The transcription factor GATA-2 plays vital roles in quite diverse developmental programs, including hematopoietic stem cell (HSC) survival and proliferation. We previously identified a vascular endothelial (VE) enhancer that regulates GATA-2 activity in pan-endothelial cells. To more thoroughly define the in vivo regulatory properties of this enhancer, we generated a tamoxifen-inducible Cre transgenic mouse line using the Gata2 VE enhancer (Gata2 VECre) and utilized it to temporally direct tissue-specific conditional loss of Gata2. Here, we report that Gata2 VECre-mediated loss of GATA-2 led to anemia, hemorrhage, and eventual death in edematous embryos. We further determined that the etiology of anemia in conditional Gata2 mutant embryos involved HSC loss in the fetal liver, as demonstrated by in vitro colony-forming and immunophenotypic as well as in vivo long-term competitive repopulation experiments. We further documented that the edema and hemorrhage in conditional Gata2 mutant embryos were due to defective lymphatic development. Thus, we unexpectedly discovered that in addition to its contribution to endothelial cell development, the VE enhancer also regulates GATA-2 expression in definitive fetal liver and adult BM HSCs, and that GATA-2 function is required for proper lymphatic vascular development during embryogenesis.

IntroductionMultiple hematopoietic cell lineages are generated from hematopoietic stem cells (HSCs) that are found in a very small fraction of the Lin Ϫ Sca1 ϩ c-Kit hi (LSK) population of BM cells. Long-term repopulating HSCs (LT-HSCs) are even more rare, and can be purified almost to homogeneity in the LSK CD150 ϩ CD48 Ϫ CD34 Ϫ Flt3 Ϫ immunophenotypic population of murine adult BM cells. [1][2][3] Less than 5% of LT-HSCs are actively cycling in the S ϩ G 2 /M phases to produce more HSCs for maintaining life-long hematopoiesis and for the generation of terminally differentiated hematopoietic cells.Conversely, approximately 75% of LT-HSCs are quiescent (in the G 0 phase), thus maintaining both stemness and proliferative capacity. 4,5 Studies using various mutant model mice support the contention that the maintenance of HSC quiescence is essential for their long-term repopulating function. 6 The balance between quiescence and cell-cycle entry is controlled by signals from the HSC niche through a variety of signaling pathways, cyclin-dependent kinases, and transcription factors. The molecular mechanisms that control HSC cell-cycle entry are not fully understood, although recent studies have identified several nuclear proteins that are involved in restricting or promoting cell-cycle entry; these include: GFI1, 7 MEF/ELF4, 8 FOXO3A, 9 GFI1B, 10 EGR1, 11 JUNB, 12 and perhaps both c-and N-Myc. 13 The GATA transcription factors all bind to a WGATAR DNA sequence motif found in the promoters and enhancers of thousands of target genes to control their transcription. Recent genome-wide ChIP-seq experiments confirmed that WGATAA is the preferred sequence bound by GATA proteins in vivo. [14][15][16][17] The vertebrate GATA family is composed of 6 members, somewhat artificially divided into the hematopoietic (GATA-1, GATA-2, and GATA-3) and endodermal (GATA-4, GATA-5, and GATA-6) subfamilies. GATA-2 and GATA-3 are both expressed in HSCs. [18][19][20][21] Whereas Gata2-null mutant embryonic stem (ES) cells fail to generate HSCs or progenitors in reconstituted chimeric mice, 22 Gata3-null ES cells are able to generate myeloid and B-lymphoid cells, but not T cells. 23 Therefore, GATA-2 is required for the generation of HSCs, but GATA-3 is not. More specifically, GATA-2 is required for HSC proliferation and viability during definitive hematopoiesis, and even haploinsufficiency of GATA-2 results in a reduced number of functional HSCs. 22,24-26 GATA-3 is vital for the development of T cells at multiple stages in T-cell development and for Th2 differentiation in peripheral organs, but is dispensable for the generation of myeloid and B cells. 27 We recently discovered that GATA-3 is required for the generation of early T-lineage progenitor (ETP) cells, the most immature cells in the thymus, which have been shown to have complete developmental potential for T-lineage development, and that GATA-3 plays a critical role immediately around the time of thymic entry of T-cell progenitors. 28 To further explore a possible role for GATA-3 in ...

