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A large gap exists in our understanding of the course of differentiation from mesoderm to definitive hematopoietic stem cells (HSCs). Previously, we reported that Runx1+cells in embryonic day 7.5 (E7.5) embryos contribute to the hemogenic endothelium in the E10.5 aorta-gonad-mesonephros (AGM) region and HSCs in the adult bone marrow. Here, we show that two Runx1+populations subdivided by Gata1 expression exist in E7.5 embryos. The hemogenic endothelium and the HSCs are derived only from the Runx1+Gata1−population. A subset of this population moves from the extra- to intraembryonic region during E7.5–E8.0, where it contributes to the hemogenic endothelium of the dorsal aorta (DA). Migration occurs before the heartbeat is initiated, and it is independent of circulation. This suggests a developmental trajectory from Runx1+cells in the E7.5 extraembryonic region to definitive HSCs via the hemogenic endothelium.

Ets family protein Etv2 (also called ER71 or Etsrp) is a key factor for initiation of vascular and blood development from mesodermal cells. However, regulatory mechanisms and inducing signals for Etv2 expression have been largely unknown. Previously, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling enhanced differentiation of vascular progenitors into endothelial cells (ECs) and hematopoietic cells (HPCs) using an embryonic stem cell (ESC) differentiation system. Here, we show that PKA activation in an earlier differentiation stage can trigger EC/HPC differentiation through Etv2 induction. We found Etv2 was markedly upregulated by PKA activation preceding EC and HPC differentiation. We identified two cAMP response element (CRE) sequences in the Etv2 promoter and 5′‐untranslated region and confirmed that CRE‐binding protein (CREB) directly binds to the CRE sites and activates Etv2 transcription. Expression of a dominant negative form of CREB completely inhibited PKA‐elicited Etv2 expression and induction of EC/HPCs from ESCs. Furthermore, blockade of PKA significantly inhibited Etv2 expression in ex vivo whole‐embryo culture using Etv2‐Venus knockin mice. These data indicated that PKA/CREB pathway is a critical regulator for the initiation of EC/HPC differentiation via Etv2 transcription. This early‐stage molecular linkage between a triggering signal and transcriptional cascades for differentiation would provide novel insights in vascular and blood development and cell fate determination. STEMCELLS2012; 30:687–696

Etv2 (Ets Variant 2) has been shown to be an indispensable gene for the development of hematopoietic cells (HPCs)/endothelial cells (ECs). However, how Etv2 specifies the mesoderm-generating HPCs/ECs remains incompletely understood. In embryonic stem cell (ESC) differentiation culture and Etv2-null embryos, we show that Etv2 is dispensable for generating primitive Flk-1+/PDGFRα+ mesoderm but is required for the progression of Flk-1+/PDGFRα+ cells into vascular/hematopoietic mesoderm. Etv2-null ESCs and embryonic cells were arrested as Flk-1+/PDGFRα+ and failed to generate Flk-1+/PDGFRα− mesoderm. Flk-1+/Etv2+ early embryonic cells showed significantly higher hemato-endothelial potential than the Flk-1+/Etv2− population, suggesting that Etv2 specifies a hemato-endothelial subset of Flk-1+ mesoderm. Critical hemato-endothelial genes were severely down-regulated in Etv2-null Flk-1+ cells. Among those genes Scl, Fli1, and GATA2 were expressed simultaneously with Etv2 in early embryos and seemed to be critical targets. Etv2 reexpression in Etv2-null cells restored the development of CD41+, CD45+, and VE-cadherin+ cells. Expression of Scl or Fli1 alone could also restore HPCs/ECs in the Etv2-null background, indicating that these 2 genes are critical downstream targets. Furthermore, VEGF induced Etv2 potently and rapidly in Flk-1+ mesoderm. We propose that Flk-1+/PDGFRα+ primitive mesoderm is committed into Flk-1+/PDGFRα− vascular mesoderm through Etv2 and that up-regulation of Etv2 by VEGF promotes this commitment.

Adult stem cells, which are characterized by their capacity for self‐renewal and differentiation, participate in tissue homeostasis and response to injury. They are thought to enter a state of relative quiescence, known as reversible cell cycle arrest, but the underlying molecular mechanisms remain poorly characterized. Previous data from our laboratory has shown that housekeeping gene expression is downregulated in melanocyte stem cells (MelSCs), suggesting a global suppression of mRNA transcription. We now show, using antibodies against specific phosphorylated forms of RNA polymerase II (RNApII), that adult MelSCs do not undergo productive mRNA transcription elongation, while RNApII is activated and initialized, ready to synthesize mRNA upon stimulation, and that the RNApII kinase CDK9 is absent in adult MelSCs. Interestingly, other adult stem cells also, including keratinocyte, muscle, spermatogonia, and hematopoietic stem cells, showed a similar absence of RNApII phosphorylation. Although it is difficult to show the functional significance of this observation in vivo, CDK9 inhibition resulted in enhanced survival of cells that are deprived from survival factors. We conclude that the absence of productive mRNA transcription is an early, specific, and conserved characteristic of adult stem cells. Downregulation of mRNA transcription may lead to decreased rates of metabolism, and protection from cellular and genetic damage. Screening heterogeneous tissues, including tumors, for transcriptionally quiescent cells may result in the identification of cells with stem cell‐like phenotypes. STEMCELLS2010; 28:1571–1580.

Hematopoietic stem cell (HSC) proliferation is tightly regulated by a poorly understood complex of positive and negative cell-cycle regulatory mechanisms. Necdin (Ndn) is an evolutionally conserved multifunctional protein that has been implicated in cell-cycle regulation of neuronal cells. Here, we provide evidence that necdin plays an important role in restricting excessive HSC proliferation during hematopoietic regeneration. We identify Ndn as being preferentially expressed in the HSC population on the basis of gene expression profiling and demonstrate that mice deficient in Ndn show accelerated recovery of the hematopoietic system after myelosuppressive injury, whereas no overt abnormality is seen in steady-state hematopoiesis. In parallel, after myelosuppression, Ndn-deficient mice exhibit an enhanced number of proliferating HSCs. Based on these findings, we propose that necdin functions in a negative feedback loop that prevents excessive proliferation of HSCs during hematopoietic regeneration. These data suggest that the inhibition of necdin after clinical myelosuppressive treatment (eg, chemotherapy, HSC transplantation) may provide therapeutic benefits by accelerating hematologic recovery.

Hematopoietic stem cell (HSC) proliferation is tightly regulated by a poorly understood complex of positive and negative cell-cycle regulatory mechanisms. Necdin (Ndn) is an evolutionally conserved multifunctional protein that has been implicated in cell-cycle regulation of neuronal cells. Here, we provide evidence that necdin plays an important role in restricting excessive HSC proliferation during hematopoietic regeneration. We identify Ndnas being preferentially expressed in the HSC population on the basis of gene expression profiling and demonstrate that mice deficient in Ndnshow accelerated recovery of the hematopoietic system after myelosuppressive injury, whereas no overt abnormality is seen in steady-state hematopoiesis. In parallel, after myelosuppression, Ndn-deficient mice exhibit an enhanced number of proliferating HSCs. Based on these findings, we propose that necdin functions in a negative feedback loop that prevents excessive proliferation of HSCs during hematopoietic regeneration. These data suggest that the inhibition of necdin after clinical myelosuppressive treatment (eg, chemotherapy, HSC transplantation) may provide therapeutic benefits by accelerating hematologic recovery.

