A correlative study of the allantois in pig and rabbit highlighting the diversity of extraembryonic tissues in four mammalian species,including mouse and man

Despite its conserved role in placenta and umbilical cord formation, the mammalian allantois shows remarkable diversity in size and form as well as in the timing of its appearance and attachment to the chorion. In the mouse, the common allantoic diverticulum is lacking; instead, the allantoic core domain is defined as a progenitor center for allantoic development. In this study, the allantoises of the pig and the rabbit as two nonrodent mammals of increasing significance in biomedical research are compared (1) morphologically using high resolution light and electron microscopy and (2) molecularly using brachyury mRNA expression as a mesodermal marker. Multiple small allantoic diverticula in the rabbit contrast with a single large cavity filling the entire allantois of the pig, but neither pig nor rabbit allantois expresses brachyury . The mesothelium on the allantois surface shows regional variability of cell contacts and microvilli, while blood vessels appear randomly around the allantoic diverticula in a mesodermal layer of variable thickness. Primordial germ cell‐like cells are found in the allantois of the pig but not of the rabbit. To understand further the relevance of this developmental and morphological diversity, we compare the allantois development of pig and rabbit with early developmental landmarks of mouse and man. Our findings suggest that (1) tissue interaction between endoderm and mesoderm is important for allantoic development and vascular differentiation in species with a rudimentary allantoic diverticulum, (2) allantoic mesothelium plays a specific role in chorioallantoic attachment, allantoic differentiation and vascularization, and (3) there is a pronounced diversity in the extraembryonic migratory pathways of primordial germ cells among mammals. Finally, the phylogenetically basal characteristics of the pig allantois are suggestive of a functional similarity in mammals with a large allantois before placentation and in (aplacental) sauropsids with a chorioallantoic membrane well‐adjusted to material exchange function.     

Sphingosine-1-phosphate signaling promotes critical migratory events in vasculogenesis

Here we have investigated the role of sphingosine-1-phosphate (S1P) signaling in the process of vasculogenesis in the mouse embryo. At stages preceding the formation of blood vessels (7.5-8 dpc) in the embryo proper, yolk sac, and allantois, the S1P receptor S1P(2) is expressed in conjunction with S1P(1) and/or S1P(3). Additionally, sphingosine kinase-2 (SK2), an enzyme that catalyzes the formation of S1P, is expressed in these tissues throughout periods of vasculogenesis. Using the cultured mouse allantois explant model of blood vessel formation, we found that vasculogenesis was dependent on S1P signaling. We showed that S1P could replace the ability of serum to promote vasculogenesis in cultured allantois explants. Instead of small poorly reticulated clusters of rounded endothelial cells that formed under serum-free conditions, S1P promoted the formation of elongated endothelial cells that arranged into expansive branched networks of capillary-like vessels. These effects could not be reproduced by vascular endothelial growth factor or basic fibroblast growth factor administration. The ability of S1P to promote blood vessel formation was not due to effects on cell survival or on changes in numbers of endothelial cells (Flk1(+)/PECAM(+)), angioblasts (Flk1(+)/PECAM(-)), or undifferentiated mesodermal cells (Flk1(-)/PECAM(-)). The S1P effect on blood vessel formation was attributed to it promoting migratory activities of angioblasts and early endothelial cells required for the expansion of vascular networks. Together, our findings suggest that migratory events critical to the de novo formation of blood vessels are under the influence of S1P, possibly synthesized via the action of SK2, with signaling mediated by S1P receptors that include S1P(1), S1P(2), and S1P(3).     

Investigation into a role for the primitive streak in development of the murine allantois

