Conditional Gata2 inactivation results in HSC loss and lymphatic mispatterning

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.

GATA factors belong to an evolutionarily conserved family of C4 zinc finger transcription factors that play demonstrably crucial roles in quite diverse developmental programs, including hematopoietic, urogenital, otic, and neuronal developmental elaboration (1–11). GATA-2 was first demonstrated to be essential for hematopoiesis, as Gata2 homozygous null mutant (Gata2^–/–) embryos die at E10.5 during embryogenesis due to a failure in initial blood cell generation (2). Analyses of null mutant embryonic stem cells and their contribution to in vitro differentiation or to definitive hematopoiesis in reconstituted chimeras supported the argument that GATA-2 is required for the proliferation and/or survival of hematopoietic stem cells (HSCs) (12). Recently, it was shown that Gata2 haploinsufficiency resulted in altered integrity of the definitive HSC compartment, leading to a reduction in the number of HSCs by essentially one-half (13, 14).

We previously identified and characterized a Gata2 intron 4 enhancer that conferred reporter gene activity in transgenic mice in both blood and lymphatic endothelial cells (LECs) as well as in poorly characterized subsets of hematopoietic cells (5). Here, we report the generation of conditionally inducible Gata2 vascular endothelial (VE) enhancer–regulated Cre transgenic lines and the consequences of their induced activity in a floxed Gata2(Gata2^fl) genetic background. To circumvent the normal E10.5 demise encountered in Gata2^–/– embryos, we utilized a version of Cre recombinase fused to a tamoxifen-sensitive (Tx-sensitive) ligand-binding domain of the estrogen receptor (CreERT², ref. 15). This strategy allowed us to administer Tx, thereby activating Cre and inactivating the Gata2^fl allele, after the time when embryos would normally encounter the first lethal block in primitive erythropoiesis. Analyses of Tx-treated doubly transgenic Gata2 compound mutant (Tg^VE:Gata2^–/fl) embryos revealed that the VE enhancer harbors what we believe to be a previously undiscovered Gata2 transcriptional regulatory activity in definitive (fetal and adult) HSCs, and unexpectedly that GATA-2 deficiency in the endothelial lineage results in edema and hemorrhage, leading to late gestational lethality. Histological examination of Tx-treated Tg^VE:Gata2^–/fl embryos revealed blood pooling in the lymphatic vasculature due to failed lymphatic-venous abscission. Thus, these data show that the Gata2-null mutant animals that survive the initial primitive erythroid embryonic lethal deficiency normally encountered at E10.5 subsequently die of LEC deficiency at E16.5, thus providing the first evidence to our knowledge that GATA-2 is required for an essential endothelial (either autonomous or non-autonomous) function.

Recently, several groups reported that human patients with GATA2 mutations presented symptoms of primary lymphedema (16–18), underscoring the vital role of transcription factor GATA-2 in lymphatic development. Through analysis of the conditionally mutant Gata2 mice described here, we hope to further elucidate the role of GATA-2 in lymphangiogenesis.

Generation of Gata2 VE enhancer–regulated mCherry/CreERT² transgenic lines. We generated transgenic mice bearing integrated copies of the VECreERT² transgene in which the Gata2 VE enhancer independently directed the transcription of an inducible Cre recombinase or the fluorescent mCherry (mCh) reporter gene (Figure 1A and ref. 19). Of the transgenic founder animals that were determined by PCR to harbor both transgenes (11 of 44; hereafter referred to as Tg^VE), some (7 of 11) stably transmitted both. Their progeny (F₂–F₅ generations) were used for Cre transgene copy number (and other, see below) analyses, and they ranged from 5 to 47 (using actin as a normalization control) (ref. 20 and Figure 1B).

Figure 1

A Gata2 vascular enhancer confers CreERT² and mCh transgene expression in the embryonic vasculature. (A) Schematic depicting the CreERT² (15) or mCh (19) cDNA driven by the HSV TK promoter (tkP) and the 1.2-kbp Gata2 VE (5) in VECreERT² or VEmCherry expression plasmid, respectively. Each minigene cassette was flanked by tandem repeats of chicken HS4 insulators (ins) (53). Both inserts were excised from the vector and coinjected (1:1) into the pronuclei of mouse oocytes to generate doubly transgenic (Tg^VE) mice. F₂–F₅ progeny were used in subsequent analyses (B–E). (B) Cre transgene copy number (normalized to Actin) was determined by qPCR to range between 5 and 47 copies. Cre mRNA level (normalized to endogenous endothelia-restricted Flk1 mRNA) in the heart (black bars) and kidney (white bars) of neonatal Tg^VE pups (n = 3 to 8) was determined by RT-qPCR. Of the 3 Tg^VE lines that showed significant endothelial mCh staining (see below), Tg^VE56 and Tg^VE62 both robustly expressed Cre mRNA, while Cre transcripts were barely detectable in Tg^VE73. qPCR primer sequences are listed in Supplemental Table 1. Data represent mean ± SD. (C) mCh epifluorescence in representative E10.5 embryos. Of 7 lines that stably transmitted both transgenes, Tg^VE62 expressed mCh most robustly in an endothelia-specific manner, while in some lines mCh was weakly expressed (e.g., Tg^VE60 and Tg^VE473). (D) Coincident expression (merge) of mCh (Tg^VE62) and eGFP (generated from Gata2^+/gfp; ref. 21) in the major and fine cranial vasculature in an E10.5 Tg^VE62:Gata2^+/gfp compound mutant embryo. mCh expression temporally and spatially parallels that of eGFP (Gata2) in the vasculature. The asterisk indicates a GFP-exclusive area of Gata2 expression in the ventral midbrain. (E) Coincidence (merge) of mCh (Tg^VE62) and eGFP (from Tg^Tie2.gfp) epifluorescence in the major and intersomitic vasculature of an E10.5 Tg^VE62:Tg^Tie2.gfp embryo, underscoring the vascular endothelial tissue specificity of the Tg^VE transgene.

When we examined the expression of the co-integrated transgenes, we found that robust mCh epifluorescence in an endothelia-restricted pattern was detected in E10.5 embryos from 3 lines (56, 62, and 73), while other lines (60, 457, and 473) exhibited no or only very faint vascular mCh fluorescence (Figure 1C and data not shown). Furthermore, in the 3 lines that displayed robust mCh staining, Cre mRNA (normalized to Flk1 mRNA) in the vascularized heart and kidneys of P0–P2 transgenic pups was easily detectable by RT-qPCR in Tg^VE56 and Tg^VE62 but not in Tg^VE73 mice (Figure 1B). Therefore, only Tg^VE56 and Tg^VE62 lines were used for subsequent studies.

