restricted to endothelial and erythroid cells during early embryogenesis. The transgenic Et(GBT-B1)tpl1 line recapitulates endogenous endothelial aqp1a expression. Because currently very few reporter lines can differentiate between arterial and venous endothelial cells, the Et(GBT-B1)tpl1 transgenic line and characterization of the aqp1a expression pattern will be useful for future studies of endothelial and arterial-venous differentiation.