Despite its importance as the source of one of three major vascular systems in the mammalian conceptus, little is known about the murine allantois, which will become the umbilical cord of the chorio-allantoic placenta. During gastrulation, the allantois grows into the exocoelomic cavity as a mesodermal extension of the posterior primitive streak. On the basis of morphology, gene expression and/or function, three cell types have been identified in the allantois: an outer layer of mesothelial cells, whose distal portion will become transformed into chorio-adhesive cells, and endothelial cells within the core. Formation of endothelium and chorio-adhesive cells begins in the distal region of the allantois, farthest from the streak. Over time, endothelium spreads to the proximal allantoic region, whilst the distal outer layer of presumptive mesothelium gradually acquires vascular cell adhesion molecule (VCAM1) and mediates chorio-allantoic union. Intriguingly, the VCAM1 domain does not extend into the proximal allantoic region. How these three allantoic cell types are established is not known, although contact with the chorion has been discounted. In this study, we have investigated how the allantois differentiates, with the goal of discriminating between extrinsic mechanisms involving the primitive streak and an intrinsic role for the allantois itself. Exploiting previous observations that the streak contributes mesoderm to the allantois throughout the latter's early development, microsurgery was used to remove allantoises at ten developmental stages. Subsequent whole embryo culture of operated conceptuses resulted in the formation of regenerated allantoises at all time points. Aside from being generally shorter than normal, none of the regenerates exhibited abnormal differentiation or inappropriate cell relationships. Rather, all of them resembled intact allantoises by morphological, molecular and functional criteria. Moreover, fate mapping adjacent yolk sac and amniotic mesoderm revealed that these tissues and their associated bone morphogenetic protein 4 (BMP4) did not contribute to restoration of allantoic outgrowth and differentiation during allantoic regeneration. Thus, on the basis of these observations, we conclude that specification of allantoic endothelium, mesothelium and chorio-adhesive cells does not occur by a streak-related mechanism during the time that proximal epiblast travels through it and is transformed into allantoic mesoderm. Rather, all three cell-types are established by mechanisms intrinsic to the allantois, and possibly include roles for cell age and cell position. However, although chorio-adhesive cells were not specified within the streak, we discovered that the streak nonetheless plays a role in establishing VCAM1's expression domain, which typically began and was thereafter maintained at a defined distance from the primitive streak. When allantoises were removed from contact with the streak, normally VCAM1-negative proximal allantoic regions acquired VCAM1. These results suggested that the streak suppresses formation of chorio-adhesive cells in allantoic mesoderm closest to it. Together with previous results, findings presented here suggest a model of differentiation of allantoic mesoderm that invokes intrinsic and extrinsic mechanisms, all of which appear to be activated once the allantoic bud has formed.     

Differential expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells

Embryonic stem (ES) cells have the potential to develop into various cell lineages including hemangioblasts (Flk1+), a common progenitor for hematopoietic and vascular endothelial cells. Previous studies indicate that Flk1+ cells, a marker for hemangioblast, can be derived from ES cell and that Flk1+ can be differentiated into hematopoietic or endothelial cells depending on culture conditions. We developed an improved in vitro system to generate Flk1+-enriched cultures from mouse ES cells and used this in vitro system to study the role of Wnt signalling in early endothelial progenitor cells. We determined the expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells. RT-PCR analyses identified significantly higher expression of non-canonical Wnt5a and Wnt11 genes in Flk1+ cells compared to Flk1- cells. In contrast, expression of canonical Wnt3a gene was reduced in Flk1+ cells. In addition, Frizzled2, Frizzled5 and Frizzled7 genes were also expressed at a higher level in Flk1+ cells. The differential expression of Wnt and Frizzled genes in Flk1+ cells provides a novel insight into the role of non-canonical Wnt signalling in vascular endothelial fate determination.     

Flt1 and Flk1 mediate regulation of intraocular pressure and their double heterozygosity causes the buphthalmia in mice

Flt1 and Flk1 are receptor tyrosine kinases for vascular endothelial growth factor-A which play a crucial role in physiological and pathological angiogenesis. To study genetic interaction between the Flt1 and Flk1 genes, we crossed between Flt1 and Flk1 heterozygous (Flt1(+/-) and Flk1(+/-)) mice. We found that Flt1; Flk1 double heterozygous (Flt1(+/-); Flk1(+/-)) mice showed enlarged eyes similar to the buphthalmia detected in human congenital glaucoma with elevation of intraocular pressure (IOP). Actually, IOP was elevated in Flt1(+/-); Flk1(+/-) mice and also in Flt1 or Flk1 single heterozygous mice. However, none of these mutants showed hallmarks of glaucoma such as ganglion cell death and excavation of optic disc. To clarify the pathological causes for enlarged eyes and elevated IOP, we investigate the mice from matings between Flt1(+/-) and Flk1(+/-) mice. Flt1(+/-) mice showed enlarged Schlemm's canal and disordered collagen fibers in the sclera, whereas Flk1(+/-) mice showed atrophied choriocapillaris in the choroid. These tissues are a part of the main outflow and alternative uveoscleral outflow pathway of the aqueous humor, suggesting that these pathological changes found in Flt1(+/-) and Flk1(+/-) mice are associated with the buphthalmia in Flt1(+/-); Flk1(+/-) mice.     

A mutant receptor tyrosine phosphatase,CD148, causes defects in vascular development

Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPeta), participates in developmental vascularization. A mutant allele, CD148(DeltaCyGFP), was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions.     

