Spred-2 Suppresses Aorta-Gonad-Mesonephros Hematopoiesis by Inhibiting MAP Kinase Activation

Hematopoietic cells are derived from multipotent progenitors, hematopoietic stem cells (HSCs). During embryogenesis, development of the hematopoietic system is dependent on a hierarchical mechanism, which is influenced by environmental niches, having contacts with stromal cells and extracellular signals including cytokines, thereby inducing expression of unique transcription factors via an intracellular signaling pathway. In the midgestation mouse embryo, definitive hematopoiesis occurs in the aorta-gonad-mesonephros (AGM) region, which contains a population that is able to repopulate irradiated mice upon direct transplantation (1, 2). Within the AGM region, the first HSCs are generated in the dorsal aorta (3). More recently, endothelial cells that overlap with expression of HSCs markers (Sca-1 and c-Kit) in midgestation mouse aorta have long-term reconstitution activities (3–5). However, intracellular signals that regulate this hematopoiesis have not been clarified.

Recently, we cloned a family of novel membrane-bound molecules, Spred-1, Spred-2, and Spred-3 (6, 7), which are related to Sprouty family proteins that act as negative regulators during development (8, 9). Spred-1, Spred-2, and Spred-3 inhibit the Ras/mitogen-activated protein (MAP) kinase cascade mediated by fibroblast growth factor receptor and epidermal growth factor receptor, by binding to Ras and consequently inhibiting phosphorylation of Raf. Spred proteins have a negative effect on extracellular signal–regulated kinase (Erk)-dependent differentiation in PC12 pheochromocytoma cells and C2C12 myoblastic cells (7). The physiological functions of Spred proteins, however, remain to be elucidated. In this study, we demonstrated the importance of Spred-2 for hematopoiesis in the midgestation mouse embryo and, in part, in adult bone marrow.

A targeting vector was constructed to delete exon 6 of Spred-2–encoding KBD and SPR domains. Targeting of embryonic stem cells and the generation of chimeric mice were performed according to a previously published procedure (10). Detailed analysis of embryonic stem clones and Spred-2–null mice will be published elsewhere.

AGM cultures and semisolid colony-forming assay were performed as previously described (11).

5 μg total RNA isolated from E11.5 AGM was reverse transcribed with Superscript II (GIBCO BRL). PCRs were performed using rTaq (Takara Biotechnology Inc.) with the following settings: 95°C for 3 min, 26 cycles at 95°C for 10 s, 55°C for 10 s, and 72°C for 1 min. The primer sets used were as follows: 5′-TGTGAGCACCGGAAGATTTATACC-3′, 5′-CGCGGCGGCTTTGTGCTT-3′ (for Spred-2); 5′-AGCACTGATTATATTCCTGG-3′, 5′-TGCTTGGCCAGTATTCTGCG-3′ (for CD45); 5′-ACCACCCGATACCCACCTAT-3′, 5′-GCCATGGCAGTCACCATGCT-3′ (for GATA-2); and 5′-ACCACAGTCCATGCCATCAC-3′, 5′-TCCACCACCCTGTTGCTGTA-3′ (for G3PDH).

We prepared mutant proteins of Spred-2 (see Fig. 2 B): ΔN, with the NH₂-terminal EVH-1 domain (residues 1–139) deletion; ΔC, with the COOH-terminal SPR domain (residues 289–410) deletion; and ΔKBD, with the c-Kit–binding domain (residues 197–287) deletion. Production of retroviruses and infection of AGM cultures were performed as previously described (11).

The nonadherent cells, which were recovered from the AGM culture, were stained with PE-conjugated rat anti–mouse CD45 (30-F11; Becton Dickinson) for 30 min on ice. After washing, stained cells were analyzed by FACSCalibur™ (Becton Dickinson). The percentage of CD45⁺ cells in the virus-infected green fluorescent protein (GFP)⁺ cells was determined.

Nonadherent cells derived from the mock-infected AGM culture were stained with PE-conjugated anti–mouse CD45 (30-F11) and allophycocyanin-conjugated anti–mouse c-Kit (2B8; Becton Dickinson). GFP⁺ CD45⁻ c-Kit⁻, GFP⁺ CD45^low c-Kit^low/+, and GFP⁺ CD45⁺ c-Kit⁻ cells were sorted by FACS Vantage™ (Becton Dickinson). Each of sorted cells (2 × 10³) in each population were seeded on OP9 stromal cells in DMEM supplemented with 10% (vol/vol) fetal calf serum, 100 ng/ml IL-3, 100 ng/ml stem cell factor (SCF), and 4 U/ml erythropoietin (EPO).

