Marian Peeters

Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo.

The aorta–gonad–mesonephros (AGM) region is a potent hematopoietic site within the mammalian embryo body, and the first place from which hematopoietic stem cells (HSCs) emerge. Within the complex embryonic vascular, excretory and reproductive tissues of the AGM region, the precise location of HSC development is unknown. To determine where HSCs develop, we subdissected the AGM into aorta and urogenital ridge segments and transplanted the cells into irradiated adult recipients. We demonstrate that HSCs first appear in the dorsal aorta area. Furthermore, we show that vitelline and umbilical arteries contain high frequencies of HSCs coincident with HSC appearance in the AGM. While later in development and after organ explant culture we find HSCs in the urogenital ridges, our results strongly suggest that the major arteries of the embryo are the most important sites from which definitive HSCs first emerge.

Hematopoietic Stem Cell Development Is Dependent on Blood Flow

During vertebrate embryogenesis, hematopoietic stem cells (HSCs) arise in the aorta-gonads-mesonephros (AGM) region. We report here that blood flow is a conserved regulator of HSC formation. In zebrafish, chemical blood flow modulators regulated HSC development, and silent heart (sih) embryos, lacking a heartbeat and blood circulation, exhibited severely reduced HSCs. Flow-modifying compounds primarily affected HSC induction after the onset of heartbeat; however, nitric oxide (NO) donors regulated HSC number even when treatment occurred before the initiation of circulation, and rescued HSCs in sih mutants. Morpholino knockdown of nos1 (nnos/enos) blocked HSC development, and its requirement was shown to be cell autonomous. In the mouse, Nos3 (eNos) was expressed in HSCs in the AGM. Intrauterine Nos inhibition or embryonic Nos3 deficiency resulted in a reduction of hematopoietic clusters and transplantable murine HSCs. This work links blood flow to AGM hematopoiesis and identifies NO as a conserved downstream regulator of HSC development.

Ventral embryonic tissues and Hedgehog proteins induce early AGM hematopoietic stem cell development

Hematopoiesis is initiated in several distinct tissues in the mouse conceptus. The aorta-gonad-mesonephros (AGM) region is of particular interest, as it autonomously generates the first adult type hematopoietic stem cells (HSCs). The ventral position of hematopoietic clusters closely associated with the aorta of most vertebrate embryos suggests a polarity in the specification of AGM HSCs. Since positional information plays an important role in the embryonic development of several tissue systems, we tested whether AGM HSC induction is influenced by the surrounding dorsal and ventral tissues. Our explant culture results at early and late embryonic day 10 show that ventral tissues induce and increase AGM HSC activity, whereas dorsal tissues decrease it. Chimeric explant cultures with genetically distinguishable AGM and ventral tissues show that the increase in HSC activity is not from ventral tissue-derived HSCs, precursors or primordial germ cells (as was previously suggested). Rather, it is due to instructive signaling from ventral tissues. Furthermore, we identify Hedgehog protein(s) as an HSC inducing signal.

Interleukin-1–mediated hematopoietic cell regulation in the aorta-gonad-mesonephros region of the mouse embryo

Hematopoiesis during development is a dynamic process, with many factors involved in the emergence and regulation of hematopoietic stem cells (HSCs) and progenitor cells. Whereas previous studies have focused on developmental signaling and transcription factors in embryonic hematopoiesis, the role of well-known adult hematopoietic cytokines in the embryonic hematopoietic system has been largely unexplored. The cytokine interleukin-1 (IL-1), best known for its proinflammatory properties, has radioprotective effects on adult bone marrow HSCs, induces HSC mobilization, and increases HSC proliferation and/or differentiation. Here we examine IL-1 and its possible role in regulating hematopoiesis in the midgestation mouse embryo. We show that IL-1, IL-1 receptors (IL-1Rs), and signaling mediators are expressed in the aorta-gonad-mesonephros (AGM) region during the time when HSCs emerge in this site. IL-1 signaling is functional in the AGM, and the IL-1RI is expressed ventrally in the aortic subregion by some hematopoietic, endothelial, and mesenchymal cells. In vivo analyses of IL-1RI-deficient embryos show an increased myeloid differentiation, concomitant with a slight decrease in AGM HSC activity. Our results suggest that IL-1 is an important homeostatic regulator at the earliest time of HSC development, acting to limit the differentiation of some HSCs along the myeloid lineage.