Nanog is a key molecule involved in the maintenance of the self-renewal of undifferentiated embryonic stem (ES) cells. In this work we investigate whether Nanog can enhance self-renewal in hematopoietic stem cells. Contrary to our expectation, no positive effect of Nanog transduction was detected in bone marrow reconstitution assays. However, recipients of Nanog-transduced (Nanog) hematopoietic stem cells (HSCs) invariantly develop a unique disorder typified by an atrophic thymus occupied by Nanog-expressing γδT-cell receptor–positive (TCR+) cells (Nanog T cells). All thymi are eventually occupied by Nanog T cells with CD25+CD44+ surface phenotype that home selectively to the thymus on transfer and suppress normal thymocyte development, which is partly ascribed to destruction of the microenvironment in the thymus cortex. Moreover, this initial disorder invariantly develops to a lymphoproliferative disorder, in which Nanog T cells undergo unlimited proliferation in the peripheral lymphoid tissues and eventually kill the host. This invariable end result suggests that Nanog is a candidate oncogene for γδT-cell malignancy.

Nanog is a key molecule involved in the maintenance of the self-renewal of undifferentiated embryonic stem (ES) cells. In this work we investigate whether Nanog can enhance self-renewal in hematopoietic stem cells. Contrary to our expectation, no positive effect of Nanog transduction was detected in bone marrow reconstitution assays. However, recipients of Nanog-transduced (Nanog) hematopoietic stem cells (HSCs) invariantly develop a unique disorder typified by an atrophic thymus occupied by Nanog-expressing γδT-cell receptor–positive (TCR+) cells (Nanog T cells). All thymi are eventually occupied by Nanog T cells with CD25+CD44+surface phenotype that home selectively to the thymus on transfer and suppress normal thymocyte development, which is partly ascribed to destruction of the microenvironment in the thymus cortex. Moreover, this initial disorder invariantly develops to a lymphoproliferative disorder, in which Nanog T cells undergo unlimited proliferation in the peripheral lymphoid tissues and eventually kill the host. This invariable end result suggests that Nanog is a candidate oncogene for γδT-cell malignancy.

During early mouse embryogenesis, fetal liver kinase-1 (Flk-1), a receptor for vascular endothelial growth factor, and Runx1, a runt domain transcription factor, have prerequisite roles in the generation of hematopoietic lineages. Flk-1 expression is maintained in successive stages from mesodermal to endothelial cells and is down-regulated in nascent hematopoietic cells, whereas Runx1 (Runt-related transcription factor 1) is expressed in embryonic sites of hematopoietic cell de novo generation and in practically all hematopoietic organs. Here we show that Runx1 represses Flk-1 during the development of hemogenic endothelial cells into hematopoietic cells. We established embryonic stem cell clones carrying the Venus gene, a modified version of yellow fluorescence protein, in the Runx1 locus and cultured them on OP9 cells. Flk-1+ cells appeared on day 3.5, and Runx1+ cells first appeared from the Flk-1+ fraction on day 4.5. The Flk-1+Runx1+ cells rapidly stopped expressing Flk-1 with further incubation and eventually gave rise to CD45+ or TER119+ cells. Runx1 repressed Flk-1 promoter transcriptional activity in an endothelial cell line, and this repression required intact DNA-binding and transactivating domains of Runx1 protein. The repressor activity of Runx1 endogenous Flk-1 was also confirmed overexpressing Runx1 in embryonic stem cell differentiation cultures. These results provide novel insight into the role Runx1 during the development of hematopoietic cell lineages.

During early mouse embryogenesis, fetal liver kinase-1 (Flk-1), a receptor for vascular endothelial growth factor, and Runx1, a runt domain transcription factor, have prerequisite roles in the generation of hematopoietic lineages. Flk-1 expression is maintained in successive stages from mesodermal to endothelial cells and is down-regulated in nascent hematopoietic cells, whereas Runx1 (Runt-related transcription factor 1) is expressed in embryonic sites of hematopoietic cell de novo generation and in practically all hematopoietic organs. Here we show that Runx1 represses Flk-1 during the development of hemogenic endothelial cells into hematopoietic cells. We established embryonic stem cell clones carrying the Venusgene, a modified version of yellow fluorescence protein, in the Runx1locus and cultured them on OP9 cells. Flk-1+cells appeared on day 3.5, and Runx1+cells first appeared from the Flk-1+fraction on day 4.5. The Flk-1+Runx1+cells rapidly stopped expressing Flk-1 with further incubation and eventually gave rise to CD45+or TER119+cells. Runx1 repressed Flk-1 promoter transcriptional activity in an endothelial cell line, and this repression required intact DNA-binding and transactivating domains of Runx1 protein. The repressor activity of Runx1 endogenous Flk-1 was also confirmed overexpressing Runx1 in embryonic stem cell differentiation cultures. These results provide novel insight into the role Runx1 during the development of hematopoietic cell lineages.

To develop a tool to obtain a high level of gene expression specifically in endothelial cells (ECs), we assessed enhancer activity of fragments in the first intron of the VE-cadherin gene using 3 different experimental systems: luciferase assay in the F2 EC line, green fluorescent protein (GFP) expression in ECs generated in embryonic stem (ES) cell differentiation culture, and GFP expression in transgenic mice. Although the 2.5-kbp (kilobase pair) 5′ flanking sequence of the VE-cadherin gene is EC specific, adding 4 kbp of the 5′ half of the first intron affected an enhancement of the gene expression level in all 3 assay systems. No other fragments tested in this study could confer such effects. Compared with other gene expression units, the unit described in this study would be the most optimum one available to date for EC-specific gene expression. Because this unit can express genes in VE-cadherin+progenitors of hematopoietic cells but not in fully committed hematopoietic cells, it will be useful to manipulate specifically the uncommitted progenitor stage during hematopoietic cell differentiation. (Blood. 2005; 105:4657-4663)

To develop a tool to obtain a high level of gene expression specifically in endothelial cells (ECs), we assessed enhancer activity of fragments in the first intron of the VE-cadherin gene using 3 different experimental systems: luciferase assay in the F2 EC line, green fluorescent protein (GFP) expression in ECs generated in embryonic stem (ES) cell differentiation culture, and GFP expression in transgenic mice. Although the 2.5-kbp (kilobase pair) 5′ flanking sequence of the VE-cadherin gene is EC specific, adding 4 kbp of the 5′ half of the first intron affected an enhancement of the gene expression level in all 3 assay systems. No other fragments tested in this study could confer such effects. Compared with other gene expression units, the unit described in this study would be the most optimum one available to date for EC-specific gene expression. Because this unit can express genes in VE-cadherin+ progenitors of hematopoietic cells but not in fully committed hematopoietic cells, it will be useful to manipulate specifically the uncommitted progenitor stage during hematopoietic cell differentiation. (Blood. 2005; 105:4657-4663)