Spred-2^+/+ and Spred-2^−/− E10.5 embryonic trunk lower than the heart was incubated in dispase II (Roche) at 37°C for 20 min. After washing in PBS, cells were treated with cell dissociation buffer (Invitrogen) at 37°C for 20 min. Dissociated cells were stained with anti–mouse vascular endothelial-cadherin (VE-cad; 11D4.1; Becton Dickinson). 2 × 10³ sorted VE-cad⁺ cells were seeded on OP9 stromal cells in the medium as described above. After 3 d, cobble stone area–forming cells were counted.

2 × 10⁶ Plat-E cells (12) were transfected with expression constructs using a Trans-IT 293 (Mirus). On the next day, cells were starved for 16 h and then treated with 100 ng/ml SCF for 5 min. Cell lysates were immunoprecipitated with anti-GFP antibody (Medical & Biological Laboratories, Co.). Immunoprecipitates or cell lysates were separated by SDS-PAGE and immunoblotted with anti–phospho-Erk1/2 antibody (E10; Cell Signaling Technology), anti-Erk2 antibody (sc-145; Santa Cruz Biotechnology, Inc.), anti–c-Kit (M-14; Santa Cruz Biotechnology, Inc.), and anti-Flag (M2; Sigma-Aldrich) antibody.

AGM cells cultured for 2 d were treated with 0, 1, 3, or 10 μM U0126 (Cell Signaling Technology) or SB203538 (Calbiochem). After 8 d of treatment, the nonadherent cells were counted and analyzed for determination of the percentage of CD45⁺ cells.

Inhibition of SCF-induced Erk phosphorylation and Elk-1 activation by Spred-2 mutants is presented in Fig. S1. Fig. S1 and Supplemental Materials and Methods are available.

First, to examine any possible role that the Ras/MAP kinase pathway has in AGM hematopoiesis at a midgestation stage, we examined the effect of U0126, a MEK inhibitor, on the appearance of CD45⁺ hematopoietic lineage cells from E11.5 primary AGM culture. In the AGM culture with SCF, basic fibroblast growth factor (bFGF), and oncostatin M (OSM), endothelial-like cells are initially observed and then the nonadherent cells, which include lineage-committed cells (e.g., CD45⁺), gradually emerge (13, 14). As shown in Fig. 1 A, U0126 dose dependently inhibited the production of CD45⁺ hematopoietic cells, whereas SB203538, a p38 inhibitor, had no effect. Next, we tested the effect of a dominant negative Ras (Ras-17N) and a dominant negative mutant of Akt (K197A/T308A/S473A; Akt-DN; reference 15), which is known to function downstream of the PI3K pathway on the generation of CD45⁺ cells in the AGM culture. We used bicistronic retrovirus vector pMY-IRES-EGFP so that the infected cells could be monitored by measuring expression of an enhanced GFP by flow cytometry. As shown in Fig. 1 B, overexpression of Ras-17N reduced the production of CD45⁺ cells, whereas Akt-DN did not strongly affect the production of CD45⁺ cells in the culture containing SCF, bFGF, and OSM (Fig. 1 B). Similar results were observed for stimulation with SCF or bFGF alone (Fig. 1 B). These data indicate that the Ras/MAP kinase cascade plays an essential role in AGM hematopoiesis.

Next, we attempted to define the role of Spreds in AGM hematopoiesis. Among Spred family proteins, we focused on Spred-2 because it has been shown to negatively regulate c-Kit signaling (16). We first examined Spred-2 expression in the AGM region. Hematopoietic (positive for either CD45 or Ter119) and nonhematopoietic (negative for both CD45 and Ter119) cells were prepared from E11.5 AGM, and RNA from each population was subjected to RT-PCR. As shown in Fig. 2 A, transcripts for Spred-2 were detectable in both populations, whereas those for hematopoietic transcription factor GATA-2 were detected only in the former population. Spred-2 protein expressed in the AGM region may play a role in the hematopoiesis in midgestation mouse embryo.

To examine whether the production of hematopoietic cells in the AGM culture was regulated by Spred-2, wild-type and truncated forms of Spred-2 and related molecules (Fig. 2 B) were overexpressed in cultured AGM cells by retrovirus gene transfer. As shown in Fig. 2 C, full-length Spred-2 exhibited a significant inhibitory effect on the appearance of CD45⁺nonadherent cells in the AGM cultures. In contrast, neither Sprouty-4, which is related to the Spred proteins, nor STAP-1, which interacts with c-Kit (17), was able to inhibit the appearance of CD45⁺cells from cultured AGM cells (Fig. 2, C and D, and unpublished data). It has been shown that Sprouty-4 has the inhibitory effect on bFGF-induced Erk phosphorylation, but not epidermal growth factor– and vascular endothelial growth factor (VEGF)-induced Erk phosphorylation (7, 18, 19).