Study of the murine allantois by allantoic explants

The murine allantois will become the umbilical artery and vein of the chorioallantoic placenta. In previous studies, growth and differentiation of the allantois had been elucidated in whole embryos. In this study, the extent to which explanted allantoises grow and differentiate outside of the conceptus was investigated. The explant model was then used to elucidate cell and growth factor requirements in allantoic development. Early headfold-stage murine allantoises were explanted directly onto tissue culture plastic or suspended in test tubes. Explanted allantoises vascularized with distal-to-proximal polarity, they exhibited many of the same signaling factors used by the vitelline and cardiovascular systems, and they contained at least three cell types whose identity, gene expression profiles, topographical associations, and behavior resembled those of intact allantoises. DiI labeling further revealed that isolated allantoises grew and vascularized in the absence of significant cell mingling, thereby supporting a model of mesodermal differentiation in the allantois that is position- and possibly age-dependent. Manipulation of allantoic explants by varying growth media demonstrated that the allantoic endothelial cell lineage, like that of other embryonic vasculatures, is responsive to VEGF(164). Although VEGF(164) was required for both survival and proliferation of allantoic angioblasts, it was not sufficient to induce appropriate epithelialization of these cells. Rather, other VEGF isoforms and/or the outer sheath of mesothelium, whose maintenance did not appear to be dependent upon endothelium, may also play important roles. On the basis of these findings, we propose murine allantoic explants as a new tool for shedding light not only on allantoic development, but for elucidating universal mechanisms of blood vessel formation, including vascular supporting cells, either in the intact organism or in existing in vitro systems.     

Stem cell-derived endothelial cells/progenitors migrate and pattern in the embryo using the VEGF signaling pathway

Endothelial precursor cells respond to molecular cues to migrate and assemble into embryonic blood vessels, but the signaling pathways involved in vascular patterning are not well understood. We recently showed that avian vascular patterning cues are recognized by mammalian angioblasts derived from somitic mesoderm through analysis of mouse-avian chimeras. To determine whether stem cell-derived endothelial cells/progenitors also recognize global patterning signals, murine ES cell-derived embryoid bodies (EBs) were grafted into avian hosts. ES cell-derived murine endothelial cells/progenitors migrated extensively and colonized the appropriate host vascular beds. They also formed mosaic vessels with avian endothelial cells. Unlike somite derived-endothelial cells, ES cell-derived endothelial cells/progenitors migrated across the host embryonic midline to the contralateral side. To determine the role of VEGF signaling in embryonic vascular patterning, EBs mutant for a VEGF receptor (flk-1(-/-)) or a signal (VEGF-A(-/-)) were grafted into quail hosts. Flk-1(-/-) EB grafts produced only rare endothelial cells that did not migrate or assemble into vessels. In contrast, VEGF-A(-/-) EB grafts produced endothelial cells that resembled wild-type and colonized host vascular beds, suggesting that host-derived signals can partially rescue mutant graft vascular patterning. VEGF-A(-/-) graft endothelial cells/progenitors crossed the host midline with much lower frequency than wild-type EB grafts, indicating that graft-derived VEGF compromised the midline barrier when present. Thus, ES cell-derived endothelial cells/progenitors respond appropriately to global vascular patterning cues, and they require the VEGF signaling pathway to pattern properly. Moreover, EB-avian chimeras provide an efficient way to screen mutations for vascular patterning defects.     

EphB4 controls blood vascular morphogenesis during postnatal angiogenesis

Guidance molecules have attracted interest by demonstration that they regulate patterning of the blood vascular system during development. However, their significance during postnatal angiogenesis has remained unknown. Here, we demonstrate that endothelial cells of human malignant brain tumors also express guidance molecules, such as EphB4 and its ligand ephrinB2. To study their function, EphB4 variants were overexpressed in blood vessels of tumor xenografts. Our studies revealed that EphB4 acts as a negative regulator of blood vessel branching and vascular network formation, switching the vascularization program from sprouting angiogenesis to circumferential vessel growth. In parallel, EphB4 reduces the permeability of the tumor vascular system via activation of the angiopoietin-1/Tie2 system at the endothelium/pericyte interface. Furthermore, overexpression of EphB4 variants in blood vessels during (i) vascularization of non-neoplastic cell grafts and (ii) retinal vascularization revealed that these functions of EphB4 apply to postnatal, non-neoplastic angiogenesis in general. This implies that both neoplastic and non-neoplastic vascularization is driven not only by a vascular initiation program but also by a vascular patterning program mediated by guidance molecules.     