The aorta-gonads-mesonephros (AGM) region of the mouse embryo has been assigned as the origin of definitive hematopoiesis. The transcription factor GATA-2 has specific but unclarified roles in early hematopoiesis. To elucidate the expression profile of GATA-2, we prepared transgenic mouse lines containing the green fluorescent protein (GFP) gene driven by GATA-2 gene regulatory elements. We also prepared a mouse line in which GFP reporter sequences were inserted into the endogenous GATA-2 gene. Both mouse mutants expressed GFP in the early hematopoietic tissues. The CD45 antigen, a marker of hematopoietic cells, was expressed in a small fraction of transgene (TG)–derived GFP+ cells. The remaining TG-GFP+/CD45- cells were adherent to plastic and produced CD45+ hematopoietic cells abundantly when cultured in vitro. Exogenous expression of GATA-2 in TG-GFP+/CD45- cells from the AGM region inhibited their differentiation into CD45+ cells. Loss of GATA-2 function through the disruption of the GATA-2 locus enhanced the earlier emergence of CD45+ cells in the yolk sac of the 9.5-day conceptus. These results demonstrated that GATA-2 is expressed in the precursor of hematopoietic cells and works as a gatekeeper to preserve their immaturity. A reduction of GATA-2 expression or activity is required for the differentiation of precursors to hematopoietic cells.

The aorta-gonads-mesonephros (AGM) region of the mouse embryo has been assigned as the origin of definitive hematopoiesis. The transcription factor GATA-2 has specific but unclarified roles in early hematopoiesis. To elucidate the expression profile of GATA-2, we prepared transgenic mouse lines containing the green fluorescent protein (GFP) gene driven by GATA-2gene regulatory elements. We also prepared a mouse line in which GFPreporter sequences were inserted into the endogenous GATA-2gene. Both mouse mutants expressed GFP in the early hematopoietic tissues. The CD45 antigen, a marker of hematopoietic cells, was expressed in a small fraction of transgene (TG)–derived GFP+cells. The remaining TG-GFP+/CD45-cells were adherent to plastic and produced CD45+hematopoietic cells abundantly when cultured in vitro. Exogenous expression of GATA-2 in TG-GFP+/CD45-cells from the AGM region inhibited their differentiation into CD45+cells. Loss of GATA-2 function through the disruption of the GATA-2locus enhanced the earlier emergence of CD45+cells in the yolk sac of the 9.5-day conceptus. These results demonstrated that GATA-2 is expressed in the precursor of hematopoietic cells and works as a gatekeeper to preserve their immaturity. A reduction of GATA-2 expression or activity is required for the differentiation of precursors to hematopoietic cells.

We demonstrated that Flk-1+cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells (ECs) and mural cells (MCs) to suffice as vascular progenitor cells (VPCs). In the present study, we investigated the importance of the stage of ES cell differentiation on effective participation in adult neovascularization. We obtained Flk-1+LacZ-expressing undifferentiated VPCs. Additional culture of these VPCs with vascular endothelial growth factor (VEGF) resulted in a mixture of ECs and MCs (differentiated VPCs). We injected VPCs subcutaneously into tumor-bearing mice. Five days after the injection, whereas undifferentiated VPCs were often detected as nonvascular cells, differentiated VPCs were more specifically incorporated into developing vasculature mainly as ECs. VPC-derived MCs were also detected in vascular walls. Furthermore, transplantation of differentiated VPCs augmented tumor blood flow in nude mice. These results indicate that a specific vascular contribution in adult neovascularization can be achieved by selective transplantation of ES cell–derived VPCs in appropriate differentiation stages, which should be the basis for vascular regeneration schemes.

We demonstrated that Flk-1+ cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells (ECs) and mural cells (MCs) to suffice as vascular progenitor cells (VPCs). In the present study, we investigated the importance of the stage of ES cell differentiation on effective participation in adult neovascularization. We obtained Flk-1+LacZ-expressing undifferentiated VPCs. Additional culture of these VPCs with vascular endothelial growth factor (VEGF) resulted in a mixture of ECs and MCs (differentiated VPCs). We injected VPCs subcutaneously into tumor-bearing mice. Five days after the injection, whereas undifferentiated VPCs were often detected as nonvascular cells, differentiated VPCs were more specifically incorporated into developing vasculature mainly as ECs. VPC-derived MCs were also detected in vascular walls. Furthermore, transplantation of differentiated VPCs augmented tumor blood flow in nude mice. These results indicate that a specific vascular contribution in adult neovascularization can be achieved by selective transplantation of ES cell–derived VPCs in appropriate differentiation stages, which should be the basis for vascular regeneration schemes.

Vascular endothelial growth factor (VEGF) is a major growth factor for developing endothelial cells (ECs). Embryonic lethality due to haploinsufficiency of VEGF in the mouse highlighted the strict dose dependency of VEGF on embryonic vascular development. Here we investigated the dose-dependent effects of VEGF on the differentiation of ES cell–derived fetal liver kinase 1 (Flk-1)/VEGF receptor 2+ (VEGFR2+) mesodermal cells into ECs on type IV collagen under a chemically defined serum-free condition. These cells could grow even in the absence of VEGF, but differentiated mostly into mural cells positive for α-smooth muscle actin. VEGF supported in a dose-dependent manner the differentiation into ECs defined by the expression of VE-cadherin, platelet-endothelial cell adhesion molecule 1 (PECAM-1)/ CD31, CD34, and TIE2/TEK. VEGF requirement was greater at late than at early phase of culture during EC development, whereas response of VEGFR2+ cells to VEGF-E, which is a virus-derived ligand for VEGFR2 but not for Flt-1/VEGFR1, was not dose sensitive even at late phase of culture. Delayed expression of VEGFR1 correlated with increased dose dependency of VEGF. These results suggested that greater requirement of VEGF in the maintenance than induction of ECs was due to the activity of VEGFR1 sequestering VEGF from VEGFR2 signal. The chemically defined serum-free culture system described here provides a new tool for assessing different factors for the proliferation and differentiation of VEGFR2+ mesodermal cells.

Vascular endothelial growth factor (VEGF) is a major growth factor for developing endothelial cells (ECs). Embryonic lethality due to haploinsufficiency of VEGF in the mouse highlighted the strict dose dependency of VEGF on embryonic vascular development. Here we investigated the dose-dependent effects of VEGF on the differentiation of ES cell–derived fetal liver kinase 1 (Flk-1)/VEGF receptor 2+(VEGFR2+) mesodermal cells into ECs on type IV collagen under a chemically defined serum-free condition. These cells could grow even in the absence of VEGF, but differentiated mostly into mural cells positive for α-smooth muscle actin. VEGF supported in a dose-dependent manner the differentiation into ECs defined by the expression of VE-cadherin, platelet-endothelial cell adhesion molecule 1 (PECAM-1)/ CD31, CD34, and TIE2/TEK. VEGF requirement was greater at late than at early phase of culture during EC development, whereas response of VEGFR2+cells to VEGF-E, which is a virus-derived ligand for VEGFR2 but not for Flt-1/VEGFR1, was not dose sensitive even at late phase of culture. Delayed expression of VEGFR1 correlated with increased dose dependency of VEGF. These results suggested that greater requirement of VEGF in the maintenance than induction of ECs was due to the activity of VEGFR1 sequestering VEGF from VEGFR2 signal. The chemically defined serum-free culture system described here provides a new tool for assessing different factors for the proliferation and differentiation of VEGFR2+mesodermal cells.

Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3), a receptor for VEGF-C, was shown to be essential for angiogenesis as well as for lymphangiogenesis. Targeted disruption of theVEGFR-3 gene in mice and our previous study using an antagonistic monoclonal antibody (MoAb) for VEGFR-3 suggested that VEGF-C/VEGFR-3 signals might be involved in the maintenance of vascular integrity. In this study we used an in vitro embryonic stem (ES) cell culture system to maintain the VEGFR-3+ endothelial cell (EC) and investigated the role of VEGFR-3 signals at the cellular level. In this system packed clusters of ECs were formed. Whereas addition of exogenous VEGF-A induced EC dispersion, VEGF-C, which can also stimulate VEGFR-2, promoted EC growth without disturbing the EC clusters. Moreover, addition of AFL4, an antagonistic MoAb for VEGFR-3, resulted in EC dispersion. Cytological analysis showed that VEGF-A– and AFL4-treated ECs were indistinguishable in many aspects but were distinct from the cytological profile induced by antagonistic MoAb for VE-cadherin (VECD-1). As AFL4- induced EC dispersion requires VEGF-A stimulation, it is likely that VEGFR-3 signals negatively modulate VEGFR-2. This result provides new insights into the involvement of VEGFR-3 signals in the maintenance of vascular integrity through modulation of VEGFR-2 signals. Moreover, our findings suggest that the mechanisms underlying AFL4-induced EC dispersion are distinct from those underlying VECD-1–induced dispersion for maintenance of EC integrity.

Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3), a receptor for VEGF-C, was shown to be essential for angiogenesis as well as for lymphangiogenesis. Targeted disruption of theVEGFR-3gene in mice and our previous study using an antagonistic monoclonal antibody (MoAb) for VEGFR-3 suggested that VEGF-C/VEGFR-3 signals might be involved in the maintenance of vascular integrity. In this study we used an in vitro embryonic stem (ES) cell culture system to maintain the VEGFR-3+endothelial cell (EC) and investigated the role of VEGFR-3 signals at the cellular level. In this system packed clusters of ECs were formed. Whereas addition of exogenous VEGF-A induced EC dispersion, VEGF-C, which can also stimulate VEGFR-2, promoted EC growth without disturbing the EC clusters. Moreover, addition of AFL4, an antagonistic MoAb for VEGFR-3, resulted in EC dispersion. Cytological analysis showed that VEGF-A– and AFL4-treated ECs were indistinguishable in many aspects but were distinct from the cytological profile induced by antagonistic MoAb for VE-cadherin (VECD-1). As AFL4- induced EC dispersion requires VEGF-A stimulation, it is likely that VEGFR-3 signals negatively modulate VEGFR-2. This result provides new insights into the involvement of VEGFR-3 signals in the maintenance of vascular integrity through modulation of VEGFR-2 signals. Moreover, our findings suggest that the mechanisms underlying AFL4-induced EC dispersion are distinct from those underlying VECD-1–induced dispersion for maintenance of EC integrity.

Accumulating evidence in various species has suggested that the origin of definitive hematopoiesis is associated with a special subset of endothelial cells (ECs) that maintain the potential to give rise to hematopoietic cells (HPCs). In this study, we demonstrated that a combination of 5′-flanking region and 3′ portion of the first intron of the Flk-1 gene (Flk-1 p/e) that has been implicated in endothelium-specific gene expression distinguishes prospectively the EC that has lost hemogenic activity. We assessed the activity of this Flk-1 p/e by embryonic stem (ES) cell differentiation culture and transgenic mice by using theGFP gene conjugated to this unit. The expression ofGFP differed from that of the endogenous Flk-1gene in that it is active in undifferentiated ES cells and inactive in Flk-1+ lateral mesoderm. Flk-1 p/e becomes active after generation of vascular endothelial (VE)–cadherin+ ECs. Emergence of GFP− ECs preceded that of GFP+ ECs, and, finally, most ECs expressed GFP both in vitro and in vivo. Cell sorting experiments demonstrated that only GFP− ECs could give rise to HPCs and preferentially expressed Runx1 and c-Myb genes that are required for the definitive hematopoiesis. Integration of both GFP+ and GFP− ECs was observed in the dorsal aorta, but cell clusters appeared associated only to GFP−ECs. These results indicate that activation of Flk-1 p/e is associated with a process that excludes HPC potential from the EC differentiation pathway and will be useful for investigating molecular mechanisms underlying the divergence of endothelial and hematopoietic lineages.

Accumulating evidence in various species has suggested that the origin of definitive hematopoiesis is associated with a special subset of endothelial cells (ECs) that maintain the potential to give rise to hematopoietic cells (HPCs). In this study, we demonstrated that a combination of 5′-flanking region and 3′ portion of the first intron of the Flk-1gene (Flk-1p/e) that has been implicated in endothelium-specific gene expression distinguishes prospectively the EC that has lost hemogenic activity. We assessed the activity of this Flk-1p/e by embryonic stem (ES) cell differentiation culture and transgenic mice by using theGFPgene conjugated to this unit. The expression ofGFPdiffered from that of the endogenous Flk-1gene in that it is active in undifferentiated ES cells and inactive in Flk-1+lateral mesoderm. Flk-1p/e becomes active after generation of vascular endothelial (VE)–cadherin+ECs. Emergence of GFP−ECs preceded that of GFP+ECs, and, finally, most ECs expressed GFP both in vitro and in vivo. Cell sorting experiments demonstrated that only GFP−ECs could give rise to HPCs and preferentially expressed Runx1and c-Mybgenes that are required for the definitive hematopoiesis. Integration of both GFP+and GFP−ECs was observed in the dorsal aorta, but cell clusters appeared associated only to GFP−ECs. These results indicate that activation of Flk-1p/e is associated with a process that excludes HPC potential from the EC differentiation pathway and will be useful for investigating molecular mechanisms underlying the divergence of endothelial and hematopoietic lineages.

Alveolar bone resorption by osteoclasts is essential for tooth eruption. Osteoclast‐deficient Csfmophomozygous (op/op) mice, which lack functional macrophage colony‐stimulating factor (M‐CSF), suffer from osteopetrosis and completely lack tooth eruption. Although osteoclasts appear, and osteopetrosis is cured with age in op/opmice, tooth eruption is never seen. This fact suggests that there is a critical period when osteoclasts are required for tooth eruption. In this study, to detect the critical period, we administered an antagonistic antibody directed against c‐Fms, a receptor for M‐CSF, to inbred C57BL/6 mice for various periods. Administration of this antibody decreased tartrate‐resistant acid phosphatase‐positive (TRAP) osteoclasts, and incisor eruption was completely inhibited by continual administration of this antibody from embryonic day 15.5 (E15.5) until postnatal day 12.5 (D12.5). A 1‐day delay of this administration abolished the inhibition of incisor eruption. The number of TRAP‐positive osteoclasts was significantly reduced between E16.5 and E18.5 in the mice treated with antibody from E15.5 compared with those treated from E16.5. These results indicate that this period, during which the number of osteoclasts decreases significantly, is critical for inhibiting incisor eruption in C57BL/6 mice.

