Placental Growth Factor Expression Is Required for Bone Marrow Endothelial Cell Support of Primitive Murine Hematopoietic Cells

Two distinct microenvironmental niches that regulate hematopoietic stem/progenitor cell physiology in the adult bone marrow have been proposed; the endosteal and the vascular niche. While extensive studies have been performed relating to molecular interactions in the endosteal niche, the mechanisms that regulate hematopoietic stem/progenitor cell interaction with bone marrow endothelial cells are less well defined. Here we demonstrate that endothelial cells derived from the bone marrow supported hematopoietic stem/progenitor cells to a higher degree than other endothelial or stromal cell populations. This support was dependant upon placental growth factor expression, as genetic knockdown of mRNA levels reduced the ability of endothelial cells to support hematopoietic stem/progenitor cells in vitro. Furthermore, using an in vivo model of recovery from radiation induced myelosuppression, we demonstrate that bone marrow endothelial cells were able to augment the recovery of the hematopoietic stem/progenitor cells. However, this effect was diminished when the same cells with reduced placental growth factor expression were administered, possibly owing to a reduced homing of the cells to the bone marrow vasculature. Our data suggest that placental growth factor elaborated from bone marrow endothelial cells mediates the regulatory effects of the vascular niche on hematopoietic stem/progenitor cell physiology.     

The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation

The mouse placenta was unveiled as an important reservoir for hematopoietic stem cells (HSCs), yet the origin of placental HSCs was unknown. By tracking developing HSCs by expression of Runx1-lacZ and CD41, we have found that HSCs emerge in large vessels in the placenta. Analysis of Ncx1(-/-) embryos, which lack a heartbeat, verified that HSC development is initiated in the placental vasculature independent of blood flow. However, fewer CD41+ hematopoietic cells were found in Ncx1(-/-) placentas than in controls, implying that some HSCs/progenitors colonize the placenta via circulation and/or HSC emergence is compromised without blood flow. Importantly, placentas from Ncx1(-/-) embryos possessed equal potential to generate myelo-erythroid and B and T lymphoid cells upon explant culture, verifying intact multilineage hematopoietic potential, characteristic of developing HSCs. These data suggest that, in addition to providing a niche for a large pool of HSCs prior to liver colonization, the placenta is a true site of HSC generation.     

Hepatoblasts comprise a niche for fetal liver erythropoiesis through cytokine production

In mammals, definitive erythropoiesis first occurs in fetal liver (FL), although little is known about how the process is regulated. FL consists of hepatoblasts, sinusoid endothelial cells and hematopoietic cells. To determine niche cells for fetal liver erythropoiesis, we isolated each FL component by flow cytometry. mRNA analysis suggested that Dlk-1-expressing hepatoblasts primarily expressed EPO and SCF, genes encoding erythropoietic cytokines. EPO protein was detected predominantly in hepatoblasts, as assessed by ELISA and immunohistochemistry, and was not detected in sinusoid endothelial cells and hematopoietic cells. To characterize hepatoblast function in FL, we analyzed Map2k4^−/− mouse embryos, which lack hepatoblasts, and observed down-regulation of EPO and SCF expression in FL relative to wild-type mice. Our observations demonstrate that hepatoblasts comprise a niche for erythropoiesis through cytokine secretion.     

Kit ligand has a critical role in mouse yolk sac and aorta–gonad–mesonephros hematopoiesis

Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta–gonad–mesonephros (AGM) niche. In‐depth analysis of loss‐of‐function and transgenic reporter mouse models show that Kitl‐deficient embryos harbor decreased numbers of yolk sac erythro‐myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)‐derived erythropoiesis, and a reduction in tissue‐resident macrophages. Pre‐HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss‐of‐function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivoAGM and yolk sac endothelial niche.     