A simple method for immobilizing endothelial cells in the channels of a microfluidic device fabricated with soft lithography is presented that requires no surface oxidation of the substrate material used in conjunction with the microfluidic device and is operable even with a reversible seal. Specifically, optimal conditions for culturing bovine pulmonary artery endothelial cells (bPAECs) to the surface of a Petri dish were investigated. The parameters investigated included fibronectin concentration, temperature, seeding density, and immobilization time. To enhance the utility of the device, all optimization studies, and studies involving platelet adhesion to the immobilized endothelium, were performed in parallel channels, thereby enabling improved throughput over a single channel device. The optimal conditions for cell immobilization included coating the petri dish with 100 μg/mL fibronectin, a seeding cell density of 1.00 × 10 5 cells mL −1 and an immobilization time of 90 min at 37 °C. The device was then employed to monitor the physical interaction (adhesion) of platelets to the immobilized endothelium in the presence of a known platelet activator (ADP) and a drug inhibitor of platelet activation. The number of platelets adhering to the endothelial cells in the channels increased from 17.0 ± 2.3 in the absence of ADP to 63.2 ± 2.4 in the presence of 5.00 μM ADP. Moreover, the data presented here also shows that inhibition of endothelium nitric oxide (NO) production, a recognized inhibitor of platelet adhesion to the endothelium, increased the number of platelets adhering to the surface to 35.4 ± 1.0. In the presence of NO inhibition and 5.00 μM ADP, the affect on platelet adhesion was further increased to 127 ± 5.2. Finally, this device was employed to investigate the effect of a drug known to inhibit platelet adhesion (clopidogrel) and, in the presence of the drug, the platelet adhesion due to activation by 5.00 μM ADP decreased to 24.0 ± 3.8. This work is the first representation of multiple cell types physically interacting in the channels of a microfluidic device and further demonstrates the potential of these devices in the drug discovery process and drug efficacy studies.

The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell-specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte-specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte-specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell-regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell-specific transcriptional activity.

Protein abundance must be precisely regulated throughout life, and nowhere is the stringency of this requirement more evident than during T-cell development: A twofold increase in the abundance of transcription factor GATA3 results in thymic lymphoma, while reduced GATA3 leads to diminished T-cell production. GATA3 haploinsufficiency also causes human HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome, often accompanied by immunodeficiency. Here we show that loss of one Gata3 allele leads to diminished expansion (and compromised development) of immature T cells as well as aberrant induction of myeloid transcription factor PU.1. This effect is at least in part mediated transcriptionally: We discovered that Gata3 is monoallelically expressed in a parent of origin-independent manner in hematopoietic stem cells and early T-cell progenitors. Curiously, half of the developing cells switch to biallelic Gata3 transcription abruptly at midthymopoiesis. We show that the monoallelic-to-biallelic transcriptional switch is stably maintained and therefore is not a stochastic phenomenon. This unique mechanism, if adopted by other regulatory genes, may provide new biological insights into the rather prevalent phenomenon of monoallelic expression of autosomal genes as well as into the variably penetrant pathophysiological spectrum of phenotypes observed in many human syndromes that are due to haploinsufficiency of the affected gene.

ATP is a recognized stimulus of nitric oxide synthase and is released from red blood cells (RBCs) upon deformation. The objective of this work is to demonstrate that RBCs stimulate nitric oxide production in platelets by employing a continuous flow analysis system in which the stream contains both RBCs and platelets. Here, two drugs known to improve blood flow in vivo (pentoxyfilline and iloprost) are shown to increase both the release of RBC-derived ATP and the production of platelet-derived NO. A flowbased chemiluminescence assay (in vitro) was employed to quantitatively determine the amount of ATP released from erythrocytes subjected to flow-induced deformation. Prior to being subjected to flow, erythrocytes were incubated in the absence or presence of 4.8 µM pentoxyfilline or 80 nM iloprost. Erythrocytes obtained from rabbits (n ) 22) that were subjected to flow released 239 ( 29 nM ATP. When treated with pentoxyfilline, the ATP released from the flowing RBCs increased to 450 ( 94 nM ATP. An increase in RBC-derived ATP was also measured for iloprost-incubated RBCs in flow (362 ( 45 nM ATP). Importantly, platelets that were loaded with diaminofluorofluorescein diacetate, an intracellular fluorescence probe for NO, exhibited increases in fluorescence intensity by 16% in the presence of RBCs treated with pentoxyfilline and a 10% increase when treated with iloprost. When the ATP release from the RBCs was inhibited with glybenclamide, the platelet fluorescence intensity decreased by 25 and 51% for RBCs incubated with pentoxyfilline and iloprost, respectively. In an experiment not involving the RBC, inhibition of the P2x receptor on the platelets (an ATP receptor) resulted in no increase in platelet NO production, suggesting that the NO production in the activated platelet is due to ATP.In addition to its well-known ability as a vasodilator, NO is also able to inhibit platelet activation and aggregation. Platelets have also been shown to produce NO upon activation. 1 Freedman et al. simultaneously measured NO production and aggregation of platelets upon activation with ATP. 2 These authors placed an electrode for NO into the cell of an aggregometer in order to simultaneously monitor the NO release from platelets and the aggregation of the platelets, thus demonstrating that NO was produced and released by these cells upon activation. From these studies, the authors concluded that NO released by the platelets was key to preventing further platelet recruitment to the activated platelets, but only mildly prevented their adhesion to an endothelium.In question concerning the production and release of plateletderived NO is the mechanism by which the NO production is stimulated. For example, ATP is a recognized stimulus of nitric oxide synthase (NOS) and subsequent production of platelet NO; moreover, it is also known that ATP is released from platelets upon activation. 3 However, it has not been established if ATP released from activated platelets has the ability to stimulate the production of NO in platelets. Here, res...