To determine whether the vascular expression pattern of the mCh reporter gene reflected genuine GATA-2 expression, we bred Tg^VE62 to a Gata2-eGFP knock-in mouse (21). As depicted in Figure 1D, coincident mCh transgenic expression precisely reflected eGFP fluorescence produced from the endogenous Gata2 locus, while eGFP alone was additionally (and exclusively) expressed in a cluster of deep ventral midbrain neurons that could be visualized in the same fields (22, 23). Finally, we asked whether mCh fluorescence matched the tissue specificity of a known vascular endothelia-specific gene, the Tie2 tyrosine kinase (24). As shown in Figure 1E, the transgenic mCh expression pattern in endothelial cells was indistinguishable from that of GFP directed by the Tie2 gene promoter and enhancer (24) in the intersomitic vasculature.

We further investigated the inducibility and tissue specificity of CreERT² activity in these two lines by examining transgene activity in the ROSA26 reporter R26R strain, in which the lacZ gene, flanked by transcriptional termination sites and loxP sequences, is integrated at the ROSA26 locus (25). In this configuration, Cre recombinase activity is reported by β-gal expression only in those tissues in which Cre becomes activated. Without Tx, we could detect no X-gal staining in the doubly transgenic Tg^VE:R26R embryo or a littermate R26R embryo (Supplemental Figure 1A; supplemental material available online with this article; doi: 10.1172/JCI61619DS1). However, RT-qPCR analyses of total RNA isolated from their respective yolk sacs confirmed that Cre (as well as mCh, normalized to Flk1 mRNA) was expressed only in the doubly transgenic Tg^VE:R26R embryo but not in its littermate R26R embryo (Supplemental Figure 1B). In the presence of Tx (administered by daily gavage of pregnant dams from E9 to E11 during embryogenesis), the Tg^VE:R26R embryo, unlike its control R26R sibling (Figure 2A), displayed strong X-gal staining in the blood vasculature, mirroring mCh expression (Figure 1C). Thus, we concluded that the Cre activity in Tg^VE is stringently ligand dependent and is functionally expressed in a tissue-specific manner, as anticipated.

Figure 2

ROSA26R-derived β-gal expression in Tx-treated Tg^VE embryos. (A) X-gal accumulation is evident in the vasculature of a Tx-treated whole-mount E11.5 Tg^VE62:R26R compound mutant embryo (right), but not in a (Tx-treated) R26R littermate that lacks the Cre transgene (left). In this experiment, embryos were administered Tx from E9 to E11 by gavage of pregnant dams. (B–G) The Tg^VE62:R26R compound mutant embryo (A, right) was sectioned to further analyze X-gal localization at the cellular level. (B) The endocardium (e) and the endocardial cushion (ec) in the E11.5 heart were positive for β-gal activity. (C) X-gal staining is also evident in the cardinal vein (cv), from which the lymphatic vascular system develops. (D–F) Transverse cryosections in the vicinity of the dorsal aorta (da) revealed budding single (arrowheads, D and E) and clustered (arrows, enlarged view, E and F) lacZ-positive (rounded) hematopoietic cells. ms, mesonephros. (G) Punctate, single-cell X-gal staining was also present in FL endothelial and hematopoietic cells. Scale bars: 1 mm (A); 100 μm (B, D and G); 50 μm (C); 10 μm (E and F).

When we examined transverse sections of the E11.5 Tg^VE:R26R embryo (Figure 2A), X-gal accumulation was observed in the endocardium and the endocardial cushions of the developing heart (Figure 2B), as well as in the cardinal vein (Figure 2C), from which a subset of endothelial cells first gain competence to become LECs as they begin to express Prox1 at E9.5 of embryogenesis (26). LECs then delaminate and migrate dorsolaterally to eventually give rise to the first bona fide lymphatic structure, the jugular lymph sac. Further remodeling and elaboration of the embryonic lymph sacs eventually give rise to the mature lymphatic vascular system that is responsible for modulating fluid homeostasis (27).

More interestingly, X-gal–stained individual rounded cells that appeared to be budding from distended endothelial cells into the lumen of the dorsal aorta were routinely observed (Figure 2, D and E, arrowheads) and also appeared in clusters (Figure 2, E and F, arrows). When we examined serial transverse sections along the anteroposterior length of Tx-treated Tg^VE:R26R embryos (n = 3; >80 serial sections per embryo), we found that 70% ± 6% of hematopoietic clusters stained with X-gal. Such rounded cells budding into the dorsal aorta at this stage have been reported to include HSCs (28, 29). Within the fetal liver (FL), which is the major hematopoietic organ during mid-embryogenesis, we also observed discrete cells displaying β-gal activity (Figure 2G). We were able to validate the previous characterization of the Gata2 VE element as a vascular endothelial enhancer (5) using this independent strategy, while at the same time discovering that the enhancer might also bear very early hematopoietic lineage specificity.

VE-regulated Gata2 loss in the FL results in depletion of definitive HSCs. To functionally assess the activity of the VE enhancer, we bred the CreERT² lines in order to conditionally ablate the floxed Gata2 (Gata2^fl) allele by removing its fifth exon, which encodes the primary DNA-binding zinc finger (10). In the absence of Tx administration, progeny from these intercrosses were recovered in a normal Mendelian distribution. In particular, Gata2^–/fl and Tg^VE:Gata2^–/fl sibling animals from litters that were not exposed to Tx were healthy and lived a normal life span. In order to avoid purported effects of Tx administration or Cre expression on embryonic survivability, we tested the progeny of 15 litters from Tg^VE:Gata2^+/– × Gata2^+/fl intercrosses, with and without different concentrations of Tx, to determine: (a) whether the Cre (or theoretically, the mCh) transgene “poisoned” normal Mendelian survival among the progeny; and (b) the precise dosage of Tx and method of administration that led to robust Cre activation accompanied by no (unusual) embryonic death. Since constitutive loss of GATA-2 results in early embryonic lethality at about E10.5 from a failure to properly execute primitive hematopoiesis (2, 3), Tx was administered after primitive hematopoiesis (beginning between E7 and E7.5) was already in process. Tx was administered to pregnant dams from Gata2^+/fl × Tg^VE:Gata2^+/– intercrosses beginning on E9 for 3 consecutive days by oral gavage. (Previous studies showed that when Tx was administered to pregnant dams, there was a lag time of 12 hours before levels peaked in the serum of treated animals [ref. 30].) We finally employed an experimentally determined optimal Tx dose of 0.07 mg/g maternal body weight.

