Early Embryonic Blood Research Articles

Morphological Study on Development of Vascular Endothelium in Chick Embryo

The vascular endothelium and hematopoietic stem cells are believed to derive from common progenitor cells called hemangioblasts. However, morphological study on the development of vascular endothelial cell has been scarce. The present study is intended to provide more morphological explanation of the differentiation process of the hemangioblast. We performed histological observation, using 10 four-day-old chick embryos. After fixation with 4% concentration paraformaldehyde, hematoxylin eosin staining, toluidine blue staining and immunostaining were performed in that order for observation of morphological changes of the detected cells. Blood corpuscles were aggregated in the dorsal aorta and the heart (that had been observed to beat in its preparation). Some red blood cells showed immunoreactivity to both anti-Flk-1(VEGFR2) and anti-CD31 antibody. Additionally, some blood cells had small vacuoles in the cytoplasm, and they fused to form one larger vacuole ultimately a balloon-like shape that was connected to endothelial cells. These cells were present in close proximity to the vascular endothelium. We reveal that some of the early embryonic blood cells differentiate into vascular endothelial cells. Keywords: Vasculogenesis, Angiogenesis, Hemangioblasts, Vascular endothelial cells

A Review of Strategies for Producing Chimeric Birds

Avian chimeras have been shown to be very useful tools for studying embryonic development. Blastodermal cells form stage X embryos and primordial germ cells (PGCs) are extensively used in chimera production. Intraspecies and interspecies germline chimeras have been successfully produced by transferring blastodermal cells or PGCs into recipient blastoderm at stage X or into the early embryonic blood. The long-term culture system established using pluripotent cells from birds provides an alternative method for preserving the endangered species source and facilitates the manipulation of avian transgenesis. A high rate of donor cell-derived progeny production has always been pursued by germline chimera studies. However, the rate of progeny production was unstable. Different methods have been used in an attempt to improve the efficiency of germline chimera production. Several methods have been shown to greatly enhance the efficiency of donor cell-derived progeny production. In this review, we focus on the strategies used for germline chimera production in birds and discuss the factors and measures affecting the efficiency of obtaining donor cell-derived progeny.

The transcriptional programme controlled by Runx1 during early embryonic blood development

Transcription factors have long been recognised as powerful regulators of mammalian development yet it is largely unknown how individual key regulators operate within wider regulatory networks. Here we have used a combination of global gene expression and chromatin-immunoprecipitation approaches during the early stages of haematopoietic development to define the transcriptional programme controlled by Runx1, an essential regulator of blood cell specification. Integrated analysis of these complementary genome-wide datasets allowed us to construct a global regulatory network model, which suggested that key regulators are activated sequentially during blood specification, but will ultimately collaborate to control many haematopoietically expressed genes. Using the CD41/integrin alpha 2b gene as a model, cellular and in vivo studies showed that CD41 is controlled by both Scl/Tal1 and Runx1 in fully specified blood cells, and initiation of CD41 expression in E7.5 embryos is severely compromised in the absence of Runx1. Taken together, this study represents the first global analysis of the transcriptional programme controlled by any key haematopoietic regulator during the process of early blood cell specification. Moreover, the concept of interplay between sequentially deployed core regulators is likely to represent a design principle widely applicable to the transcriptional control of mammalian development.

Early embryonic blood cells collect antigens and induce immunotolerance in the hatched chicken

Earlier experimental data in our laboratory showed that introduction of an exogenous protein into early chicken embryonic blood leads to immunotolerance of hatched chicken to that protein. However, the underlying mechanism is yet unknown. In the present study, we show that the blood cells collecting circulating antigen might contribute to the establishment of immunotolerance. In this experiment, most of the chicken embryo blood cells took up injected fluorescein isothiocyanate-BSA at approximately embryonic d 3. At the same stage, 1 microL of embryo blood was taken out and incubated with BSA. After being loaded with BSA in vitro and washed, these cells were injected back into the original embryo. The BSA-specific lymphocytes were depleted in chickens whose early embryo cells had been loaded with BSA, as evidenced by a significant decrease in anti-BSA antibody after challenge with BSA when the chickens were 3 wk old. In addition, by direct injection of BSA to embryonic d 3 embryo blood, the hatched chickens had decreased amounts of anti-trinitrophenol antibody after the chickens were challenged with trinitrophenol-BSA, indicating that the helper function of BSA-specific T cells was impaired. In conclusion, these observations suggest that some early embryo blood cells possibly collect and store antigen for the establishment of self-tolerance before the maturation of B and T cells.