Nitric oxide (NO) is quantitatively determined in platelets prior to, and after, stimulation with adenosine triphosphate (ATP) or activation with adenosine diphosphate (ADP). Platelets obtained from the whole blood of rabbits were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent NO production was measured as a fluorescent benzotriazole. Experiments were performed to determine the effect of probe concentration and probe incubation time in the platelets prior to measurement of the fluorescence. This information, combined with the method of multiple standard additions, was then employed to determine the moles of intracellular NO in the platelets (2.7 +/- 0.3) x 10(-16) mol of NO/platelet and the basal level of extracellular NO in the platelet sample (9.9 +/- 2.2) x 10(-18) mol of NO/platelet. Moreover, this method was used to quantitatively determine the amount of NO released from platelets whose NO production was stimulated with ATP (a nitric oxide synthase stimulus) or ADP, a substance known to result in NO production through platelet aggregation. When stimulated with ATP, the NO released from the platelets was determined to be (2.0 +/- 0.1) x 10(-17) mol of NO/platelet. When activated with ADP, the platelets released (2.8 +/- 0.3) x 10(-17) mol of NO/platelet. The difference between the extracellular basal levels of NO and that after stimulation with either ATP or ADP is in agreement with current estimates of NO release from platelets. Therefore, we conclude that a fluorescence determination of NO using the DAF family of probes, in combination with the method of multiple standard additions, can be employed to quantitatively determine the basal levels of NO in platelets, as well as the amount of NO released from stimulated and/or activated platelets.

Patient groups subject to higher occurrence of stroke (e.g., people with diabetes, cystic fibrosis, pulmonary hypertension) have reduced release of ATP from their erythrocytes (ERYs) when subjected to flow-induced deformation or pharmacological stimuli. These same groups also have platelets that are more adhesive in comparison to controls. Here we show platelet aggregation, and inhibition of that aggregation, is affected by free Ca(2+) entering the platelet through the ATP-gated P2X1 receptor. The addition of ATP (10 microM) increased the platelet NO by 26.7 +/- 7.7%. This value was decreased significantly to below basal levels in the presence of NF 449 (p < 0.001), an inhibitor of the P2X1 receptor on the platelet. Aggregation profiles measured in the presence of ATP revealed that when the P2X1 receptor was blocked, or when the measurements were performed in Ca(2+) free buffer, platelet aggregation was nearly eliminated. Our findings employing standard aggregation measurements suggest that ATP behaves as a platelet inhibitor below 1.6 x 10(-19) moles ATP per platelet; however, above this value, ATP behaves as a platelet activator. These findings suggesting a dual nature of ATP with regard to platelet behavior were confirmed by passing platelets over endothelial cells that were coated in the channels of a microfluidic device. Importantly, it was determined that ERY-derived ATP release was a major determinant of platelet adhesion to the endothelium. These findings may have implications in anti-platelet drug design as most current therapies focus on the inhibition of P2Y-type receptors. Moreover, through the use of microfluidic technologies, we have provided in vitro evidence for a possible relationship between ERY properties and platelet behavior in vivo.