Of 89 Tx-treated embryos recovered from 10 litters that were analyzed between gestational days 13.5 and 15.5, Tg^VE:Gata2^–/fl embryos were recovered at the anticipated Mendelian ratios (Table 1). However, we noticed that compared with their littermates, Tx-treated Tg^VE:Gata2^–/fl embryos and yolk sacs appeared to be anemic, with death occurring by E16.5 (see below). We were curious to determine whether the observed pale appearance of the Tx-treated Tg^VE:Gata2^–/fl embryos might be attributable to disruption of a function for GATA-2 in erythropoiesis, since Grass et al (31) independently identified the same Gata2 enhancer and reported that the VE element was able to potentiate reporter gene expression in transfected murine erythroleukemia cells. Alternatively, the VE enhancer could regulate Gata2 transcription at some very early stage of hematopoiesis. Our earlier observations, of rounded X-gal–stained cells apparently budding from the dorsal aorta (Figure 2, D–F), lend credence to the latter hypothesis.

Table 1

Distribution of embryos from Gata2^+/fl × TgVE:Gata2^+/– intercrosses

To delve more deeply into the etiology of the observed anemia in these embryos, we examined Tx-treated FL cells from E14.5 embryos in triplicate in colony-forming assays in order to determine their ability to generate CFU of either erythroid (burst-forming unit–erythroid [BFU-E]), myeloid (granulocytic, macrophage, or both [CFU-G, CFU-M, or CFU-GM]), or mixed (CFU-GEMM) lineages. Compared with Tx-treated E14.5 wild-type control embryos (Gata2^+/+ with or without Tg^VE, n = 11), the total FL cell number in Tx-treated heterozygotes (Gata2^+/–, Tg^VE:Gata2^+/fl, n = 13) and Tg^VE:Gata2^–/fl embryos (n = 6) was correspondingly reduced with increasing allelic loss of GATA-2 (Figure 3A). In the latter group of embryos, total FL cell number was reduced to 19% of that in wild-type embryos.

Figure 3

Tx-treated Tg^VE:Gata2^–/fl FL cells fail to generate hematopoietic colonies in vitro. (A–D) FLs recovered from Tx-treated (see Methods) E14.5 embryos were dissociated into single cells. The total FL cell number in Gata2 heterozygous (Gata2^+/–, Tg^VE:Gata2^+/fl [+/–]; n = 13) and Tg^VE:Gata2^–/fl embryos (Tg:–/fl; n = 6) was significantly and correspondingly reduced with increasing loss of GATA-2 activity compared with wild-type embryos (Gata2^+/+, Tg:Gata2^+/+ [+/+]; n = 11). (B) Total FL cells from the embryos shown in A were seeded into methylcellulose medium (M3434). For control embryos, 2 × 10⁴ total FL cells were seeded per plate. For Tg^VE:Gata2^–/fl embryos, cells were seeded at multiple concentrations (2 × 10⁴, 5 × 10⁴, and 1 × 10⁵ per plate) in triplicate. Colonies were assessed after 7–12 days in culture. Results represent mean ± SD of 4 independent experiments. Colony numbers were normalized to 10⁵ FL cells for graphical illustration. Compared with wild-type FL cells, Gata2 heterozygous and Tg^VE:Gata2^–/fl cells generated fewer and no CFU colonies, respectively. (C) Total RNA was prepared from FL cells of Tx-treated embryos (E9–E11; see A) and then quantified (in triplicate) for Gata2 mRNA (normalized to endogenous Hprt mRNA) by RT-qPCR. Gata2 mRNA was reduced by 85% in Tx-treated Tg^VE:Gata2^–/fl (Tg:–/fl; n = 6) compared with wild-type embryos (Gata2^+/+ [+/+]; n = 6). qPCR primer sequences are listed in Supplemental Table 1. (D) Individual hematopoietic colonies (n = 35) isolated from methylcellulose cultures seeded with Tx-treated FL cells from different Tg^VE:Gata2^+/fl embryos from separate experiments (see B) were used for genomic DNA extraction, and then qPCR (in triplicate) was performed using primers that detected Gata2 exon 5 and Actin (normalization control; see Supplemental Table 1). Gata2 wild-type (arbitrarily set at 100%) and heterozygous genomic DNAs from tail snips of adult mice were included to validate the assay. Overall, in Tg^VE:Gata2^+/fl cells, 84% of the single Gata2 floxed allele was successfully deleted by Tx-induced Cre recombinase. Data represent mean ± SD

FL cells from these Tx-treated embryos were then seeded into methylcellulose medium supplemented with growth factors (M3434, Stem Cell Technologies). Gata2 heterozygotes (Gata2^+/–, Tg^VE:Gata2^+/fl, n = 13) and Tg^VE:Gata2^–/fl embryos (n = 6) also generated lower numbers of erythroid, myeloid, and mixed-lineage colonies (Figure 3B). Notably, Tx-treated Tg^VE:Gata2^–/fl FL cells failed to generate BFU-E, CFU-G/M/GM, or CFU-GEMM colonies (Figure 3B), initially suggesting that a very early definitive hematopoietic progenitor compartment was severely compromised in Tx-treated Tg^VE:Gata2^–/fl embryos.

Successful Cre-mediated excision of the Gata2 floxed allele was determined at the RNA and genomic DNA levels. Remaining GATA-2 transcript abundance (if any) in Tx-treated FL samples that were used for CFU assays was examined by RT-qPCR. In Tg^VE:Gata2^–/fl FL cells, Gata2 mRNA (relative to endogenous Hprt mRNA) was reduced to 15% of that in wild-type embryos (Figure 3C). We also quantified Cre-mediated excision of the floxed Gata2 allele using qPCR assay to detect the presence of exon 5 (the one flanked by loxP sites in the floxed Gata2 allele, normalized to Actin) in genomic DNAs extracted from individual colonies isolated from Tx-treated Tg^VE:Gata2^+/fl methylcellulose cultures. Of the 35 Tg^VE:Gata2^+/fl individual colonies analyzed, Gata2 exon 5 was detected at 58% ± 6% relative to the wild-type control. Hence, the calculated excision efficiency of the single floxed Gata2 allele achieved was 84% (Figure 3D and Supplemental Figure 2). Together, our results confirmed that the in vivo induction of CreERT² activity led to effective Gata2 inactivation in Tg^VE:Gata2^–/fl embryos.