Alveolar bone resorption by osteoclasts is essential for tooth eruption. Osteoclast‐deficient Csfmophomozygous (op/op) mice, which lack functional macrophage colony‐stimulating factor (M‐CSF), suffer from osteopetrosis and completely lack tooth eruption. Although osteoclasts appear, and osteopetrosis is cured with age in op/opmice, tooth eruption is never seen. This fact suggests that there is a critical period when osteoclasts are required for tooth eruption. In this study, to detect the critical period, we administered an antagonistic antibody directed against c‐Fms, a receptor for M‐CSF, to inbred C57BL/6 mice for various periods. Administration of this antibody decreased tartrate‐resistant acid phosphatase‐positive (TRAP) osteoclasts, and incisor eruption was completely inhibited by continual administration of this antibody from embryonic day 15.5 (E15.5) until postnatal day 12.5 (D12.5). A 1‐day delay of this administration abolished the inhibition of incisor eruption. The number of TRAP‐positive osteoclasts was significantly reduced between E16.5 and E18.5 in the mice treated with antibody from E15.5 compared with those treated from E16.5. These results indicate that this period, during which the number of osteoclasts decreases significantly, is critical for inhibiting incisor eruption in C57BL/6 mice.

Platelet-derived growth factor (PDGF), a potent chemotactic and proliferation factor for mesenchymal-derived cells, has been demonstrated to play critical roles in kidney development. Two receptors for PDGF, PDGFR-α and PDGFR-β, have been identified and we previously analyzed the effects of blockade of PDGFR-α signal in neonatal mice. In the current study, we examined the role of PDGFR-β in glomerular development by blocking PDGFR-β signal in neonatal mice by administration of antagonistic anti-PDGFR-β monoclonal antibody. Unlike the mice injected with anti-PDGFR-α antibody, the mice injected daily with anti-PDGFR-β antibody could be kept alive at least for 2 weeks after birth but showed severe disruption of the glomerular structure, whereas no apparent deformation was observed in the collecting ducts. In the disrupted glomeruli, the number of the mesangial cells was reduced markedly. Electron microscopic analysis and immunohistochemical studies with terminal deoxynucleotidyl transferase nick-end labeling staining revealed that the capillary endothelial cells of the glomeruli in the outer cortex region underwent apoptosis. However, the glomeruli located near the medulla were less affected. Because PDGFR-β is not expressed in the endothelial cells, the effects of the blockade of PDGFR-β might have caused glomerular endothelial cell apoptosis by inducing the loss of mesangial cells and/or pericytes.

Using polymerase chain reaction–coupled subtractive hybridization, we have isolated genes expressed during the in vitro differentiation of murine embryonic stem cells into endothelial cells (ECs). Among the genes obtained, we identified one gene that was inducible by vascular endothelial growth factor (VEGF) in the murine EC line MSS31. Analysis of the nucleotide and deduced amino acid sequences revealed that the protein was composed of 930 amino acids, including an HEXXH(X)18E consensus sequence of the M1 aminopeptidase family, and is thought to be a mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase (mPILSAP). The recombinant protein hydrolyzed N-terminal leucyl and methionyl residues from synthetic substrates. Immunohistochemical analysis revealed that mPILSAP was expressed in ECs during postnatal angiogenesis. Specific elimination of mPILSAP expression by antisense oligodeoxynucleotide (AS-ODN) attenuated VEGF-stimulated proliferation, migration, and network formation of ECs in vitro. Moreover, AS-ODN to mPILSAP inhibited angiogenesis in vivo. These results suggest a novel function of mPILSAP, which is expressed in ECs and plays an important role in angiogenesis.

Using polymerase chain reaction–coupled subtractive hybridization, we have isolated genes expressed during the in vitro differentiation of murine embryonic stem cells into endothelial cells (ECs). Among the genes obtained, we identified one gene that was inducible by vascular endothelial growth factor (VEGF) in the murine EC line MSS31. Analysis of the nucleotide and deduced amino acid sequences revealed that the protein was composed of 930 amino acids, including an HEXXH(X)18E consensus sequence of the M1 aminopeptidase family, and is thought to be a mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase (mPILSAP). The recombinant protein hydrolyzed N-terminal leucyl and methionyl residues from synthetic substrates. Immunohistochemical analysis revealed that mPILSAP was expressed in ECs during postnatal angiogenesis. Specific elimination of mPILSAP expression by antisense oligodeoxynucleotide (AS-ODN) attenuated VEGF-stimulated proliferation, migration, and network formation of ECs in vitro. Moreover, AS-ODN to mPILSAP inhibited angiogenesis in vivo. These results suggest a novel function of mPILSAP, which is expressed in ECs and plays an important role in angiogenesis.

The number of molecules identified as being involved in the development of the vascular system has been increasing recently. Among those are secretory molecules and their receptors that collaborate in concert to regulate the process. Of note, however, is that we know little about the response of normal endothelial cells to different stimuli. In this study, we established a method of producing a normal endothelial population from embryonic stem cells. This new culture system was used to analyze the behavior of endothelial cells to various angiogenic stimuli. Our study demonstrated clearly that this culture system is extremely useful in revealing the behavior of developing endothelial cells. Implications derived from our observations are discussed.

Melanocytes are derived from neural crest and migrate along the dorsolateral pathway to colonize the final destination in the skin. Stem cell factor and its receptor c-kit were identified as gene products of Sl and W mutant loci; both of them were known to have defects in melanocytes survival. In this review, we focus on the function of stem cell factor and c-kit in melanocyte migration and survival, which has become clearer in the last decade. By analysis of both molecules in wild-type and white spotting mutant mice, ligand and receptor set were shown to play multiple roles in the development of melanocytes in mouse ontogeny. Functional blockade of c-kit by specific monoclonal antibody illustrated distinct c-kit dependent and independent stages in melanocyte development. Finally, SCF transgene expression demonstrated that part of the c-kit dependent step is regulated by spatiotemporally specific ligand expression and also indicated the presence of c-kit independent melanocyte stem cells in postnatal skin.Journal of Investigative Dermatology Symposium Proceedings (2001) 6, 1–5; doi:10.1046/j.0022-202x.2001.00006.x

Mice deficient in lymphotoxin β receptor (LTβR) or interleukin 7 receptor α (IL-7Rα) lack Peyer's patches (PPs). Deficiency in CXC chemokine receptor 5 (CXCR5) also severely affects the development of PPs. A molecular network involving these three signaling pathways has been implicated in PP organogenesis, but it remains unclear how they are connected during this process. We have shown that PP organogenesis is initiated at sites containing IL-7Rα+ lymphoid cells and vascular cell adhesion molecule (VCAM)-1/intercellular adhesion molecule (ICAM)-1 expressing nonlymphoid elements. Here we characterize these lymphoid and nonlymphoid components in terms of chemokine signals. The lymphoid population expresses CXCR5 and has a strong chemotactic response to B lymphocyte chemoattractant (BLC). Importantly, chemokines produced by VCAM-1+ICAM-1+ nonlymphoid cells mediate the recruitment of lymphoid cells. Furthermore, we show that these VCAM-1+ICAM-1+ cells are mesenchymal cells that are activated by lymphoid cells through the LTβR to express adhesion molecules and chemokines. Thus, promotion of PP development relies on mutual interaction between mesenchymal and lymphoid cells.