Cytokeratins 8 and 19 in the mouse placental development

To investigate the expression and biological roles of cytokeratin 19 (K19) in development and in adult tissues, we inactivated the mouse K19 gene (Krt1-19) by inserting a bacterial beta-galactosidase gene (lacZ) by homologous recombination in embryonic stem cells, and established germ line mutant mice. Both heterozygous and homozygous mutant mice were viable, fertile, and appeared normal. By 7.5-8.0 days post coitum (dpc), heterozygous mutant embryos expressed lacZ in the notochordal plate and hindgut diverticulum, reflecting the fact that the notochord and the gut endoderm are derived from the axial mesoderm-originated cells. In the adult mutant, lacZ was expressed mainly in epithelial tissues. To investigate the possible functional cooperation and synergy between K19 and K8, we then constructed compound homozygous mutants, whose embryos died approximately 10 dpc. The lethality resulted from defects in the placenta where both K19 and K8 are normally expressed. As early as 9. 5 dpc, the compound mutant placenta had an excessive number of giant trophoblasts, but lacked proper labyrinthine trophoblast or spongiotrophoblast development, which apparently caused flooding of the maternal blood into the embryonic placenta. These results indicate that K19 and K8 cooperate in ensuring the normal development of placental tissues.     

Altered cell-surface targeting of stem cell factor causes loss of melanocyte precursors in Steel17H mutant mice.

The normal products of the murine Steel (Sl) and Dominant white spotting (W) genes are essential for the development of melanocyte precursors, germ cells, and hematopoietic cells. The Sl locus encodes stem cell factor (SCF), which is the ligand of c-kit, a receptor tyrosine kinase encoded by the W locus. One allele of the Sl mutation, Sl17H, exhibits minor hematopoietic defects, sterility only in males, and a complete absence of coat pigmentation. The Sl17H gene encodes SCF protein which exhibits an altered cytoplasmic domain due to a splicing defect. In this paper we analyzed the mechanism by which the pigmentation phenotype in Sl17H mutant mice occurs. We show that in embryos homozygous for Sl17H the number of melanocyte precursors is severely reduced on the lateral neural crest migration pathway by e11.5 and can no longer be detected by e13.5 when they would enter the epidermis in wildtype embryos. The reduced number of dispersing melanocyte precursors correlates with a reduction of SCF immunoreactivity in mutant embryos in all tissues examined. Regardless of the reduced amount, functional SCF is present at the cell surface of fibroblasts transfected with Sl17H mutant SCF cDNA. Since SCF immunoreactivity normally accumulates in basolateral compartments of SCF-expressing embryonic epithelial tissues, we analyzed the localization of wildtype and Sl17H mutant SCF protein in transfected epithelial (MDCK) cells in vitro. As expected, wildtype forms of SCF localize to and are secreted from the basolateral compartment. In contrast, mutant forms of SCF, which either lack a membrane anchor or exhibit the Sl17H altered cytoplasmic tail, localize to and are secreted from the apical compartment of the cultured epithelium. We suggest, therefore, that the loss of melanocyte precursors prior to epidermal invasion, and the loss of germ cells from mature testis, can be explained by the inability of Sl17H mutant SCF to be targeted to the basolateral compartment of polarized epithelial keratinocytes and Sertoli cells, respectively.     

Fibulin-5 expression in the human placenta

Fibulin-5 is a secreted extracellular matrix glycoprotein and displays a diverse panel of biological functions, which can be segregated into elastogenic as well as extra-elastogenic functions. While elastogenic functions of fibulin-5 include essential roles in early steps of elastic fibre assembly, extra-elastogenic functions are widespread. Depending on the cell type used, fibulin-5 mediates cell adherence via a subset of integrins, antagonizes angiogenesis and inhibits migration as well as proliferation of endothelial and smooth muscle cells. In this study, we focused on the spatiotemporal expression of fibulin-5 in the human placenta. With progressing gestation, placental fibulin-5 expression increased from first trimester towards term. At term, placental fibulin-5 mRNA expression is lower when compared with other well-vascularized organs such as lung, kidney, heart, uterus and testis. In first trimester, placenta immunohistochemistry localized fibulin-5 in villous cytotrophoblasts and extravillous cytotrophoblasts of the proximal cell column. In term placenta, fibulin-5 was detected in the endothelial basement membrane and adventitia-like regions of vessels in the chorionic plate and stem villi. Cell culture experiments with the villous trophoblast-derived cell line BeWo showed that fibulin-5 expression was downregulated during functional differentiation and intercellular fusion. Moreover, cultivation of BeWo cells under low oxygen conditions impaired intercellular fusion and upregulated fibulin-5 expression. The spatiotemporal shift from the trophoblast compartment in first trimester to the villous vasculature at term suggests a dual role of fibulin-5 in human placental development.     