Since the in vitro colony-forming assays could not be used to pinpoint where the defect was in the affected progenitor population (i.e., in multipotent hematopoietic progenitors or even more primitive HSCs), more precise and quantitative hematopoietic lineage analysis was undertaken using flow cytometry. To investigate in greater depth the hypothesis that the VE enhancer regulates Cre-mediated inactivation of Gata2, thereby ablating a very early hematopoietic progenitor compartment, we isolated E14.5 FLs from embryos that had been administered Tx daily from E9 through E11 and then performed flow cytometry to determine the immunophenotype corresponding to different early progenitor populations. The combinatorial analysis of a subset of Slam family receptors (CD150 and CD48) in conjunction with conventional HSC surface markers (mature hematopoietic lineage–negative, Sca-1–positive, and c-Kit–positive [LSK]) facilitates the identification of a highly enriched HSC cellular fraction in which 1 in every 2.1 recovered cells is capable of conferring long-term multilineage reconstitution in lethally irradiated host animals (32, 33).

We found that Tx-treated embryos bearing a single Gata2 null allele (Tg^VE:Gata2^+/–, Tg^VE:Gata2^+/fl, Gata2^–/fl; n = 9) had reduced LSK and LSKS (lin^–Sca-1⁺c-Kit⁺CD150⁺CD48^–) cells when compared with wild-type or pseudo-wild-type (Gata2^+/+, Tg^VE:Gata2^+/+, Gata2^+/fl; n = 6) embryos (Figure 4). The total number of HSCs recovered from Tx-treated embryos bearing two wild-type alleles was between 2.2 × 10⁴ to 5.7 × 10⁴ (LSK) and 1.2 × 10³ to 4.6 × 10³ (LSKS), whereas only 1.3 × 10⁴ to 4.0 × 10⁴ (LSK) or 0.5 × 10³ to 1.6 × 10³ (LSKS) cells were recovered from Tx-treated embryos bearing a single Gata2 null allele. These data mirrored previous reports showing that Gata2 haploinsufficiency results in compromised HSC homeostasis (13, 14). Intriguingly, we found that when Tg^VE:Gata2^–/fl embryos (n = 4) were administered Tx between E9 and E11, both the LSK and the more highly HSC-enriched LSKS populations displayed an exponential decline (Figure 4, C–E). A small number of LSK cells (i.e., 98–477) were recovered from Tx-treated Tg^VE:Gata2^–/fl (Figure 4C; n = 4) embryos, and likewise a minuscule number of LSKS cells were recovered from these FLs (Figure 4D). We obtained the same results when Tx was administered later during gestation (between E11 and E13), at a time during embryogenesis when definitive hematopoiesis was already fully engaged (Figure 4B). This is consistent with the observation that in embryos hemizygous for an active Tg^VE transgene, a majority of both LSK and LSKS cells were mCh⁺ (64% ± 4% and 87% ± 6%, respectively, n = 7; Figure 4E and data not shown). We conclude that the loss of GATA-2 activity in those tissues regulated by the Gata2 VE enhancer results in a virtually complete loss of HSCs in the most highly refined fraction of fetal HSCs, revealing for the first time to our knowledge the localization of a transcriptional regulatory element that is capable of controlling HSC activity of the Gata2 gene.

Figure 4

Reduced HSC numbers in Tx-treated Tg^VE:Gata2^–/fl FLs. FLs recovered from E14.5 embryos that had been exposed to Tx in utero were mechanically disrupted, individually processed, and stained with various antibodies prior to analysis by flow cytometry. (A and B) Representative contour plots for the LSK fraction of 3 Tx-treated Gata2 control (Tg^VE:Gata2^+/fl, Tg^VE:Gata2^+/–, and Gata2^–/fl in A; Gata2^+/+, Tg^VE:Gata2^+/+, and Gata2^–/+ in B) and 3 Tx-treated compound mutant Tg^VE:Gata2^–/fl embryos, which were administered Tx in utero on E9–E11 (A) or E11–E13 (B). The fraction of LSK cells in the gated areas used to quantify the HSC compartment (boxed) is shown. LSK cells were essentially absent in the Tx-treated Tg^VE:Gata2^–/fl FLs whether Cre was induced during or after the initiation of definitive hematopoiesis. The total number of LSK (C) and LSKS (LSK Slam or LSKCD150⁺CD48^–; D) cells recovered from the livers of E14.5 embryos, which had been exposed to Tx from E9 to E11, of various Gata2 genotypes are represented (the ordinate axis is on log scale). Gata2 wild-type and pseudo-wild-type (Gata2^+/+, Tg^VE:Gata2^+/+, Gata2^+/fl [+/+]; n = 6), Gata2 heterozygous (Gata2^–/fl, Tg^VE:Gata2^+/–, Tg^VE:Gata2^+/fl [+/–]; n = 9), and Tg^VE:Gata2^–/fl (Tg:–/fl; n = 4). Data were compiled from two independent experiments; horizontal black bars represent the mean number of LSK (C) or LSKS (D) cells of each group of embryos. Statistical significance was determined by Student’s t test. (E) Prominent mCh expression in FL LSK and LSKS cells. FL cells from Tx-treated (from E9 to E11) E14.5 embryos were analyzed for mCh expression by flow cytometry. A large fraction of E14.5 FL LSK or LSKS cells express mCh in Tg^VE-positive Gata2 wild-type (Tg:+/+; n = 2) as well as Tg^VE-positive Gata2 heterozygous (Tg^VE:Gata2^+/–, Tg^VE:Gata2^+/fl [Tg:+/–]; n = 5) embryos. Compared with the LSK cells recovered from Tg:+/+ and Tg:+/– FLs (2.2 × 10⁴ to 5.7 × 10⁴ and 1.3 × 10⁴ to 4 × 10⁴, respectively), very few LSK cells (98–477) were recovered from Tg^VE:Gata2^–/fl (Tg:–/fl; n = 4) embryos. Compared with the LSKS cells recovered from Tg:+/+ and Tg:+/– FLs (1.2 × 10³ to 4 × 10³ and 0.5 × 10² to 1.6 × 10², respectively), a minuscule number of LSKS cells were recovered from Tg^VE:Gata2^–/fl FLs. Although there appears to be little difference in mCh⁺ LSK and LSKS percentages in Tx-treated Tg^VE:+/+ and Tg^VE:+/– FLs, the absolute number indeed drops by half.

