Search

During angiogenesis, VEGF acts as an attractive cue for endothelial cells (ECs), while Sema3E mediates repulsive cues. Here, we show that the small GTPase RhoJ integrates these opposing signals in directional EC migration. In the GTP‐bound state, RhoJ interacts with the cytoplasmic domain of PlexinD1. Upon Sema3E stimulation, RhoJ released from PlexinD1 induces cell contraction. PlexinD1‐bound RhoJ further facilitates Sema3E‐induced PlexinD1‐VEGFR2 association, VEGFR2 transphosphorylation at Y1214, and p38 MAPK activation, leading to reverse EC migration. Upon VEGF stimulation, RhoJ is required for the formation of the holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin‐1, thereby preventing degradation of internalized VEGFR2, prolonging downstream signal transductions via PLCγ, Erk, and Akt, and promoting forward EC migration. After conversion to the GDP‐bound state, RhoJ shifts from PlexinD1 to VEGFR2, which then terminates the VEGFR2 signals. RhoJ deficiency in ECs efficiently suppressed aberrant angiogenesis in ischemic retina. These findings suggest that distinct Rho GTPases may act as context‐dependent integrators of chemotactic cues in directional cell migration and may serve as candidate therapeutic targets to manipulate cell motility in disease or tissue regeneration. Synopsis: Semaphorin 3E (Sema3E)/PlexinD1 and VEGF/VEGFR2 signalling pathways regulate endothelial cell migration in an opposite fashion. In this study, GTPase RhoJ is shown to integrate attractive and repulsive chemotactic cues by mediating interaction between VEGFR2 and PlexinD1. Sema3E releases GTP‐RhoJ from PlexinD1 to promote cell contractility.RhoJ facilitates Sema3E‐induced VEGFR2‐PlexinD1 association and p38 MAPK activation.GTP‐RhoJ sustains VEGF‐induced signalling by mediating VEFR2 interaction with PlexinD1/Neuropilin 1 complex and preventing VEGFR2 degradation.GDP‐RhoJ promotes VEGFR2 degradation, thus terminating signalling via internalized VEGFR2.RhoJ deficiency suppresses aberrant angiogenesis in ischemic retina. [ABSTRACT FROM AUTHOR]

Etv2 is a critical determinant for the commitment of endothelial ( EC) and hematopoietic ( HPC) cells from mesoderm. Etv2 is assumed to be transiently required for EC commitment but dispensable after most ECs differentiate around E9.5. To confirm the time window of Etv2 requirement, Etv2 was ablated at different time points using ROSA26Cre ER mice. Unexpectedly, Etv2 ablation at E9.5 caused vascular remodeling defects in cranial and yolk sac vasculature. Immunostaining showed that Etv2+/ VE-cadherin ( VECAD)− cells were present around forming vasculature, mostly co-expressing Flk-1 with a small number of Etv2+/ VECAD+ cells, indicating that Etv2+/Flk-1+/ VECAD− cells are the major Etv2+ population promoting vascular remodeling around E9.5. Gene expression analysis showed up-regulation of Fgf proteins, Il-6, Glypican-3 and matrix metalloproteases in Etv2+/ VEDAC− cells over Etv2−/ VECAD+ mature ECs. Blockade of those factors caused reduced EC sprouting in ex vivo explant culture from E9.5 embryos, suggesting the functional significance of environmental factors derived from Etv2+ cells. Altogether, we propose that Etv2+/ VEDAC− cells around E9.5-E10.5 provide extracellular factors to complete vascular morphogenesis in addition to becoming differentiated ECs incorporated into vessels. This insight for the new role of Ets protein in perivascular Flk-1+/ VECAD−/(Etv2+) cells to induce expression of angiogenic factors may provide another strategy to control angiogenesis. [ABSTRACT FROM AUTHOR]

Background: Early mesoderm can be classified into Flk-1+ or PDGF receptor alpha (PDGFRα)+ population, grossly representing lateral and paraxial mesoderm, respectively. It has been demonstrated that all endothelial (EC) and hematopoietic (HPC) cells are derived from Flk-1+ cells. Although PDGFRα+ cells give rise to ECs/HPCs in in vitro ES differentiation, whether PDGFRα+ population can become hemato-endothelial lineages has not been proved in mouse embryos. Results: Using PDGFRαMerCreMer mice, PDGFRα+ early mesoderm was shown to contribute to endothelial cells including hemogenic ECs, fetal liver B lymphocytes, and Lin-Kit+Sca-1+ (KSL) cells. Contribution of PDGFRα+ mesoderm into ECs and HPCs was limited until E8.5, indicating that PDGFRα+/Flk-1+ population that exists until E8.5 may be the source for hemato-endothelial lineages from PDGFRα+ population. The functional significance of PDGFRα+ mesoderm in vascular development and hematopoiesis was confirmed by genetic deletion of Etv2 or restoration of Runx1 in PDGFRα+ cells. Etv2 deletion and Runx1 restoration in PDGFRα+ cells resulted in abnormal vascular remodeling and rescue of fetal liver CD45+ and Lin-Kit+Sca-1+ (KSL) cells, respectively. Conclusions: Endothelial and hematopoietic cells can be derived from PDGFRα+ early mesoderm in mice. PDGFRα+ mesoderm is functionally significant in vascular development and hematopoiesis from phenotype analysis of genetically modified embryos. Developmental Dynamics 242:254-268, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