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.     

Regulation of 17-beta hydroxysteroid dehydrogenase type 2 in human placental endothelial cells

17-beta hydroxysteroid dehydrogenase type 2 (HSD17B2) oxidizes estradiol to estrone, testosterone to androstenedione, and 20 alpha-dihydroprogesterone to progesterone. HSD17B2 is highly expressed in human placental tissue where it is localized to placental endothelial cells lining the fetal compartment. The aim of this study was to investigate the effects of potential regulatory factors including progesterone, estradiol, and retinoic acid (RA) onHSD17B2 expression in primary human placental endothelial cells in culture.HSD17B2 mRNA expression was not regulated by progesterone, the progesterone agonist R5020, or estradiol treatment. RA significantly induced HSD17B2 mRNA levels and enzyme activity in a dose- and time-dependent manner. Maximal stimulation occurred at Hour 48 at an RA concentration of 10(-6) M. Both retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA) were readily detected by immunoblotting in isolated placental endothelial cells. RNA interference directed against RARA or RXRA led to reduced basal levels of HSD17B2 mRNA levels and significantly abolished RA-stimulated HSD17B2 expression. Together, these data indicate that regulation of HSD17B2 mRNA levels and enzymatic activity by RA in the placenta is mediated by RARA and RXRA.     

Regulation of cellular function through stem cell factor and c‐Kit receptor in the cerebellar cell culture

Evidence that stem cell factor (SCF) and c‐Kit receptor tyrosine kinase expressed in the cerebellum during postnatal development, suggests a possible contribution of the SCF/Kit signaling pathway in the cerebellar development. In the present study, we prepared cerebellar cultures from C57Bl/6J mouse at postnatal day 6 to investigate the role of c‐Kit receptor and SCF in regulation of cell growth and viability in the postnatal cerebellar cells. SCF increased the number of survival cells and density of calbindin and GFAP expression in the immunoblot analysis. Treatment with c‐Kit antibody accelerated cellular loss in serum‐free media and decreased the expression of calbindin and GFAP. The recovery effects of SCF on the cellular proliferation and the expression of functional proteins in the cultures containing c‐Kit antibody suggest an involvement of SCF/Kit pathways in the control of postnatal development of cerebellar cells.     

Regulation of hematopoietic development in the aorta-gonad-mesonephros region mediated by Lnk adaptor protein

Development of hematopoietic cells in the aorta-gonad-mesonephros (AGM) region in the midgestation mouse embryo involves a multistep process, sequentially changing from endothelial cell-like cells, including hemangioblasts, into hematopoietic stem cells, progenitors, and/or lineage-committed cells. An adaptor molecule, Lnk, is known to negatively control the production of pro- and pre-B cells and hematopoietic progenitor cells in adult bone marrow. Here we show a role of Lnk in hematopoietic development in the AGM region. Lnk was predominantly expressed in the endothelial cells lining the dorsal aorta at embryonic day 11.5 (E11.5). Overexpression of Lnk in the primary culture of the AGM region at E11.5 suppressed the emergence of CD45+ hematopoietic cells. Point mutation in the SH2 domain of Lnk, which abolishes the binding capability of Lnk to c-Kit upon stimulation with stem cell factor (SCF), led to loss of Lnk-dependent inhibition of hematopoietic cell development in AGM cultures, suggesting Lnk-mediated inhibition of the SCF/c-Kit signaling pathway. In cultured AGM cells from Lnk homozygous mutant mouse embryos, the number of emerged CD45+ cells was 2.5-fold larger than that from heterozygous littermates. Furthermore, aorta cells of E11.5 Lnk homozygous mutant mice also showed enhanced hematopoietic colony-forming activity. Thus, Lnk is a negative regulator of hematopoiesis in the AGM region.     