VE-regulated Gata2 loss in Tx-treated mice depletes BM HSCs. To determine whether the Gata2 VE enhancer displays stage-specific differential activity among definitive HSCs, we asked whether the Tg^VE transgene would ablate Gata2 expression in adult BM HSCs as it did in FL HSCs, distinct but closely related definitive HSC populations (34). Gata2^–/fl and Gata2^+/fl adult mice (without or with the Tg^VE, from 8 to 12 weeks of age) were gavaged with Tx (5 mg/d) for 5 consecutive days. Two days after the final Tx administration, BM cells were harvested and analyzed by flow cytometry. Once again, we employed the immunophenotype defined by Kiel et al. (32) to define the most enriched definitive HSC population (LSKS cells).

In comparing the absolute LSK and LSKS cell numbers between Tg^VE:Gata2^+/fl and Tg^VE:Gata2^–/fl adult mice that had not been administered Tx (Figure 5B and data not shown) and between Gata2^+/fl and Gata2^–/fl mice exposed to Tx (Figure 5B and data not shown), we again noted that reduced Gata2 gene dosage correlated perfectly with diminished LSKS cell number. This is reminiscent of the same phenomenon observed in E14.5 FL HSCs (Figure 4) that was also previously reported by others (14). When Gata2^+/fl and Gata2^–/fl mice bearing the Tg^VE were exposed to Tx, we saw a further precipitous decline in the LSKS population as a consequence of reduced GATA-2 activity (Figure 5, A–C). Explicitly, in Tx-treated adult Tg^VE:Gata2^–/fl mice, the LSKS population was depleted by approximately 2 orders of magnitude within a week after the first Tx exposure (Figure 5, B and C). Thus, the Gata2 VE enhancer is functional in both FL and adult BM definitive HSCs, and loss of GATA-2 adversely impacts HSC survival and/or proliferation.

Figure 5

Severely attenuated HSC recovery from Tx-treated Tg^VE:Gata2^–/fl adult BM. (A) Adult mice (8–12 weeks of age) were gavaged with Tx (5 mg/d) or sunflower oil for 5 consecutive days. BM cells were isolated 48 hours after the final Tx administration and then stained with various antibodies directed against cell surface antigens prior to analyses. Representative contour plots for the LSKS (LSK Slam; LSKCD150⁺CD48^–) fractions in Tx-treated control (2 Tg^VE:Gata2^+/fl and 1 Tg^VE:Gata2^+/–) mice and in 3 Tx-treated Tg^VE:Gata2^–/fl compound heterozygous adult mice are shown. The percentage of LSKS cells in each gated area (boxed) is shown. (B) Distribution of highly purified HSCs isolated from Tx-treated (white) or mock-treated (black) control or test mice (2 tibias plus 2 femurs). Gata2^+/fl and Gata2^–/fl adult mice without or with Tg^VE were gavaged with Tx (5 mg/d) or sunflower oil for 5 consecutive days. The 4 groups of mice are designated as follows: Gata2^+/fl, +/fl (n = 2); Tg^VE:Gata2^+/fl, Tg:+/fl (n = 8); Gata2^–/fl, –/fl (n = 3); and Tg^VE:Gata2^–/fl, Tg:–/fl (n = 10). Data were compiled from 3 independent experiments. The ordinate axis is on a log scale; the horizontal bars represent the mean number of LSKS cells in Tx-treated mice of each genotype. Statistical significance was determined by Student’s t test. (C) Absolute number of adult BM LSKS cells in each mouse (2 tibias plus 2 femurs) expressing mCh (ordinate axis is log scale). The number of mCh-expressing cells in the Tg^VE:Gata2^+/fl versus Tg^VE:Gata2^–/fl fractions differs by 2–3 orders of magnitude. The horizontal bars represent the mean number of mCh-positive LSKS cells in Tx-treated mice of each genotype. Statistical significance was determined by Student’s t test.

Defective long-term hematopoietic reconstitution from Tg^VE:Gata2^–/fl FL cells. To functionally detect HSCs in Tx-treated mice, we performed competitive repopulation assays. Total CD45.2 FL cells (5 × 10⁵) from control (Gata2^+/fl [n = 1] or Tg^VE:Gata2^+/+ [n = 3]) or from Tg^VE:Gata2^–/fl (n = 2) E14.5 embryos, all of which had been exposed to Tx from E9 to E11, were coinjected at a 1:1 ratio with wild-type CD45.1 adult BM cells (5 × 10⁵) into lethally irradiated CD45.1 animals (5 mice per group; Figure 6A). Peripheral blood reconstitution from CD45.2 donor FL-derived cells was monitored from week 4 to 16 after transplantation. In all animals receiving Gata2^+/fl (n = 5) or Tg^VE:Gata2^+/+ (n = 15) FL cell transplants, the FL-derived donor cells contributed to the myeloid (>80%), B (>80%), and T (>60%) lymphoid lineages in the peripheral blood (Figure 6B and Supplemental Figure 3A). In contrast, in transplant recipients injected with Tx-treated Tg^VE:Gata2^–/fl FL cells, only a weak multilineage reconstitution was observed in most animals (8 of 10), while some level (<30%) of multilineage reconstitution was seen in the remaining two animals (Figure 6B, Supplemental Figure 3A, and data not shown).

Figure 6

Long-term hematopoietic reconstitution of Tx-treated E14.5 Tg^VE:Gata2^–/fl FL cells is severely compromised. (A) Strategy for competitive reconstitution of lethally irradiated CD45.1 adult mice. 5 × 10⁵ E14.5 total FL cells from control (Gata2^+/fl [n = 1],Tg^VE:Gata2^+/+ [n = 3], or Tg^VE:Gata2^–/fl [n = 2]) Tx-treated CD45.2 embryos were transplanted together with 5 × 10⁵ CD45.1 adult BM cells. FL cells from each embryo were transplanted into 5 recipients. (B) Donor chimerism in the peripheral blood was assessed by flow cytometry using anti-CD45.1–PECy7 and anti-CD45.2–APC antibodies from 4 to 16 weeks after transplantation. CD45.2 donor cell contribution (mean ± SD) from 2 independent experiments is shown. Unlike the robust peripheral blood reconstitution by control FL cells in transplant recipients, Tg^VE:Gata2^–/fl FL cells barely reconstituted their irradiated hosts. Transplant recipients: Gata2^+/fl (n = 5), Tg^VE:Gata2^+/+ (n = 15), Tg^VE:Gata2^–/fl (n = 10). (C and D) Sixteen weeks after transplantation, BM cells were harvested from each mouse (2 tibias plus 2 femurs), and the ratios of CD45.1 to CD45.2 LSKS cells were determined by flow cytometry. Representative contour plots of irradiated CD45.1 recipients that received Gata2^+/fl (+/fl) or Tg:Gata2^–/fl (Tg:–/fl) FL cells are shown (C). Note the conspicuous absence of CD45.2 FL donor-derived LSKS cells in the latter representative recipient. The average ratio of CD45.1 to CD45.2 LSKS cells in transplant recipients that received Gata2^+/fl (+/fl; n = 5), Tg:Gata2^+/+ (Tg:+/+; n = 5), or Tg:Gata2^–/fl (Tg:–/fl; n = 10) is shown (D). Note that in the latter group of recipients, the majority of LSKS cells are not derived from FL. Data represent mean ± SD.