As the ability to detect and define stem cells (SCs) has increased, attention is turning towards the definition of the niche and the identification of the signals that induce SC quiescence or proliferation. The melanocyte stem–niche system, in which the SCs and their progeny occupy geographically distinct domains within the hair follicle, provides one of the best models for studying the complex interplay between environmental cues and transcription factors that underpin cell fate. This review discusses what is known of the origin and molecular characteristics of melanocyte SCs and proposes a series of temporal events that are likely to contribute to the establishment of melanocyte SCs in the hair follicle. We also highlight the possibility of in vitro systems capable of directing cultured melanocytes/melanoblasts to a SC fate in response to specific extrinsiccues. [ABSTRACT FROM AUTHOR]

Hemangioblasts are thought to be one of the sources of hematopoietic progenitors, yet little is known about their localization and fate in the mouse embryo. We show here that a subset of cells co-expressing the hematopoietic marker GATA-1 and the endothelial marker VE-cadherin localize on the yolk sac blood islands at embryonic day 7.5. Clonal analysis demonstrated that GATA-1+ cells isolated from E7.0–7.5 embryos include a common precursor for hematopoietic and endothelial cells. Moreover, this precursor possesses primitive and definitive hematopoietic bipotential. By using a transgenic complementation rescue approach, GATA-1+ cell-derived progenitors were selectively restored in Runx1-deficient mice. In the rescued mice, definitive erythropoiesis was recovered but the rescued progenitors did not display multilineage hematopoiesis or intra-aortic hematopoietic clusters. These results provide evidence of the presence of GATA-1+ hemangioblastic cells in the extra-embryonic region and also their functional contribution to hematopoiesis in the embryo. [ABSTRACT FROM AUTHOR]

Definitive hematopoiesis has been proposed to arise from hemogenic endothelial cells during mouse embryogenesis. The c- myb proto-oncogene is essential for the development of definitive hematopoiesis and was reported to be activated in hemogenic endothelial cells. To investigate whether c-Myb is involved in regulating the development of hemogenic endothelial cells, we conditionally induced c- myb over-expression during the in vitro differentiation of embryonic stem cells. VE-cadherin+ CD45− cells inducibly expressing c-Myb showed an increase in multilineage colony formation as well as an augmented capacity of the colony forming cells to self-renew in vitro under the condition that only the endogenous c- myb gene was expressed during differentiation of hematopoietic cells. Over-expression of c-Myb in the endothelial population led to activation of genes associated with definitive hematopoiesis such as Runx1, Hoxb4, Mll and Etv6. Our data provide evidence that c-Myb is able to exert an effect in endothelial cells which fosters the establishment of their hemogenic potential. [ABSTRACT FROM AUTHOR]

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3′:5′-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with hepatocyte growth factor (HGF) from 14 days (keratinocyte depletion). The HGF increased the number of melanoblasts and melanocytes, but not the percentage of differentiated melanocytes in the melanoblast–melanocyte population in the absence of keratinocytes. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and/or G2/M phases of the cell cycle were increased by the treatment with HGF. Moreover, an anti-HGF antibody supplemented to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes, but not the differentiation of melanocytes. These results suggest that HGF is a keratinocyte-derived factor involved in regulating the proliferation of epidermal melanoblasts and melanocytes from newborn mice in cooperation with cAMP elevators and/or bFGF. [ABSTRACT FROM AUTHOR]

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3′:5′-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were then further cultured with MDMD/MDMDF supplemented with steel factor (SLF) (keratinocyte depletion). SLF increased the number of melanoblasts and melanocytes as well as the proportion of differentiated melanocytes in the absence of keratinocytes. Flow cytometric analysis showed that melanoblasts and melanocytes in the S and G2/M phases of the cell cycle were increased by treatment with SLF. Moreover, an anti-SLF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes as well as the differentiation of melanocytes. These results suggest that SLF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF. [ABSTRACT FROM AUTHOR]