It has been widely accepted that hematopoietic and endothelial cell lineages diverge from a common progenitor referred to as the hemangioblast. Recently, analyses of the potential of progenitor cells purified from mouse embryos as well as embryonic stem cells differentiating in vitro resolved intermediate stages between mesodermal cells and committed precursors for hematopoietic and endothelial cell lineages. There are two distinct hematopoietic cell lineages which have different origins, i.e., primitive hematopoietic lineage derived from mesoderm or hemangioblasts and definitive hematopoietic lineage derived from endothelial cells. The endothelium is suggested to provide a milieu in which the definitive hematopoietic lineage acquires multiple potentials.

Osteoclasts are hematopoietic cells essential for bone resorption. To understand the process of osteoclastogenesis, we have developed a culture system that employs a stromal cell line, in which differentiation of osteoclasts from single embryonic stem (ES) cells occurs. This culture, which did not require any cell passaging or other manipulations, enabled us to investigate the temporal and spatial localization of the osteoclast lineage in the colonies formed from ES cells. Cells expressing tartrate-resistant acid phosphatase, a specific marker of the osteoclast lineage, were first detected on day 8, and subsequently became localized at the periphery of colonies and matured into multinucleated cells to resorb bone. Addition of macrophage colony-stimulating factor and osteoprotegerin-ligand, which are produced by stromal cells, promoted osteoclastogenesis in whole colonies, indicating that the location and maintenance of mature osteoclasts as well as the growth and differentiation of osteoclast precursors are regulated by these two factors.

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21ras in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21Asn-17 Ha-ras was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21ras activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21ras activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21ras mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.

Vascular endothelial growth factor (VEGF) plays a major role in tumor angiogenesis. VEGF-C, however, is thought to stimulate the growth of lymphatic vessels because an expression of its specific receptor, VEGF receptor-3 (VEGFR-3), was demonstrated to be restricted to lymphatic vessels. Here we demonstrate that the inactivation of VEGFR-3 by a novel blocking monoclonal antibody (mAb) suppresses tumor growth by inhibiting the neo-angiogenesis of tumor-bearing tissues. Although VEGFR-3 is not expressed in adult blood vessels, it is induced in vascular endothelial cells of the tumor-bearing tissues. Hence, VEGFR-3 is another receptor tyrosine kinase involved in tumor-induced angiogenesis. Micro-hemorrhage in the tumor-bearing tissue was the most conspicuous histologic finding specific to AFL4 mAb-treated mice. Scanning microscopy demonstrated disruptions of the endothelial lining of the postcapillary venule, probably the cause of micro-hemorrhage and the subsequent collapse of the proximal vessels. These findings suggest the involvement of VEGFR-3 in maintaining the integrity of the endothelial lining during angiogenesis. Moreover, our results suggest that the VEGF-C/VEGFR-3 pathway may serve another candidate target for cancer therapy.

Small GTP-binding protein GDP dissociation stimulator (Smg GDS) regulates GDP/GTP exchange reaction of Ki-Ras and the Rho and Rap1 family members and inhibits their binding to membranes. In fibroblasts, Smg GDS shows mitogenic and transforming activities in cooperation with Ki-Ras. However, the physiological function of Smg GDS remains unknown. Here we show that mice lacking Smg GDS died of heart failure shortly after birth, not resulting from developmental heart defects but from enhanced apoptosis of cardiomyocytes triggered by cardiovascular overload. Furthermore, neonatal thymocytes and developing neuronal cells underwent apoptotic cell death. Smg GDS−/− thymocytes were susceptible to apoptotic inducers, such as etoposide and UV irradiation. Smg GDS−/− thymocytes were protected from etoposide-induced cell death by ex vivo transduction of the Smg GDS cDNA. These phenotypes partly coincide with those observed in Ki-Ras-deficient mice, suggesting that Smg GDS is involved in antiapoptotic cell survival signaling through Ki-Ras.

Transcription factors belonging to the basic helix-loop-helix (bHLH) family are involved in various cell differentiation processes. We report the isolation and functional characterization of a novel bHLH factor, termed OUT. OUT, structurally related to capsulin/epicardin/Pod-1 and ABF-1/musculin/MyoR, is expressed mainly in the adult mouse reproductive organs, such as the ovary, uterus, and testis, and is barely detectable in tissues of developing embryos. Physical association of OUT with the E protein was predicted from the primary structure of OUT and confirmed by co-immunoprecipitation. However, unlike other bHLH factors, this novel protein failed to bind E-box or N-box DNA sequences and inhibited DNA binding of homo- and heterodimers consisting of E12 and MyoD in gel mobility shift assays. In luciferase assays, OUT inhibited the induction of E-box-dependent transactivation by MyoD-E12 heterodimers. Deletion studies identified the domain responsible for the inhibitory action of OUT in its bHLH and C-terminal regions. Moreover, terminal differentiation of C2C12 myoblasts was inhibited by exogenous introduction of OUT. These inhibitory functions of OUT closely resemble those of the helix-loop-helix inhibitor Id proteins. Based on these findings, we propose that this novel protein functions as a negative regulator of bHLH factors through the formation of a functionally inactive heterodimeric complex.

A primitive vascular plexus is formed through coordinated regulation of differentiation, proliferation, migration, and cell-cell adhesion of endothelial cell (EC) progenitors. In this study, a culture system was devised to investigate the behavior of purified EC progenitors in vitro. Because Flk-1+ cells derived from ES cells did not initially express other EC markers, they were sorted and used as EC progenitors. Their in vitro differentiation into ECs, via vascular endothelial-cadherin (VE-cadherin)+ platelet-endothelial cell adhesion molecule-1 (PECAM-1)+ CD34-to VE-cadherin+ PECAM-1+CD34+ stage, occurred without exogenous factors, whereas their proliferation, particularly at low cell density, required OP9 feeder cells. On OP9 feeder layer, EC progenitors gave rise to sheet-like clusters of Flk-1+ cells, with VE-cadherin concentrated at the cell-cell junction. The growth was suppressed by Flt-1-IgG1 chimeric protein and dependent on vascular endothelial growth factor (VEGF) but not placenta growth factor (PIGF). Further addition of VEGF resulted in cell dispersion, indicating the role of VEGF in the migration of ECs as well as their proliferation. Cell-cell adhesion of ECs in this culture system was mediated by VE-cadherin. Thus, the culture system described here is useful in dissecting the cellular events of EC progenitors that occur during vasculogenesis and in investigating the molecular mechanisms underlying these processes.