When we analyzed the highly HSC-enriched LSKS fraction in the BM of transplant recipients, we found that in control animals, CD45.2 FL–derived LSKS constituted the major population (60%–80%; Figure 6, C and D). In contrast, 8 of 10 animals that received Tg^VE:Gata2^–/fl FL cells had more than 90% of the LSKS fraction derived from wild-type CD45.1 BM cells. The two remaining transplant animals, in which some CD45.2 progenitor cells were detected in the peripheral blood (Figure 6B and Supplemental Figure 3A), had some (20% and 40%) CD45.2 FL-derived LSKS in their BM (Figure 6, C and D, Supplemental Figure 3B). When we analyzed FACS-sorted CD45.2-positive LSK cells from the latter two animals by qPCR, we found that these cells indeed contained an intact Gata2^fl allele (data not shown), indicating that approximately 20% of LSKS cells per Tg^VE:Gata2^–/fl embryo had escaped VECre-mediated Gata2 inactivation, in good agreement with the efficiency of conditional GATA-2 loss (Figure 3).

Within the adult hematopoietic cell hierarchy, we noted that mCh epifluorescence was absent in mature lymphoid, myeloid, and erythroid lineages isolated from the BM, spleen, or thymi of wild-type adult mice harboring Tg^VE (Figure 7), in stark contrast to endogenous GATA-2 expression in the erythroid and myeloid compartments (35, 36). Most conspicuously, mCh expression was almost entirely restricted to the BM LSK fraction that contained all stem cells. These additional data further underscored the highly restricted and early-acting (within hematopoietic lineages) activity of the Gata2 VE enhancer within the hematopoietic cell hierarchy. Thus, these data demonstrate that induction of VE-regulated Cre leads to functional depletion of HSCs and that the Gata2 VE element possesses HSC- and early progenitor–restricted enhancer activity.

Figure 7

mCh expression is restricted predominantly to the LSK fraction in adult BM. Cells were isolated from BM, spleen, and thymus of wild-type adult mice without Tg^VE (n = 2; gray bars) or hemizygous for Tg^VE (n = 4; black bars) and then stained with various antibodies directed against developmental cell surface antigens prior to flow cytometric analyses. The percentages of mCh-positive cells within the immature (LSK and lin^–Sca-1^–c-Kit^hi) and committed erythroid, myeloid, B lymphoid, or T lymphoid cell compartments are summarized. The latter 4 committed lineages were characterized using pairs of antibodies (CD71 and TER-119, Mac1 and Gr-1, B220 and CD19, CD4 and CD8), respectively. Most conspicuously, mCh is expressed at 30- to 1,000-fold lower abundance in all mature hematopoietic lineages, but is robustly expressed exclusively in adult BM LSK cells.

Physiological consequences of Gata2 conditional loss of function during embryogenesis. We noted that live Tg^VE:Gata2^–/fl embryos began to appear edematous by E13.5, and they invariably developed severe subcutaneous edema, hemorrhage, and anemia and died by E16.5 (Figure 8A, arrows). The identical phenotype was encountered when we employed a second, independent conditional Cre transgenic line (Tg^VE56; Figure 1B and legend to Figure 8). Importantly, we did not observe these phenotypes in Tx-treated embryos that did not bear a Cre transgene, and therefore we were confident that the phenotypes were not due to Tx toxicity; the acquisition of these phenotypes correlated only with Cre induction in the embryos that co-inherited Gata2 null and floxed alleles. Further experiments performed with only a single Tx delivery on day 9 after fertilization or after 3 consecutive daily doses from E11 to E13, instead of the generally employed Tx regimen of 3 consecutive daily doses from E9 to E11, generated the identical phenotypes of anemia, edema, and hemorrhaging in Tg^VE:Gata2^–/fl embryos. PCR genotyping of yolk sac genomic DNAs confirmed the conversion of the Gata2 floxed allele to the Gata2 exon 5–deleted allele (Gata2^Δ, Figure 8B). The Gata2^Δ amplicon was detected exclusively in embryos bearing both the Gata2 floxed allele and Tg^VE from dams that had been treated with Tx. Since CreERT² expression is restricted to endothelial cells in the yolk sac, the (unaltered) Gata2 floxed allele was still detected in total yolk sac genomic DNA of Tx-treated Tg^VE:Gata2^–/fl embryos (Figure 8B). Thus, the final phenotype revealed by employing this binary conditional inactivation strategy was that loss of GATA-2 in the endothelial lineage leads to massive mid-gestational hemorrhage, edema, and anemia, followed shortly thereafter by embryonic demise by around E16.5.

