Stuart Fraser

Requirement of Runx1/AML1/PEBP2alphaB for the generation of haematopoietic cells from endothelial cells.

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.

Recombination signal sequence-binding protein Jκ alters mesodermal cell fate decisions by suppressing cardiomyogenesis

The transcription factor recombination signal sequence-binding protein Jκ (RBP-J) is a key downstream element in the signaling pathway of all four mammalian Notch receptors that are critically involved in the control of embryonic and adult development. RBP-J-deficient mice display complex defects and die around day 9.5 postcoitum. Here, we investigate the function of RBP-J in the development of mesodermal cell lineages by using the OP9 stroma coculture system. RBP-J-deficient embryonic stem (ES) cells gave rise to cardiomyocytes, endothelial cells, and primitive and definitive hematopoietic cells. Thus, RBP-J-mediated signals are not required for generation of these cell types. However, when compared with parental RBP-J-expressing ES cells, cardiomyogenesis derived from RBP-J-deficient ES cells was increased. Repression over the cardiogenic pathway was restored by expressing RBP-J in RBP-J-deficient ES cells. Our data indicate that Notch signaling via RBP-J plays an important role for the correct specification of myocardial cell fates.

Compartmentalization of Peyer’s Patch Anlagen Before Lymphocyte Entry

We have shown that Peyer’s patch (PP) first develops as a simple and even cell aggregation during embryogenesis. To investigate when and how such a simple cell aggregation forms the complex PP architecture, we analyzed the distribution of cells expressing IL-7Rα (PP inducer cells), VCAM-1 (mesenchymal cells), CD11c (dendritic cells), and mature lymphocytes by whole-mount immunostaining of 17.5 days postcoitus to 2 days postpartum mouse gut. Our results show that compartmentalization of PP anlagen commences at day 18.5 of gestation by clustering and subsequent follicle formation of IL-7Rα+, VCAM-1+, and CD11c+ cells. This process adds the primitive architecture of PP anlage with several follicles in which IL-7Rα+ cells localize in the center, while VCAM-1+ and CD11c+ cells localize at the fringe. This follicle formation is accompanied by the establishment of PP-specific vascular network expressing mucosal addressin cellular adhesion molecule-1. Mature B and T lymphocytes entering in the PP anlage are distributed promptly to their own target zones; B cells to the follicle and T cells to nonfollicular zones. Our analysis of scid/scid mouse indicate that the initial processes including formation of PP-specific vascular network occur in the absence of lymphocytes. These observations indicate that the basic architecture of PP is formed by a set of cell lineages assembled during the initial phase of induction of PP anlagen before entry of mature lymphocytes.

Definitive hematopoietic commitment within the embryonic vascular endothelial-cadherin+ population

OBJECTIVE The aim of this study was to assess the potential of FLK1(+) and vascular endothelial (VE)-cadherin(+) populations from different stages of embryonic development to generate hematopoietic cells ex vivo and to contribute to the hematopoietic systems of recipient mice. MATERIALS AND METHODS FLK1(+) of VE-cadherin(+) cells were isolated from 7.5- to 9.5-dpc concepti and cultured ex vivo on OP9 stromal cells and hematopoietic development examined. VE-cadherin(+)CD45(-) cells from 8.5- and 9.5-dpc concepti were injected intrahepatically into newborn busulfan-treated SCID recipients and engraftment monitored. RESULTS VE-cadherin(+) cells from 7.5- and 8.5-dpc concepti can readily generate hematopoi-etic cells ex vivo compared to FLK1(+) VE-cadherin(-) cells. Similar hematopoietic potential can be found in the VE-cadherin(+) cells from the 8.5-dpc yolk sac. When VE-cadherin(+)CD45(-) cells were injected into SCID recipients, long-term engraftment, particularly within the lymphoid system, was observed. This potential was observed in VE-cadherin(+)CD45(-) cells from 9.5-dpc embryo or yolk sac but from tissues from younger concepti. CONCLUSIONS FLK1(+)VE-cadherin(-) cells, possibly representing the lateral plate mesoderm, are not as effective at generating hematopoietic cells compared to similarly staged VE-cadherin(+) cells. VE-cadherin(+)CD45(-) cells can also contribute to the hematolymphoid system of intrahepatically injected newborn SCID recipients, suggesting that cells bearing an endothelial phenotype are capable of generating long-term hematopoietic precursors.

Neural crest and the origin of ectomesenchyme: neural fold heterogeneity suggests an alternative hypothesis.