Ontogeny of haematopoiesis: recent advances and open questions

Unravelling the embryonic origins of the haematopoietic system has been the subject of sustained research for more than a century. Nevertheless, many important questions are still either unanswered or remain a matter of intense debate. Recent progress in mouse and embryonic stem cell model systems as well as imaging and post-genomic technologies has provided new insights into many of these open questions. Here we place into context recent reports on the anatomical site of blood stem cell emergence and, using red blood cells as an example, illustrate how the development of stem cells and the other blood lineages is both temporally and spatially decoupled. In addition, we outline how embryonic stem cell assays are increasingly used as a powerful surrogate for studying lineage relationships and developmental potential of early embryonic blood progenitors. Finally, we review how recent progress in the reconstruction of transcriptional regulatory networks is beginning to define the connectivity between key regulators that control early blood development. In light of these rapid recent advances, research into the embryonic origins of the haematopoietic system should remain one of the most vibrant disciplines within the wider field of haematology for the foreseeable future.

Efficient system for preservation and regeneration of genetic resources in chicken: concurrent storage of primordial germ cells and live animals from early embryos of a rare indigenous fowl (Gifujidori)

The unique accessibility of chicken primordial germ cells (PGCs) during early development provides the opportunity to combine the reproduction of live animals with genetic conservation. Male and female Gifujidori fowl (GJ) PGCs were collected from the blood of early embryos, and cryopreserved in liquid nitrogen for >6 months until transfer. Manipulated GJ embryos were cultured until hatching; fertility tests indicated that they had normal reproductive abilities. Embryos from two lines of White Leghorn (24HS, ST) were used as recipients for chimera production following blood removal. The concentration of PGCs in the early embryonic blood of 24HS was significantly higher than in ST (P < 0.05). Frozen-thawed GJ PGCs were microinjected into the bloodstream of same-sex recipients. Offspring originating from GJ PGCs in ST recipients were obtained with a higher efficiency than those originating from GJ PGCs in 24HS recipients (23.3% v. 3.1%). Additionally, GJ progeny were successfully regenerated by crossing germline chimeras of the ST group. In conclusion, the cryogenic preservation of PGCs from early chicken embryos was combined with the conservation of live animals.

Human Embryonic Stem Cells as an In Vitro Model for Human Vascular Development and the Induction of Vascular Differentiation

Early embryonic blood vessels are typically composed of fragile tubes of endothelial cells encircled by vascular smooth muscle cells. Early human vasculogenesis was explored in spontaneous and directed differentiation models derived from human embryonic stem (HES) cells. In a 3-dimensional (3D) model, HES cells were studied for their potential for vascular differentiation during the spontaneous formation of embryoid bodies. Directed differentiation was investigated by means of a 2-dimensional (2D) differentiation method to promote vascular differentiation from HES cells (without the formation of embryoid bodies). Using this latter approach, up-regulation of early lineage markers of endothelial progenitors were induced. Additional culture under strict conditions and exposure to angiogenic growth factors resulted in a prolonged differentiation pathway into mature endothelial cells and up-regulation of vascular smooth muscle cell markers. The use of 3D collagen gels and Matrigel assays for the induction and inhibition of human vascular sprouting in vitro further established the vascular potential of the cells generated by the 2D differentiation system. Our study shows that HES cells can provide useful models to study early differentiation and development of blood vessels. Moreover, the 2D differentiation model facilitates both the production of vascular lineage cells from HES cells for various potential therapeutic applications and also provides a model for studying the mechanisms involved in early human embryonic blood vessel development.