FGF ligand family mRNA expression profile for mouse preimplantation embryos, early gestation human placenta, and mouse trophoblast stem cells

Signaling by fibroblast growth factor (FGF) is essential is for trophoblast stem (TS) cells and preimplantation embryos. FGF4 provides essential signaling, but the expression of the complete set of 23 FGF family members has not been analyzed. Here, semi-quantitative RT-PCR and microarray analyses were used to define expression of all FGF ligand mRNA. RT-PCR was done for developmentally important FGF subfamilies, FGF10/FGF22 and FGF8/FGF17/FGF18 as well as FGF11. FGF4 and FGF18 are detected at highest levels by RT-PCR and microarrays. FGF10 was detected at low levels in both assays. FGF11 was detected at moderate levels by microarray, but not by RT-PCR. FGF17 was detected at low levels by array and moderate levels by RT-PCR. FGF8 and FGF22 were detected by RT-PCR, but not by microarrays during late cleavage divisions. FGF8, FGF5, and FGF9 were detected in the oocyte by microarray. FGF2, FGF3, and FGF7 were not detected by RT-PCR or microarrays and FGF13, FGF14, and FGF23 were not detected by microarray. Since a major role of FGF is to maintain TS cells, we tested human and mouse placental cell lines and early gestation human placenta for expression of FGF ligands. Expression in mouse TS cells was compared with preimplantation embryos, and human placental cell line expression was compared with human placenta, to infer which ligands are expressed in placental lineage vs. other cell lineages. The data suggest that human and mouse placenta share FGF18 and its high expression suggests preimplantation and early placental function.     

A recombinant ectodomain of the receptor for the stem cell factor (SCF) retains ligand-induced receptor dimerization and antagonizes SCF-stimulated cellular responses.

The stem cell factor (SCF) is a polypeptide ligand that is essential for the development of germ cells, hematopoietic progenitor cells, and melanocyte precursors. It binds to a tyrosine kinase membrane receptor that is encoded by the c-kit proto-oncogene. We have constructed an expression vector that directs the synthesis of the entire extracellular ligand-binding domain of the Kit/SCF receptor. When expressed and amplified in Chinese hamster ovary cells, a secreted 90-kDa glycoprotein could be harvested from the growth medium of the cells in a soluble form. This extracellular portion of the Kit/SCF receptor, denoted Kit-X, was recognized by antibodies specific to the SCF receptor; and when injected into animals, it raised antibodies that were reactive with the complete membrane form of the receptor. Direct binding and covalent cross-linking of radiolabeled SCF showed that Kit-X fully retained high affinity ligand binding and also underwent efficient dimerization in the presence of the ligand. The capacity of Kit-X to act as an antagonist of SCF was assayed on cultured cells that overexpress the receptor. Simultaneous addition of SCF and Kit-X to these cells resulted in a stoichiometric inhibition of SCF binding and a consequent decrease in autophosphorylation of the SCF receptor on tyrosine residues. The inhibition extended to later SCF-mediated responses, including the association of the receptor with phosphatidylinositol 3'-kinase and coupling to the Raf1 protein kinase. These results indicate that the recombinant ectodomain of the Kit-SCF receptor can be used as a specific antagonist of SCF actions and may enable detailed molecular analysis of ligand-receptor interactions.     

The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential.

The chorio-allantoic placenta forms through the fusion of the allantois (progenitor tissue of the umbilical cord), with the chorionic plate. The murine placenta contains high levels of hematopoietic stem cells, and is therefore a stem cell niche. However, it is not known whether the placenta is a site of hematopoietic cell emergence, or whether hematopoietic cells originate from other sites in the conceptus and then colonize the placenta. Here, we show that the allantois and chorion, isolated prior to the establishment of circulation, have the potential to give rise to myeloid and definitive erythroid cells following explant culture. We further show that the hematopoietic potential of the allantois and chorion does not require their union, indicating that it is an intrinsic property of these tissues. These results suggest that the placenta is not only a niche for, but also a source of, hematopoietic cells.