The striking similarity between mesodermally derived fibroblasts and ectomesenchyme cells, which are thought to be derivatives of the neural crest, has long been a source of interest and controversy. In mice, the gene encoding the alpha subunit of the platelet‐derived growth factor receptor (PDGFRα) is expressed both by mesodermally derived mesenchymal cells and by ectomesenchyme. Whole‐mount immunostaining previously revealed that PDGFRα is present in the cephalic neural fold epithelium of early murine embryos (Takakura et al. [ 1997 ] J Histochem Cytochem 45:883–893). We now show that, within the neural fold, a sharp boundary exists between E‐cadherin–expressing non‐neural epithelium and the neural epithelium of the dorsal ridge. In addition, we found that cells coexpressing E‐cadherin and PDGFRα are present in the non‐neural epithelium of the neural folds. These observations raise the possibility that at least some PDGFRα+ ectomesenchyme originates from the lateral non‐neural domain of neural fold epithelium. This inference is consistent with previous reports (Nichols [ 1981 ] J Embryol Exp Morphol 64:105–120; Nichols [ 1986 ] Am J Anat 176:221–231) that mesenchymal cells emerge precociously from an epithelial neural fold domain resembling the primitive streak in the early embryonic epiblast. Therefore, we propose the name “metablast” for this non‐neural epithelial domain to indicate that it is the site of a delayed local delamination of mesenchyme similar to involution of mesoderm during gastrulation. We further propose the testable hypothesis that neural crest and ectomesenchyme are developmentally distinct progenitor populations and that at least some ectomesenchyme is metablast‐derived rather than neural crest‐derived tissue. Developmental Dynamics 229:118–130, 2004.

Putative intermediate precursor between hematogenic endothelial cells and blood cells in the developing embryo

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.

Origin of Hematopoietic Progenitors during Embryogenesis

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.

In Vitro Differentiation of Mouse Embryonic Stem Cells: Hematopoietic and Vascular Cell Types

Publisher Summary Embryonic stem cells have been posited as sources of differentiated cell types for regenerative medicine. One of the most enticing cell types is the recently described endothelial progenitor cell (EPC) which can contribute to blood vessels and is a candidate for therapy against vascular diseases. This chapter describes an in vitro differentiation system, which results in the generation of endothelial, smooth muscle, and hematopoietic cells from ES cells. An ES-derived equivalent of the lateral plate mesoderm (LPM) has been generated, a critical source of vascular and hematopoietic cells during embryonic development. This in vitro differentiation system is useful for the analysis of developmental pathways and is highly amenable for the analysis of gene expression at distinct stages of the development of the vascular and hematopoietic lineages from Flk-1+ precursors.

All B cells are progeny of endothelial cells : a new perspective

Summary: 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 hematopoietic cells (HPC). Emerging evidence, including our own, and 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 satisfactorily 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.

Disruption of the mouse rbp-j gene alters differentiation of cell lineages derived from mesoderm

Abstract Notch proteins are transmembrane receptors which influence differentiation, proliferation and apoptosis in many developmental systems. Four different mammalian Notch receptors have been described. After ligand binding and activation, the Notch intracellular domain (N IC ) is released from the cytoplasmic membrane and translocates into the nucleus to act as a transcription factor. N IC binds to DNA via the adapter protein RBP-J (also termed CBF-1) which is an essential component for the signalling of all 4 Notch receptors. Binding of N IC converts the transcriptional repressor RBP-J into a transcriptional activator. RBP-J −/− mice die early in development from multiple defects. To investigate the role of RBP-J in the development of mesoderm-derived cell lineages, we analysed the differentiation of RBP-J −/− embryonic stem cells (ES) using the OP9 stromal co-culture system. RBP-J −/− ES differentiated normally into mesodermal Flk-1(VEGF-R2) + cells but further differentiation of these cells was drastically altered. RBP-J −/− ES generated 60 times more cardiac muscle colonies and nearly twice as many endothelial colonies as RBP-J −/+ or RBP-J +/+ ES. RBP-J −/− cells generated c-Kit + Sca-1 + AA4.1 + hematopoietic stem sells and mature erythroid, myeloid, B-lymphoid cells in vitro. Furthermore, transplantation experiments revealed that RBP-J −/− ES can also give rise to B cells in vivo. Our data demonstrate that the RBP-J signalling pathway is an important regulator of myogenic and endothelial differentiation and/or proliferation. Moreover, we show that RBP-J is not required for the generation of erythroid, myeloid and B lymphoid cells from embryonic stem cells.