Expression of the Ly-6A (Sca-1) lacZ transgene in mouse haematopoietic stem cells and embryos

Summary. The Sca‐1 surface glycoprotein is used routinely as a marker for haematopoietic stem cell enrichment. Two allelic genes, Ly‐6A and Ly‐6E, encode this marker and appear to be differentially regulated in haematopoietic cells and haematopoietic stem cells. The Sca‐1 protein has been shown to be expressed at a greater frequency in these cells from Ly‐6A strains of mice. To study the specific expression pattern and haematopoietic regulation of the Ly‐6A gene, we constructed a 14 kb cassette from a genomic Ly‐6A fragment, inserted a lacZ reporter gene and created transgenic mice. We found that the Ly‐6A lacZ transgene was expressed in the haematopoietic tissues and predominantly in the T‐lymphoid lineage. Some expression was also found in the B‐lymphoid and myeloid lineages. We demonstrated functional haematopoietic stem cell enrichment by sorting for β‐galactosidase‐expressing cells from the bone marrow. In addition, we found an interesting embryonic expression pattern in the AGM region, the site of the first haematopoietic stem cell generation. Surprisingly, when compared with data from Ly‐6E lacZ transgenic mice, our results suggest that the Ly‐6A cassette does not improve lacZ marker gene expression in haematopoietic cells.

Elevated expression of phospholipid transfer protein in bone marrow derived cells causes atherosclerosis

Background Phospholipid transfer protein (PLTP) is expressed by various cell types. In plasma, it is associated with high density lipoproteins (HDL). Elevated levels of PLTP in transgenic mice result in decreased HDL and increased atherosclerosis. PLTP is present in human atherosclerotic lesions, where it seems to be macrophage derived. The aim of the present study is to evaluate the atherogenic potential of macrophage derived PLTP. Methods and Findings Here we show that macrophages from human PLTP transgenic mice secrete active PLTP. Subsequently, we performed bone marrow transplantations using either wild type mice (PLTPwt/wt), hemizygous PLTP transgenic mice (huPLTPtg/wt) or homozygous PLTP transgenic mice (huPLTPtg/tg) as donors and low density lipoprotein receptor deficient mice (LDLR−/−) as acceptors, in order to establish the role of PLTP expressed by bone marrow derived cells in diet-induced atherogenesis. Atherosclerosis was increased in the huPLTPtg/wt→LDLR−/− mice (2.3-fold) and even further in the huPLTPtg/tg→LDLR−/− mice (4.5-fold) compared with the control PLTPwt/wt→LDLR−/− mice (both P<0.001). Plasma PLTP activity levels and non-HDL cholesterol were increased and HDL cholesterol decreased compared with controls (all P<0.01). PLTP was present in atherosclerotic plaques in the mice as demonstrated by immunohistochemistry and appears to co-localize with macrophages. Isolated macrophages from PLTP transgenic mice do not show differences in cholesterol efflux or in cytokine production. Lipopolysaccharide activation of macrophages results in increased production of PLTP. This effect was strongly amplified in PLTP transgenic macrophages. Conclusions We conclude that PLTP expression by bone marrow derived cells results in atherogenic effects on plasma lipids, increased PLTP activity, high local PLTP protein levels in the atherosclerotic lesions and increased atherosclerotic lesion size.

Neurulation in the pig embryo

Neurulation is based on a multitude of factors and processes generated both inside and outside the neural plate. Although there are models for a general neurulation mechanism, specific sets of factors and processes have been shown to be involved in neurulation depending on developmental time and rostro-caudal location at which neurulation occurred in the species under investigation. To find a common thread amongst these apparently divergent modes of neurulation another representative mammalian species, the pig, was studied here by scanning electron microscopy. The data are compared to a series of descriptions in other species. Furthermore, the relation of axial curvature and neural tube closure rate is investigated. In the pig embryo of 7 somites, the first apposition of the neural folds occurs at somite levels 5–7. This corresponds to closure site 1 in the mouse embryo. At the next stage the rostral and caudal parts of the rhombencephalic folds appose, leaving an opening in between. Therefore, at this stage four neuropores can be distinguished, of which the anterior and posterior ones will remain open longest. The two rhombencephalic closure sites have no counterpart in the mouse, but do have some resemblance to those of the rabbit. The anterior neuropore closes in three phases: (1) the dorsal folds slowly align and then close instantaneously, the slow progression being likely due to a counteracting effect of the mesencephalic flexure; (2) the dorso-lateral folds close in a zipper-like fashion in caudo-rostral direction; (3) the final round aperture is likely to close by circumferential growth. At the stage of 22 somites the anterior neuropore is completely closed. In contrast to the two de novo closure sites for the anterior neuropore in the mouse embryo, none of these were detected in the pig embryo. The posterior neuropore closes initially very fast in the somitic region, but this process almost stops thereafter. We suggest that the somites force the neural folds to elevate precociously. Between the stages of 8– 20 somites the width of the posterior neuropore does not change, while the rate of closure gradually increases; this increase may be due to a catch-up of intrinsic neurulation processes and to the reduction of axial curvature. At the stage of 20–22 somites the posterior neuropore suddenly reduces in size but thereafter a small neuropore remains for 5 somite stages. The closure of the posterior neuropore is completed at the stage of 28 somites.