Embryonic stem cells can differentiate in vitro into hematopoietic cells through two intermediate stages; the first being FLK1+ E-cadherin- proximal lateral mesoderm and the second being CD45- VE-cadherin+endothelial cells. To further dissect the CD45-VE-cadherin+ cells, we have examined distribution of ?4-integrin on this cell population, because ?4-integrin is the molecule expressed on hematopoietic stem cells. During culture of FLK1+ E-cadherin- cells, CD45- VE-cadherin+?4-integrin- cells differentiate first, followed by ?4-integrin+ cells appearing in both CD45- VE-cadherin+ and CD45-VE-cadherin- cell populations. In the CD45-VE-cadherin+ cell population, ?4-integrin+ subset but not ?4-integrin- subset had the potential to differentiate to hematopoietic lineage cells, whereas endothelial cell progenitors were present in both subsets. The CD45-VE-cadherin- ?4-integrin+ cells also showed hematopoietic potential. Reverse transcription-polymerase chain reaction analyses showed that differential expression of the Gata2 and Myb genes correlated with the potential of the ?4-integrin+ cells to give rise to hematopoietic cell differentiation. Hematopoietic CD45-VE-cadherin+ ?4-integrin+ cells were also present in the yolk sac and embryonic body proper of 9.5 day postcoitum mouse embryos. Our results suggest that the expression of ?4-integrin is a marker of the earliest precursor of hematopoietic cell lineage that was diverged from endothelial progenitors.

Melanocytes (Mc) and their progenitors melanoblasts (Mb) are derived from the neural crest and migrate along the dorsolateral pathway to colonize the dermis, the epidermis, and finally the hair matrix. To examine the involvement of cadherins in the migration of Mc lineage cells, we combined flow cytometric analysis of dissociated live cells with immunohistochemical staining of tissue sections to quantify the level of cadherin expression on the surface of Mb/Mc. At 11.5 days postcoitum, Mb are in the dermis and are E-cadherin−P-cadherin−(E-cad−P-cad−). During the next 48 h, a 200-fold increase of E-cadherin expression is induced on the surface of Mb prior to their entry into the epidermis, thereby forming a homogeneous E-cadhighP-cad−/lowpopulation. The cadherin expression pattern then diversifies, giving rise to three populations, an E-cad−P-cad−dermal population, E-cadhighP-cadlowepidermal population, and E-cad−P-cadmed-highfollicular population. In all three populations, the patterns of expression are region-specific, being identical with those of surrounding cells such as keratinocytes and fibroblasts, and are preserved before and after pigmentation. While most of the epidermal Mb/Mc disappear after the neonatal stage in normal mice, forced expression of steel factor in the epidermis of transgenic mice promotes survival of epidermal Mb/Mc, maintaining epidermal-type cadherin expression pattern (E-cadhighP-cadlow) throughout the postnatal life. These findings indicate the involvement of extrinsic cues in coordinating the cadherin expression pattern of Mb/Mc and suggest a role for E- and P-cadherins in guiding Mc progenitors to their final destinations.

We demonstrated previously that a single injection of recombinant human macrophage colony-stimulating factor (rhM-CSF) is sufficient for osteoclast recruitment and survival in osteopetrotic (op/op) mice with a deficiency in osteoclasts resulting from a mutation in M-CSF gene. In this study, we show that a single injection of recombinant human vascular endothelial growth factor (rhVEGF) can similarly induce osteoclast recruitment in op/op mice. Osteoclasts predominantly expressed VEGF receptor 1 (VEGFR-1), and activity of recombinant human placenta growth factor 1 on osteoclast recruitment was comparable to that of rhVEGF, showing that the VEGF signal is mediated through VEGFR-1. The rhM-CSF–induced osteoclasts died after injections of VEGFR-1/Fc chimeric protein, and its effect was abrogated by concomitant injections of rhM-CSF. Osteoclasts supported by rhM-CSF or endogenous VEGF showed no significant difference in the bone-resorbing activity. op/op mice undergo an age-related resolution of osteopetrosis accompanied by an increase in osteoclast number. Most of the osteoclasts disappeared after injections of anti-VEGF antibody, demonstrating that endogenously produced VEGF is responsible for the appearance of osteoclasts in the mutant mice. In addition, rhVEGF replaced rhM-CSF in the support of in vitro osteoclast differentiation. These results demonstrate that M-CSF and VEGF have overlapping functions in the support of osteoclastic bone resorption.

A primitive vascular plexus is formed through coordinated regulation of differentiation, proliferation, migration, and cell-cell adhesion of endothelial cell (EC) progenitors. In this study, a culture system was devised to investigate the behavior of purified EC progenitors in vitro. Because Flk-1+cells derived from ES cells did not initially express other EC markers, they were sorted and used as EC progenitors. Their in vitro differentiation into ECs, via vascular endothelial-cadherin (VE-cadherin)+platelet-endothelial cell adhesion molecule-1 (PECAM-1)+CD34−to VE-cadherin+PECAM-1+CD34+stage, occurred without exogenous factors, whereas their proliferation, particularly at low cell density, required OP9 feeder cells. On OP9 feeder layer, EC progenitors gave rise to sheet-like clusters of Flk-1+cells, with VE-cadherin concentrated at the cell-cell junction. The growth was suppressed by Flt-1-IgG1 chimeric protein and dependent on vascular endothelial growth factor (VEGF) but not placenta growth factor (PIGF). Further addition of VEGF resulted in cell dispersion, indicating the role of VEGF in the migration of ECs as well as their proliferation. Cell-cell adhesion of ECs in this culture system was mediated by VE-cadherin. Thus, the culture system described here is useful in dissecting the cellular events of EC progenitors that occur during vasculogenesis and in investigating the molecular mechanisms underlying these processes.

Embryonic stem cells can differentiate in vitro into hematopoietic cells through two intermediate stages; the first being FLK1+E-cadherin−proximal lateral mesoderm and the second being CD45−VE-cadherin+endothelial cells. To further dissect the CD45−VE-cadherin+cells, we have examined distribution of α4-integrin on this cell population, because α4-integrin is the molecule expressed on hematopoietic stem cells. During culture of FLK1+E-cadherin−cells, CD45−VE-cadherin+α4-integrin−cells differentiate first, followed by α4-integrin+cells appearing in both CD45−VE-cadherin+and CD45−VE-cadherin−cell populations. In the CD45−VE-cadherin+cell population, α4-integrin+subset but not α4-integrin−subset had the potential to differentiate to hematopoietic lineage cells, whereas endothelial cell progenitors were present in both subsets. The CD45−VE-cadherin−α4-integrin+cells also showed hematopoietic potential. Reverse transcription-polymerase chain reaction analyses showed that differential expression of the Gata2and Mybgenes correlated with the potential of the α4-integrin+cells to give rise to hematopoietic cell differentiation. Hematopoietic CD45−VE-cadherin+α4-integrin+cells were also present in the yolk sac and embryonic body proper of 9.5 day postcoitum mouse embryos. Our results suggest that the expression of α4-integrin is a marker of the earliest precursor of hematopoietic cell lineage that was diverged from endothelial progenitors.