Figure 8

Tx-treated Tg^VE:Gata2^–/fl embryos exhibit anemia, edema, hemorrhage, and late-embryonic lethality. (A) Gata2^+/fl female mice were intercrossed with Tg^VE:Gata2^+/– compound transgenic males. Embryos were collected from pregnant dams that were gavaged with Tx between E9 and E11. Anemia, edema, and hemorrhage were detected in Tx-treated Tg^VE:Gata2^–/fl (Tg:–/f) embryos, but not in any other genotype control littermates, of which some are shown here (Gata2^–/fl, Tg^VE:Gata2^+/–, Tg^VE:Gata2^+/fl [–/f, Tg:+/–, Tg:+/f, respectively]), beginning around E13.5 and progressively increasing in severity before they succumbed to lethality and necrosis by E15.5–E16.5. Arrows: subcutaneous edema in Tg^VE:Gata2^–/fl embryos. Note the visibly paler FLs (magenta asterisks) in Tx-treated E13.5 and E15.5 Tg^VE:Gata2^–/fl embryos in comparison to Tx-treated controls. (A patch of hemorrhage in the E14.5 Tg^VE:Gata2^–/fl embryo visually obscured its pallid liver). These phenotypes were reproducible using either of two transgenic lines (Tg^VE56: E14.5 embryos; Tg^VE62: E13.5 and E15.5 embryos). Further experiments performed with only a single Tx delivery on E9 (data not shown) or after 3 consecutive doses from E11 to E13 instead of E9 to E11 generated the same phenotypes (anemia, edema, hemorrhaging) in Tg^VE:Gata2^–/fl embryos. Scale bar: 0.5 mm. (B) Genotyping of a (large) representative E13.5 litter collected from a Tx-gavaged Gata2^–/fl female intercrossed with a Tg^VE:Gata2^+/fl adult male. Yolk sac genomic DNAs were used in separate PCR reactions to specifically detect the Cre and mCh transgenes (top), 3 Gata2 alleles (fl, wild-type [+], or knockout [–]; middle) and the Cre-mediated Gata2 exon 5–deleted allele (Δ; bottom). The Gata2^Δ amplicon was only detected in embryos bearing both the Gata2^fl allele and Tg^VE. Since CreERT² expression is restricted to endothelial cells in the yolk sac, the PCR amplicon derived from the unrecombined Gata2^fl allele was detected in total yolk sac genomic DNA of Tx-treated Tg^VE:Gata2^–/fl embryos. To generate the Gata2^Δ allele as a positive control, the Gata2^+/fl mouse was interbred with the ubiquitously expressed AyuI-Cre transgenic mouse (21). Sequences of primers used for PCR genotyping are listed in Supplemental Table 1.

We noted massive blood pooling in the vicinity of the jugular lymph sacs in E14.5 Tx-treated mutant, but not control, embryos (Figure 9, A and B). Detailed examination of serial histological sections of E13.5 Tx-treated (from E9 to E11) embryos revealed that in the control embryo, the jugular lymph sac was distinctly separated from the jugular vein, while in the mutant only a single vessel lumen could be found (Figure 9, C–E and I). When we examined serial sections of Tg^VE:Gata2^–/fl embryos more anteriorly and posteriorly, we saw that the lymph sacs appeared hypoplastic compared with that of the control embryo (Figure 9, E and F). Additionally, erythrocytes were aberrantly present in the lymph sacs (Figure 9, G and H) of the E13.5 Tg^VE:Gata2^–/fl mutant embryo, which typically had not presented visibly outward signs of gross hemorrhage by this gestational day (Figure 8). Thus, employing this binary conditional inactivation strategy revealed yet another striking phenotype: GATA-2 function is required for proper lymphatic morphogenesis during embryonic development.

Figure 9

Aberrant lymphatic development in Tg^VE:Gata2^–/fl embryos. (A and B) Tx-treated E14.5 Gata2^+/fl (A) and Tg^VE:Gata2^–/fl (B) embryos were fixed in 4% paraformaldehyde and then gradually cleared in benzyl benzoate:benzyl alcohol for whole-mount photography. Note the blood-engorged jugulo-axillary lymph sac (arrow) in the Tg^VE:Gata2^–/fl, but in not in the control, embryo. (C–F) Transverse paraffin sections (6 μM) of an E13.5 Tx-treated (E9–E11) Tg^VE:Gata2^+/+ (C and E) or Tg^VE:Gata2^–/fl (D and F–J) embryos were stained with H&E. While in the control embryo, the jugular vein (v) is distinctly separated from the jugular lymph sac (ls) at the axial level of the thyroid gland (th, C; an enlarged view of boxed area is shown in E), only a single vessel lumen is observed in the Tx-treated Tg^VE:Gata2^–/fl embryo (D; enlarged view of boxed area is shown in F). C and D were taken at the same magnification; the tissue edema in Tg^VE:Gata2^–/fl embryo did not permit coverage of the entire area in the microscopic field. By following serial sections anteriorly (G and H) and posteriorly (I and J) of the section shown in F, it appeared that the jugular vein remained abnormally connected to the lymphatic system. Note the interstitial edema (indicated by the arrow; B) and the aberrant presence of erythrocytes in the lymph sacs (indicated by asterisks) before the onset of visible hemorrhage (e.g., see Figure 8) of E13.5 Tx-treated Tg^VE:Gata2^–/fl embryo. a, carotid artery; eo, esophagus; sg, sympathetic ganglion; tr, trachea; va, vagal nerve trunk. Scale bars: 1 mm (A and B), 200 μm (C and D), 100 μm (E–J).

The study of Gata2 transcriptional regulation in hematopoiesis has a long and storied history. More than a decade ago, we discovered that a 271-kbp Gata2 yeast artificial chromosome (YAC) transgene, which circumscribed genomic sequences from –198 kbp to +73 kbp (with respect to the translational start site) of the Gata2 locus, could fully rescue the embryonic lethality in Gata2^–/– embryos (3). Hence, this YAC functionally defined the genomic limits of the Gata2 locus that contained all of the regulatory element(s) required for fully elaborating primitive and definitive hematopoiesis, but the YAC only incompletely rescued all of the GATA-2 functions, since the P0 pups died from incomplete patterning of the urogenital system (3, 9, 20).

In contemporaneous studies, Kobayashi-Osaki et al. (37) reported that the Gata2 GATA-2–EHRD element, located 3.1 kbp 5′ to the Gata2 1S first exon, was capable of directing GFP reporter gene expression within the para-aortic splanchnopleura, the immediate developmental precursor of the aorta-gonad-mesonephros (AGM), from which definitive HSCs first emerge in mice (38). This promoter-proximal element was later shown to modulate GATA-2 abundance in E12.5 FL HSCs and its immature progenitor populations, but not in adult HSCs (39).

Suspicions regarding the potential complexity of Gata2 hematopoietic transcriptional regulation were underscored when Bresnick and colleagues identified 5 GATA motif–bearing regions (–77, –3.9, –2.8, –1.8, and +9.5 relative to the Gata2 1S transcription start site) that are bound by GATA-2 but are replaced by GATA-1 during erythroid differentiation (40), suggesting that each of these “GATA switch” sites might also play roles in regulating Gata2 expression during hematopoiesis. When either the –1.8 or –2.8 site was systemically deleted from the germ line, GATA-2 transcripts were altered in HSCs, but this did not translate into a detectable change in HSC number (39, 41); this implies that other regulatory element(s) within the Gata2 locus are responsible for its HSC activity.