10 – Development of the Vertebrate Hematopoietic System

The adult hematopoietic system is a heterogeneous group of fully differentiated blood cells and their precursors. At its foundation are hematopoietic stem cells (HSCs) from which terminally differentiated blood cells are continuously renewed. The HSCs divide to yield immature progenitors, which in turn progress through many intermediate stages of commitment in a cascade of differentiation events. The large number of intermediate cell types and the fact that blood cells circulate throughout the embryo present many challenges to studying the embryonic origins and generation of the adult hematopoietic system. The knowledge of embryonic hematopoiesis is drawn from studies in both mammalian and nonmammalian vertebrates. The ability to perform fate mapping experiments, particularly in avian and amphibian embryos, contributes substantially to understand the mesodermal origins of hernatopoietic cells beginning at gastrulation, and has firmly established that multiple independent origins of hematopoiesis occur in the developing embryo. Hematopoiesis in vertebrates originates in multiple anatomically distinct sites in the embryo, including the yolk sac, the vitelline and umbilical arteries, and in the intraembryonic region.

Interleukin-1 regulates hematopoietic progenitor and stem cells in the midgestation mouse fetal liver

Interleukin-1 is known to play a role in modulating the hematopoietic stem cell activity in the aorta-gonad-mesonephros region of the developing embryo. This study extends this notion, showing that IL-1 and its receptor are also involved in the physiological regulation of the proliferation and differentiation of the hematopoietic stem/progenitor cells of murine fetal liver. Background Hematopoietic progenitors are generated in the yolk sac and aorta-gonad-mesonephros region during early mouse development. At embryonic day 10.5 the first hematopoietic stem cells emerge in the aorta-gonad-mesonephros. Subsequently, hematopoietic stem cells and progenitors are found in the fetal liver. The fetal liver is a potent hematopoietic site, playing an important role in the expansion and differentiation of hematopoietic progenitors and hematopoietic stem cells. However, little is known concerning the regulation of fetal liver hematopoietic stem cells. In particular, the role of cytokines such as interleukin-1 in the regulation of hematopoietic stem cells in the embryo has been largely unexplored. Recently, we observed that the adult pro-inflammatory cytokine interleukin-1 is involved in regulating aorta-gonad-mesonephros hematopoietic progenitor and hematopoietic stem cell activity. Therefore, we set out to investigate whether interleukin-1 also plays a role in regulating fetal liver progenitor cells and hematopoietic stem cells. Design and Methods We examined the interleukin-1 ligand and receptor expression pattern in the fetal liver. The effects of interleukin-1 on hematopoietic progenitor cells and hematopoietic stem cells were studied by FACS and transplantation analyses of fetal liver explants, and in vivo effects on hematopoietic stem cell and progenitors were studied in Il1r1−/− embryos. Results We show that fetal liver hematopoietic progenitor cells express the IL-1RI and that interleukin-1 increases fetal liver hematopoiesis, progenitor cell activity and promotes hematopoietic cell survival. Moreover, we show that in Il1r1−/− embryos, hematopoietic stem cell activity is impaired and myeloid progenitor activity is increased. Conclusions The IL-1 ligand and receptor are expressed in the midgestation liver and act in the physiological regulation of fetal liver hematopoietic progenitor cells and hematopoietic stem cells.

Spatio-temporal curvature pattern of the caudal body axis for non-mutant and curly tail mouse embryos during the period of caudal neural tube closure

Abstract During the period of early organogenesis the mouse embryo has a curved body shape, which is thought to interact with ongoing developmental processes. Curly tail is a mouse mutant causing spina bifida, in which aberrant axial curvature is considered to be responsible for a delay in the closure of the posterior neuropore (PNP). Since detailed descriptions of axial curvature have never been made in either the normal or the mutant embryo, the onset and development of the aberrant axial curvature in the curly tail embryo are unknown. In the present study, axial curvature and segmental growth during closure of the PNP are described using circle segments at each somite level in two non-mutant mouse strains. Using the radius and angle of the segments as parameters, CD-1 and Balb/c mouse embryos showed maxima of curvature at the levels of the limb buds. Throughout development, a general axial unbending occurred that was due to a level-specific combination of general outgrowth and other factors. A marked additional decrease in the axial curvature was spatially and temporally related to the final closure of the PNP, indicating that this decrease of curvature facilitates the final closure of the PNP. In the curly tail embryo the segment parameter radius was used to relate the axial curvature to an aberrant neural tube closure pattern. These embryos exhibited an enhanced curvature over the entire neuropore region as soon as a delay in the PNP closure could be distinguished. A steep decrease in curvature during final closure of the PNP did also occur, but at a more caudal level. Both the axial level of straightening and the rate of curvature were normalized at advanced developmental stages. The aberrant spatio-temporal curvature pattern in the curly tail mouse embryo indicates that both the rate of curvature and the axial level of unbending are important for a correct PNP closure.