Mouse interleukin‐7 (IL‐7) cDNA was cloned from mouse thymic stromal cell clone MRL104.8a using a polymerase chain reaction (PCR) technique and expressed in COS‐7 cells. The resulting recombinant interleukin‐7 (rlL‐7) supported the proliferation of mouse antigen‐specific helper T cell (Th) clone 9–16 in the absence of IL‐2 and antigen as well as mouse pre‐B cell line DW34. It was also found that high levels of the mRNA for IL‐7 were constitutively expressed in the MRL104.8a cells, and a potent amount of IL‐7 was produced in its culture supernatant. These results provide the evidence for constitutive expression of IL‐7 mRNA and for production of IL‐7 by thymic stromal cells that have a critical role in intrathymic T cell development. The results are discussed in the context of the functional and molecular relationship between IL‐7 and the previously described cytokines produced by thymic stromal cells.

Macrophage populations resident in tissues and at sites of inflammation are heterogeneous and with local proliferation sometimes evident. Using the convenient murine peritoneal cavity as an inflammation model, the appearance of macrophage lineage cells was followed with time in both thioglycollate- and sodium periodate-induced exudates. The cells were characterized by their proliferative response in vitro in response to colony-stimulating factor-1 (CSF-1) (or macrophage colony-stimulating factor [M-CSF]), particularly by their ability to form colonies in agar, in combination with flow cytometry (surface marker expression and forward and side scatter characteristics). We propose that c-Fms (CSF-1 receptor), unlike other markers, is a uniformly expressed and specific marker suitable for the detection of macrophage-lineage cells in tissues, both in the steady state and after the initiation of an inflammatory reaction. It was shown that the bone marrow myeloid precursor markers, ER-MP58 and ER-MP20 (Ly-6C), but not ER-MP12 (PECAM-1), are expressed by a high proportion of macrophage-lineage cells in the inflamed peritoneum. The macrophage colony-forming cells (M-CFCs) in a 16-hour thioglycollate-induced exudate were phenotyped as c-Fms+ERMP12-20+58+, properties consistent with their being more mature than bone marrow M-CFCs. It is proposed that ER-MP58, as well as ER-MP20, may be a useful marker for distinguishing inflammatory macrophage-lineage cells from the majority of those residing normally in tissues. © 1998 by The American Society of Hematology.

The cells that express Steel factor (SLF) in the gastrointestinal (GI) tract were studied using SLF-lacZ transgenic mice. Expression, detected by β-galactosidase histochemistry, was evident in cells between the circular and longitudinal muscle layers in the GI tract. Double staining with antibodies specific for the neural markers, PGP 9.5, MAP2 and c-Ret, showed that SLF-lacZ positive cells were enteric neurons. Enteroglia did not express SLF-lacZ. The distribution of expressing cells was complimentary to the expression of c-Kit in myenteric interstitial cells.

Immunohistochemical techniques using an anti‐mouse panmacrophage monoclonal antibody and anti‐mouse monoclonal antibodies specific for marginal metallophilic macrophages or marginal zone macrophages were used to detect red pulp macrophages, marginal metallophilic macrophages, and marginal zone macrophages in the spleen of op/opmice. In the mutant mice, the red pulp macrophages were reduced to about 60% of those in the normal littermates and the marginal metallophilic macrophages and marginal zone macrophages were absent. After administration of recombinant human macrophage colony‐stimulating factor (rhM‐CSF), numbers of red pulp macrophages increased rapidly, reaching levels found in normal littermates 1 week later. In contrast, the marginal metallophilic macrophages as well as the marginal zone macrophages appeared slowly after rhM‐CSF administration and their numbers were less than half of the baseline level of normal littermates even at 12 weeks of administration. The distribution of marginal metallophilic macrophages and marginal zone macrophages appearing after M‐CSF administration was irregular in the spleen of the op/opmice. These splenic macrophage subpopulations differed in their responses to rhM‐CSF, suggesting that distinct mechanisms may be involved in their development and differentiation. The splenic red pulp macrophages present in unmanipulated op/opmice are an M‐CSF–independent macrophage population. Although the marginal metallophilic macrophages and marginal zone macrophages are thought to be M‐CSF‐dependent, their development and differentiation appear to be influenced by locally produced M‐CSF or other cytokines. J. Leukoc. Biol. 55: 581‐588; 1994.

In gastrulating embryos, various types of cells are generated before differentiation into specific lineages. The mesoderm of the gastrulating mouse embryo represents a group of such intermediate cells. PDGF receptor alpha (PDGFRα), c-Kit and fetal liver kinase 1 (Flk1) are expressed in distinctive mesodermal derivatives of post-gastrulation embryos. Their expressions during gastrulation were examined by whole mount immunostaining with monoclonal antibodies against these three receptors. The antibodies stained different mesodermal subsets in gastrulating embryos. Flow cytometry of head fold stage embryos revealed that Flk1⁺ mesodermal cells could be further classified by the level of c-Kit expression. To examine the possibility that hematopoietic cell differentiation is initiated from the Flk1⁺ mesoderm, embryonic stem (ES) cells were cultured on the OP9 or PA6 stromal cell layer; the former but not the latter supported in vitro hematopoiesis from ES cells. Flk1⁺ cells were detected only on the OP9 cell layer from day 3 of differentiation before the appearance of hematopoietic cells. Thus, Flk1⁺ cells will be required for in vitro ES cell differentiation into hematopoietic cells. The results suggest that these three receptor tyrosine kinases will be useful for defining and sorting subsets of mesodermal cells from embryos or in vitro cultured ES cells.

Recent data have indicated that resident mouse peritoneal macrophages (PMø) transcribed the interleukin 6 (Il6) and granulocyte–macrophage colony-stimulating factor (Csfgm) genes in response to stimulation with the monocyte–macrophage colony-stimulating factor (CSF-1) but onlyIl6mRNA was translated into secreted protein. In this paper, we extend these observations. It is shown that resident PMø incubated with protein kinase (PK)C inhibitors, staurosporine (SP) and its derivative GF109203-X, showed a several fold increase in the levels ofIl6mRNA in control and CSF-1-primed PMø and a parallel release of large amounts of protein. In contrast, SP was shown to have no effect on the release of GM-CSF from control or CSF-1-primed PMø, although it increased by approximately twofold the amount ofCsfgmmRNA in CSF-1-primed Mø. When SP was added 4 h after CSF-1 priming to block CSF-1-induced protein kinase pathways, an increased amount of IL-6 release was again seen but without any increase inIl6mRNA levels. Under these conditions,Csfgmgene expression was relatively unaffected. Activation of PKC by phorbol myristate acetate (PMA) also resulted in increasedIl6gene expression by control and CSF-1-primed PMø. PMA had no apparent effect onCsfgmtranscription but appeared to influence translation at a low level, as measured by the release of small amounts of GM-CSF protein. The addition of lipopolysaccharide (LPS) to CSF-1-primed PMø resulted in a synergistic increase in the expression of both genes at the levels of transcription and protein release. The addition of SP to CSF-1-primed Mø before LPS, however, further enhanced IL-6 release but not GM-CSF release from the cells. The data indicate that CSF-1-priming drives a number of pathways involved in the regulation of expression of both genes and renders PMø highly susceptible to appropriate secondary stimulatory agents that transform the PMø into secretory inflammatory cells.