Next, we examined the function of each domain in the Spred-2 protein by using deletion mutants. ΔC could not inhibit the production of CD45⁺ cells in AGM cultures, whereas ΔN and ΔKBD exhibited inhibitory effect though the latter showed weaker effect (Fig. 2, C and D). These results suggest that the SPR domain of Spred-2 plays a critical role in the suppression of hematopoietic cell development in AGM cultures. We then examined the role of each domain of Spred-2 in the SCF/c-Kit signaling pathway. Full-length Spred-2 and ΔN interacted with c-Kit upon SCF stimulation of AGM cells. ΔC and ΔKBD showed no detectable binding to c-Kit (Fig. S1 A). SCF-induced Erk phosphorylation was suppressed by Spred-2, ΔN, and ΔKBD, though the suppression by the latter two was incomplete. By contrast, ΔC and Sprouty-4 show little or almost no inhibition of Erk activation (Fig. 2 E). Similar results were obtained by SCF-induced Elk-1 activation in 293 cells (Fig. S1 B). These results indicated that the COOH-terminal region of Spred-2 is required for the interaction with c-Kit and for efficient suppression of SCF-induced Erk-2 phosphorylation. In a previous study, we found that the COOH-terminal region of Spred-2 or Sprouty alone was sufficient for the suppression of Erk activation induced by VEGF in 293 cells (18). Therefore, VEGF and SCF may have a similar activation mechanism for MAP kinase. It is interesting to note that Spred-2•ΔN does not inhibit Erk activation induced by epidermal growth factor or nerve growth factor (16).

Next, we sought to examine the role of Spred-2 in the development of hematopoietic lineage cells expressing CD45. Nonadherent cells from the AGM culture, which had been infected with mock viruses, were gated for GFP expression and then analyzed for CD45 and c-Kit expression. As shown in Fig. 2 F, middle, the cells could be divided into three populations: CD45⁻ c-Kit⁻, CD45^low c-Kit^low/+, and CD45⁺ c-Kit⁻. In Spred-2 virus–infected AGM cells (Fig. 2 F, right), the size of the CD45^low c-Kit^low/+ population was dramatically reduced (6.4-fold) and that of the CD45⁺ c-Kit⁻ population was moderately reduced (2.5-fold). To characterize the nature of the CD45^low c-Kit^low/+ population, which was dramatically affected by Spred-2 expression, the above mentioned three populations of cells were cultured on the monolayer of a stromal cell line, OP9. As shown in Fig. 2 G, only the CD45^low c-Kit^low/+ population produced nonadherent hematopoietic cells, indicating that the CD45^low c-Kit^low/+ population could be in a more immature stage and that the other two populations could not produce hematopoietic cells on OP9. These results suggest that Spred-2 is a negative regulator of hematopoiesis in AGM.

To define the role of endogenous Spred-2 in hematopoiesis, we generated mice having Spred-2 gene mutation by homologous recombination, in which exons encoding the KBD and SPR domains were deleted. Offspring were born within the Mendelian expectation ratio from intercrosses of heterozygotes as well as incrosses of homozygotes. This indicates that Spred-2 is not necessary for fertility and development. Adult Spred-2^−/− mice appeared to be healthy and showed no apparent abnormalities in most organs (unpublished data).

To examine the effect of Spred-2 deficiency on AGM hematopoiesis at the midgestation stage, we compared the colony-forming activity in methylcellulose of E11.5 aortic cells from Spred-2^−/− with that from wild-type littermates (Fig. 3 A, +/+). The number of granulocyte and macrophage colonies formed from Spred-2^−/− aortic cells was significantly higher than that from Spred-2^+/+ littermate aortic cells, indicating that Spred-2 has an inhibitory effect on colony-forming potential of hematopoietic progenitors. Next, we examined the effect of Spred-2 deficiency on embryonic hematopoiesis using the AGM culture. As shown in Fig. 3 B, the number of nonadherent cells produced in the culture of AGM from Spred-2^−/− was greater than that from Spred-2^+/+ littermates, and the ability of the AGM region of Spred-2^−/− to generate CD45⁺hematopoietic cells in this culture was threefold higher than that of Spred-2^+/+ (Fig. 3 C). These results indicate that loss of Spred-2 function leads to an increase in hematopoiesis in the AGM region of midgestation mouse.