In our continuing efforts to elucidate the transcriptional regulation of GATA-2, we have in the past examined reporter gene expression using large (yeast and bacterial artificial chromosomes) and small (plasmid) expression vectors in transgenic studies. As a result of these analyses, we previously identified and characterized an intronic Gata2 VE enhancer predominantly as an endothelial regulatory element using lacZ-tagged plasmid expression vectors in founder transgenic studies (5). The data presented here demonstrate that the Gata2 VE enhancer is transcriptionally active in both FL and adult BM HSCs and that VE-regulated conditional Gata2 loss leads to massive depletion of the most highly purified immunophenotypic fetal and adult definitive HSCs. On closer scrutiny, we noted that VE-regulated mCh expression co-segregated with most (87% ± 6%) of the most highly purified fraction of Tg^VE-bearing wild-type E14.5 FL LSKS HSCs (Figure 4E), while only about 60% of the Tg^VE-bearing wild-type LSKS population recovered from adult BM expressed mCh (Figure 5C). This difference in mCh expression in the most highly purified fraction of FL versus adult BM HSCs could reflect the differential activity of the Gata2 VE enhancer in the HSC cellular milieu at different developmental stages; alternatively, the data could as easily be interpreted to support the hypothesis that the adult BM HSC compartment is intrinsically more heterogeneous than the fetal compartment, a contention strongly supported by recent literature (42–44). The data could also be explained simply as a consequence of diminished levels of mCh expression in adult LSKS HSC or altered stability of the protein in (perhaps longer-lived) adult LSKS cells.

The constitutive loss of GATA-2 that leads to early embryonic lethality by E10.5 has been attributed to primitive hematopoietic failure (2, 3). Further insight into a role for GATA-2 in definitive hematopoiesis was gleaned from the examination of null mutant embryonic stem cells and their contribution to in vitro differentiation or to definitive hematopoiesis in reconstituted chimeras, where it was deductively concluded that GATA-2 is required for the proliferation and/or survival of HSCs (12). More recent analyses of Gata2 heterozygous mutant mice clearly demonstrated a quantitative reduction in HSC number and compromised HSC function in competitive BM reconstitution experiments (13, 14). Here, using a combinatorial conditional loss-of-function strategy, we were able to directly and formally demonstrate that GATA-2 is vital for immunophenotypic as well as functional HSC recovery from both mid-gestational embryos and adults. In light of the negligible effects of the –1.8 and –2.8 kb promoter Gata2 mutations on HSCs (above), it will be interesting to determine the consequence of deleting the Gata2 VE enhancer singly or in combination with other putative Gata2 regulatory elements to ascertain their individual and combinatorial contribution to Gata2 transcription in HSCs and, in particular, whether the VE HSC activity described here is a unique or redundant function within the locus.

In our previous and in current studies, we noted the presence of X-gal–stained hematopoietic cells that appeared to be budding into the lumen of the dorsal aorta in E10.5 embryos, possibly hinting that this enhancer might be active in hemogenic endothelium or hemangioblasts, the presumptive progenitor cell common to the endothelial and hematopoietic lineages. Indeed, using blast colony–forming cells derived from embryonic stem cells, Lugus et al. showed that temporally sensitive, conditionally forced induction of GATA-2 led to a transient expansion of hemangioblasts in cell culture (45). Whether it is required for the development of an even earlier-stage, common progenitor cell (30, 45) will require additional study.

While GATA-2 appears not to be required for endothelial patterning into the initial primary vasculature (as evident in E10.5 Gata2 homozygous null embryos), it may be necessary for the elaboration of the mature vasculature later during embryogenesis. At the time of embryonic demise at E16.5, the binary conditionally inactivated Gata2 mutant mice displayed severe subcutaneous edema and extensive hemorrhage. We show that the VE is active in the cardinal vein, where endothelial precursor cells with potential to differentiate into LECs are generated (26), and in nascent LECs that have sprouted from the cardinal vein (5). Robust GATA-2 expression has also been reported in E16.5 and adult murine lymphatic vessel and lymphatic valves (17). The abnormal accumulation of blood in the jugular lymph sacs in Tx-treated Tg^VE:Gata2^–/fl embryos prompted us to histologically examine their embryonic lymphatic vasculature. Curiously, we found that in Tx-treated E13.5 Tg^VE:Gata2^–/fl embryos, the lymphatic and blood vascular systems remained aberrantly connected, unlike in control embryos. Interestingly, there have been several recent reports of primary lymphedema in patients with either Emberger or monocytopenia and mycobacterial infection (MonoMAC) syndrome due to GATA2 mutations (16–18).

Much progress has been made over the last decade with regard to understanding embryonic lymphatic vascular development, and numerous genes that impact this morphogenic process have now been identified (46). In particular, the blood-lymphatic mixing phenotype had been reported in mice with mutations that affect podoplanin (47), Ruxn1 (26), Meis1 (48), Syk (49), Slp76 (50), and Plcg2 (51). A number of these mutants have platelet deficiency or dysfunction, as there is accumulating evidence that platelets play a central role in lympho-venous partitioning (46). Whether this pathophysiological developmental aberration in Tx-treated Tg^VE:Gata2^–/fl embryos is due to loss of GATA-2 expression in the hematopoietic and/or lymphatic endothelial lineage will require further investigation. It will be of interest to now investigate the role and mechanism(s) by which GATA-2 intersects with the developmental programs specifying lymphangiogenic differentiation.

View Supplemental data

We gratefully acknowledge the helpful advice and assistance of, and spirited discussion with, members of our laboratory, as well as our colleagues Stephen J. Weiss, Sean Morrison, Jun-Lin Guan, and Ivan Maillard for critical evaluation of the manuscript. This work was supported initially by NIH grant R01 GM28896 and subsequently by the G. Carl Huber Professorship and NIH grant R01 AI94642 (to J.D. Engel).

Address correspondence to: James Douglas Engel, The University of Michigan Medical School, Cell and Developmental Biology, 109 Zina Pitcher Place, 3072 BSRB, Ann Arbor, Michigan 48109-2200, USA. Phone: 734.615.7509; Fax: 734.615.8500; E-mail: engel@umich.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2012;122(10):3705–3717. doi:10.1172/JCI61619.

See the related article at Cis-element mutated in GATA2-dependent immunodeficiency governs hematopoiesis and vascular integrity.

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