What guides early embryonic blood vessel formation?

Survival of vertebrate embryos depends on their ability to assemble a correctly patterned, integrated network of blood vessels to supply oxygen and nutrients to developing tissues. The arrangement of larger caliber intraembryonic vessels, specification of arterial-venous identity, and proper placement of major branch points and arterial-venous connections are all precisely determined. A number of recent studies in both mammalian and nonmammalian vertebrate species, reviewed here, have now begun to reveal the major role played by genetically predetermined extrinsic cues in guiding the formation of early embryonic blood vessels and determining the global pattern of the vasculature. Dev Dyn 1999;215:2–11. © 1999 Wiley-Liss, Inc.

What guides early embryonic blood vessel formation?

Survival of vertebrate embryos depends on their ability to assemble a correctly patterned, integrated network of blood vessels to supply oxygen and nutrients to developing tissues. The arrangement of larger caliber intraembryonic vessels, specification of arterial-venous identity, and proper placement of major branch points and arterial-venous connections are all precisely determined. A number of recent studies in both mammalian and nonmammalian vertebrate species, reviewed here, have now begun to reveal the major role played by genetically predetermined extrinsic cues in guiding the formation of early embryonic blood vessels and determining the global pattern of the vasculature.

Adenoviral-mediated expression of antisense RNA to fibroblast growth factors disrupts murine vascular development.

Fibroblast growth factors (FGFs) are expressed in the developing embryo and are postulated to regulate embryonic and vascular growth. The goal of this study was to elucidate the role of basic fibroblast growth factor (FGF-2) in early murine embryonic cardiovascular development in the mouse embryo. Gestation day 7.5 embryos were harvested and placed in culture, and 12 hr later replication-defective adenovirus (0.5 x 10(6) plaque forming units) encoding either beta-galactosidase or antisense FGF-2 RNA was injected into the sinus venosus of the cultured embryos. Embryos receiving only replication-defective adenovirus expressing the beta-galactosidase gene continued to develop normally over the next 12 hr. In contrast, those receiving adenovirus encoding antisense FGF-2 RNA displayed marked morphogenetic abnormalities, including cessation of growth and abnormal yolk sac vascular development, even though the embryonic hearts continued to beat. Abnormal development of the yolk sac vasculature was confirmed by microangiography and by histologic examination. Coinjection of virus carrying FGF-2 cDNA in the sense orientation reversed the effects of antisense FGF-2 RNA expression. These results confirm the efficacy of the replication-defective adenovirus for targeting gene expression to the developing vasculature and provide evidence for a critical role of FGF in the normal vascular assembly in the early embryo. Cessation of embryonic growth on expression of antisense FGF-2 RNA was most likely attributable to failure of efficient circulation of the early embryonic blood cells from the yolk sac into the embryo.

On the association of Poland anomaly and primary microcephaly

In this report we describe a moderately mentally retarded female child with the association of Poland's anomaly and primary microcephaly with marked neuronal migration disturbances, cortical atrophy and white substance hypoplasia. This observation further supports the suggestion that the subclavian artery supply disruption sequence may be part of a broader event of interruption of the early embryonic blood supply also involving the cerebral arterial vessels.

Subclavian artery supply disruption sequence: hypothesis of a vascular etiology for Poland, Klippel-Feil, and Möbius anomalies.

A hypothesis is presented to explain the pathogenesis of the Poland, Klippel-Feil, and Möbius anomalies, isolated absence of the pectoralis major with breast hypoplasia, isolated terminal transverse limb defects, and the Sprengel anomaly. We propose that these conditions are the result of an interruption of the early embryonic blood supply in the subclavian arteries, the vertebral arteries and/or their branches, and hypothesize that the occlusions occur at specific locations in these vessels during or around the sixth week of embryologic development and produce predictable patterns of defects. The term subclavian artery supply disruption sequence (SASDS) is suggested for the group of birth defects represented by the above conditions. Possible causes for interruption of embryonic blood supply are discussed.