It has been reported that hematopoietic cells develop from VE-cad⁺ cells (20). When VE-cad⁺ cells in E10.5 embryo were cultured on a monolayer of OP9 stromal cells, we observed cobble stone area–forming cells, which are suggested to produce HSCs (21). The number of cobble stone areas found in the cultured VE-cad⁺ cells from E10.5 Spred-2^−/− embryo was 2.2-fold larger than that from wild-type littermates (Fig. 3 D, +/+). Colony-forming ability of VE-cad⁺ cells sorted from E10.5 Spred-2^−/− and Spred-2^+/+ littermates was assayed in methylcellulose media. As shown in Fig. 3 E, VE-cad⁺ cells from Spred-2^−/− formed a 3.6-fold larger number of colonies. These results suggest that Spred-2 negatively regulates hematopoiesis in AGM.

Finally, we examined whether a deficiency of Spred-2 causes hematopoietic abnormality in adult mice. As shown in Fig. 4 A, marked increase in the number of megakaryocytes was observed in the spleen of Spred-2–deficient mice, although no qualitative or quantitative abnormalities in peripheral blood hematopoietic cells were observed. Colony-forming assay of adult bone marrow cells in methylcellulose revealed that approximately twofold more hematopoietic cell colonies were produced from bone marrow cells of Spred-2^−/− mice (381.9 ± 33.1 colonies/3 × 10⁴ cells) as compared with those from wild-type littermates (250.7 ± 18.9 colonies/3 × 10⁴ cells; Fig. 4 B). These data suggest that Spred-2 is at least in part involved in the regulation of hematopoiesis in vivo.

In this study, Spred-2–deficient mice displayed an enhanced ability of their AGM cells to differentiate into hematopoietic cells at ontogeny. The phenotype in Spred-2–deficient mice resembles that in Lnk-deficient mice whose E11.5 AGM exhibited more potent capacity of hematopoietic differentiation in the culture and in the colony-forming assay (11). It has been reported that a stable transformant of mast cell line MC9 with Lnk cDNA showed a marked attenuation of the SCF-mediated phosphorylation of Gab-2 and the subsequent phosphorylation of Erk-1/2 using the mast cell line MC9 (22). Thus, these proteins having a negative effect on the Ras/MAP kinase cascade demonstrate inhibitory activity on AGM hematopoiesis. On the other hand, Spred-2 deficiency caused a significant increase in the number of megakaryocytes in the adult spleen (Fig. 4 A). This phenotype is very similar to that observed in mice with homozygous deletion of Lnk (23). It should be noted that the maturation of progenitor cells to megakaryocytes is induced by activation of Erk MAP kinase, which is consistent with our finding (24, 25). Therefore, elevation of megakaryopoiesis in the spleen of Spred-2^−/− mice supports our idea that Spred-2 is a negative regulator of the Ras/MAP kinase pathway in hematopoiesis.

In conclusion, here we have shown that Spred-2 plays an inhibitory role in AGM hematopoiesis. Our results suggest that Spred-2 has negative effects on differentiation of vascular endothelial cells into hematopoietic cells (Fig. 3, D and E). To further support our idea, time lapse analysis or single cell fate analysis of VE-cad⁺ cells may need to be undertaken.

We thank Dr. Y. Gotoh for providing Akt-DN and GFP-Erk2 cDNAs, Dr. Y. Kanakura for c-Kit cDNAs, Dr. T. Kitamura for pMY-IRES-GFP and Plat-E cells, Dr. T. Nakano for OP9 cells, and Drs. T. Kondo, T. Kagawa, and K. Nakashima for valuable discussions. We also thank Ms. Y. Noguchi and Ms. M. Ohta for secretarial assistance, and Mr. N. Ohtsu, Ms. K. Kaneko, and Ms. Y. Kawabata for technical help.

Part of this work was supported by Grant-in-Aid for 21st Century COE Research from Ministry of Education, Culture, Sports, Science and Technology “Cell Fate Regulation Research and Education Unit” (to T. Taga), grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan (to I. Nobuhisa, A. Yoshimura, and T. Taga), the Japan Health Science Foundation (to A. Yoshimura), the Human Frontier Science Program (to A. Yoshimura and T. Taga), the Japan Research Foundation for Clinical Pharmacology (to A. Yoshimura), Uehara Memorial Foundation (to A. Yoshimura), and the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim (to T. Taga).