Summary: The discovery that lymphotoxin α (LTα) knockout mice lack peripheral lymphoid tissues reformed the study of organogenesis of peripheral lymphoid tissues from a research field that was solely descriptive and dependent on histological methods to one requiring all modern technologies. The concepts of inducer cells for organogenesis of peripheral lymphoid tissues as a separate hematopoietic lineage and of mesenchymal organizer cells have been established through this progress. These discoveries led to the comprehension of the basic framework of the events during organogenesis of peripheral lymphoid tissues. However, many important questions remain unanswered. This review discusses those questions which have arisen from our studies on the organogenesis of Peyer's patches. [ABSTRACT FROM AUTHOR]

Orally applied antigens generate systemic unresponsiveness by induction of anergy and deletion of specific T cells at high antigen doses, and induction of regulatory T cells at low doses of antigen. These different immune reactions have been attributed to different types of antigen-presenting cells (APC) and/or different secondary lymphoid organs participating in the induction of the immune response. We used high-sensitivity immunofluorescence to directly identify for the first time the cells presenting orally applied antigen in vivo. At low peptide doses (<1 mg) peptide presentation was exclusively detected on dendritic cells (DC) of the Peyer's patches (PP) and mesenteric lymph nodes (mLN). At high doses (>1 mg) peptides were presented systemically and by all types of APC but presentation was still maximal on DC of the PP (up to 65%). Nevertheless, at limiting antigen doses T cell activation in the gut-associated lymphoid tissue occurs preferentially in the mLN but not in PP. PP-deficient mice have the same frequencies of peptide-presenting cells in mLN, peripheral lymph nodes and spleen and activation of naive T cells in vivo is not affected. Therefore, PP are not critical for antigen presentation as well as for T cell activation in response to orally applied soluble antigens. [ABSTRACT FROM AUTHOR]

Hematopoiesis in most vertebrate species occurs in two distinct phases, primitive and definitive, which diverge from FLK1+VE-cadherin- mesoderm and FLK1+VE-cadherin+ endothelial cells (EC), respectively. This study aimed at determining the stage at which hematopoietic lineage fate is determined by manipulating the SCL/tal-1 expression that is known to be essential for the early development of the primitive and definitive hematopoietic systems. We established SCL-null ES cell tines in which SCL expression is rescued by tamoxifen-inducible Cre recombinase-loxP site-mediated recombination. White no hematopoietic cells (HPC) were detected in SCL-null ES cell differentiation cultures, SCL gene reactivation from day 2 to day 4 after initiation of differentiation could rescue both primitive and definitive hematopoiesis. SCL reactivation at later phases was ineffective. Moreover, generation of VE-cadherin+ EC that can give rise to definitive HPC required SCL reactivation prior to VE-cadherin expression. These results indicated that the competence to become HPC is acquired at the mesodermal stage by a SCL-dependent process that takes place independently of determination of endothelial fate. [ABSTRACT FROM AUTHOR]

BackgroundThe developmental processes leading from the mesoderm to primitive and definitive haematopoietic and endothelial lineages, although of great importance, are still poorly defined. Recent studies have suggested a model in which common precursors give rise to endothelial progenitors and haematopoietic progenitors, the latter subsequently generating both primitive and definitive haematopoietic lineages. However, this model is contradicted by findings that suggest the emergence of haematopoietic cells from the endothelial lineage. ResultsWe found sequential steps in the differentiation of FLK1+ mesoderm into haematopoietic and endothelial lineages in an in vitro differentiation system of embryonic stem (ES) cells: (i) the GATA-1+ subset of FLK1+ mesodermal cells loses the capacity to give rise to endothelial cells and is restricted to primitive erythroid, macrophage and definitive erythroid progenitors; (ii) the remaining GATA-1- cells give rise to VE-cadherin+ endothelial cells; and subsequently (iii) multiple definitive haematopoietic progenitors and endothelial cells branch off from a subset of VE-cadherin+ cells. ConclusionsThese observations strongly suggest that the divergence of primitive and multilineage definitive haematopoietic/endothelial lineages occurs first, and then multilineage definitive haematopoietic progenitors arise from VE-cadherin+ endothelial cells in the development of haematopoietic and endothelial cells. [ABSTRACT FROM AUTHOR]

Examines the monoclonal antibody-mediated manipulation as a tool for dissecting genetic pathways underlying specific embryonic processes. How the antibody is transferred to the fetus; How monoclonal antibody (Ab) works; Processes suitable for Ab-mediated manipulation.

Recent studies revealing that endothelial cells derived from E8.5-E10.5 mouse embryos give rise to haematopoietic cells appear to correspond to previous histological observations that haematopoietic cell clusters are attached to the ventral aspect of dorsal aorta in such a way as if they were budding from the endothelial cell layer. Gene disruption studies have revealed that Runx1/AML1 is required for definitive haematopoiesis but not for primitive haematopoiesis, but the precise stage of gene function is not yet known. We found that mice deficient in Runx1/AML1 (an α subunit of the transcription factor PEBP2/CBF) lack c-Kit+ haematopoietic cell clusters in the dorsal aorta, omphalomesenteric and umbilical arteries, as well as yolk sac vessels. Moreover, endothelial cells sorted from the embryo proper and the yolk sac of AML1–/– embryos are unable to differentiate into haematopoietic cells on OP9 stromal cells, whereas colonies of AML1–/– endothelial cells can be formed in culture. These results strongly suggest that the emergence of haematopoietic cells from endothelial cells represents a major pathway of definitive haematopoiesis and is an event that also occurs in the yolk sac in vivo, as suggested by earlier in vitro experiments. [ABSTRACT FROM AUTHOR]

Id proteins are thought to be negative regulators of cell differentiation and positive regulators of cell proliferation. Mammary glands of Id2-/- female mice reveal severely impaired lobulo-alveolar development during pregnancy. Id2-/- mammary epithelia show no precocious maturation, but instead exhibit intrinsic defects in both cell proliferation and cell survival, implying that the role of Id2 in pregnant mammary epithelia is mainly stimulation of cell proliferation and support of cell viability. Expression studies of genes required for mammary gland development suggest Id2 to be a downstream or parallel factor of these genes. A decrease in the DNA binding activity of Stat5 was also observed in Id2-/- mammary glands at 7 days post-coitus. Our results indicate an indispensable role of Id2 in pregnant mammary glands. [ABSTRACT FROM AUTHOR]

We present here a speculative view of embryonic hematopoiesis. We do this with the hope of finding directions for future study, keeping in mind that our model may diverge from the real situation. However, we want to emphasize that previous models have neglected the possibility that endothelial cells (EC) represent a progenitor of hematopoeitic cells (HPC). Emerging evidence, including our own,a nd previous histological studies argue for the presence of hemogenic EC. We discuss seemingly contradictory points of view of embryonic hematopoiesis, such as the origin of lymphogenic progenitors, and have shown that they may be resolved more satisfactory by introducing this notion. Obviously, a good model should be a testable one, so we are currently developing experimental systems to demonstrate that all B cells are, indeed, the progeny of EC. [ABSTRACT FROM AUTHOR]

Presents findings of a study which explained cardiomyocyte differentiation mechanisms by using a novel in vitro cardiomyocyte differentiation system using embryonic stem cells. Induction and purification of cardiomyocytes from Flk1+ mesoderm cells; Role of OP9 stroma cells in cardiomyocyte induction; Identification of cardiac progenitor cell populations; Contribution of Noggin and wnt inhibitors to cardiomyocyte induction.

The article discusses research breakthroughs in identifying niche components at the molecular and cellular levels. It highlights a study investigating niches for intestinal stem cells which later showed that the efficiency of in vitro reconstitution of this stem cell system became nearly complete when stem cells were cocultured with Paneth cells. Another breakthrough yielded by a study of environmental molecules affecting the activity of melanocytes stem cells (MCSC) is also noted.

During embryogenesis, endothelial cells are a source of hematopoietic cells. Vascular endothelial (VE)-cadherin modulates adherens junctions between endothelial cells. How endothelial cells, integrated into the vascular bed via adherens junctions, give rise to free-floating hematopoietic cells has been examined. Contrary to our previous reports, in this report a cell type simultaneously expressing VE-cadherin and the hematopoietic marker CD45 was identified, without rigorous enzymatic dissociation of embryonic tissues. In spite of expressing several other endothelial markers such as endothelial cell nitrous oxide synthase (ECNOS) and MECA-32, this newly defined population failed to produce endothelial colonies when cultured on OP9 stroma, in direct contrast to enzymatically dissociated VE-cadherin+ cells. When isolated from 9.5 days post coitus (d.p.c.) embryos, VE-cadherin+ CD45+ cells generated erythroid, myeloid, but not B lymphoid, cells, also in contrast to VE-cadherin+ cells obtained by enzymatic dissociation. Runx1 null mutant embryos lacked this novel population. Collectively, these results introduce a novel VE-cadherin+ population within the developing embryo, which may represent an intermediate cell type in the transition of hemogenic endothelial cells into blood. [ABSTRACT FROM AUTHOR]