Study of normal and pathological blood vessel morphogenesis in Flt1-tdsRed BAC Tg mice

Blood vessel development and network patterning are controlled by several signaling molecules, including VEGF, FGF, TGF‐ß, and Ang‐1,2. Among these, the role of VEGF‐A signaling in vessel morphogenesis is best understood. The biological activity of VEGF‐A depends on its reaction with specific receptors Flt1 and Flk1. Roles of VEGF‐A signaling in endothelial cell proliferation, migration, survival, vascular permeability, and induction of tip cell filopodia have been reported. In this study, we have generated Flt1‐tdsRed BAC transgenic (Tg) mice to monitor Flt1 gene expression during vascular development. We show that tdsRed fluorescence is observed within blood vessels of adult mice and embryos, indicative of retinal angiogenesis and tumor angiogenesis. Flt1 expression recapitulated by Flt1‐tdsRed BAC Tg mice overlapped well with Flk1, while Flt1 was expressed more abundantly in endothelial cells of large blood vessels such as dorsal aorta and presumptive stalk cells in retina, providing a unique model to study blood vessel development. genesis 50:561–571, 2012. © 2012 Wiley Periodicals, Inc.     

Regulation of Substances in Allantoic and Amniotic Fluid of the Chicken Embryo

Traditionally, the avian allantois has been considered a respiratory organ and a dumping ground for metabolic wastes. We tested the hypothesis that the allantoic fluid is also a depot for free amino acids and related compounds. To gain further insight in the specific role of the allantoic fluid, we included plasma and the amniotic fluid in this study. The work was carried out in 13- and 14-day-old chicken embryos. Using an HPLC-fluorometric technique, 40 of the 41 amino acids and related compounds investigated were detected. The amniotic fluid contained 32 compounds, while plasma and allantoic fluid contained 38 and 39 compounds, respectively. The glucose concentration was determined with a hexokinase technique. It was highest in plasma and lowest in the amniotic fluid. We identified three barriers that hyper- and hyporegulate a number of compounds: (1) a blood/allantois barrier, (2) a blood/amnion barrier, and (3) an allantois/amnion barrier. Compared with plasma and allantoic fluid, the amniotic fluid is a mostly hyporegulated environment.     

Evidence for novel fate of Flk1+ progenitor: contribution to muscle lineage

Flk1 is one of the specific cell surface receptors for vascular endothelial growth factor and one of the most specific markers highlighting the earliest stage of hematopoietic and vascular lineages. However, recent new evidence suggests that these Flk1(+) mesodermal progenitor cells also contribute to muscle lineages. All evidence is based on the experiments using in vitro differentiation and in vivo transplantation systems. Although this approach revealed a differentiation potential range of Flk1(+) cells that is wider than previously expected, it fails to determine whether Flk1(+) cells contribute to muscle lineage as part of the normal developmental process. To obtain direct evidence for the fate of Flk1(+) cells in development, we used a knock-in mouse line where Cre is expressed in Flk1(+) cells. Studies with these Cre lines provide direct evidence that Flk1(+) cells are progenitors for muscles, in addition to hematopoietic and vascular endothelial cells.     

Wnt2 coordinates the commitment of mesoderm to hematopoietic, endothelial, and cardiac lineages in embryoid bodies

Our recent gene expression profiling analyses demonstrated that Wnt2 is highly expressed in Flk1(+) cells, which serve as common progenitors of endothelial cells, blood cells, and mural cells. In this report, we characterize the role of Wnt2 in mesoderm development during embryonic stem (ES) cell differentiation by creating ES cell lines in which Wnt2 was deleted. Wnt2(-/-) embryoid bodies (EBs) generated increased numbers of Flk1(+) cells and blast colony-forming cells compared with wild-type EBs, and had higher Flk1 expression at comparable stages of differentiation. Although Flk1(+) cells were increased, we found that endothelial cell and terminal cardiomyocyte differentiation was impaired, but hematopoietic cell differentiation was enhanced and smooth muscle cell differentiation was unchanged in Wnt2(-/-) EBs. Later stage Wnt2(-/-) EBs had either lower or undetectable expression of endothelial and cardiac genes compared with wild-type EBs. Consistently, vascular plexi were poorly formed and neither beating cardiomyocytes nor alpha-actinin-staining cells were detectable in later stage Wnt2(-/-) EBs. In contrast, hematopoietic cell gene expression was upregulated, and the number of hematopoietic progenitor colonies was significantly enhanced in Wnt2(-/-) EBs. Our data indicate that Wnt2 functions at multiple stages of development during ES cell differentiation and during the commitment and diversification of mesoderm: as a negative regulator for hemangioblast differentiation and hematopoiesis but alternatively as a positive regulator for endothelial and terminal cardiomyocyte differentiation.     

Expression of genes associated with allantois emergence in ovine and bovine conceptuses

In the development of ruminant embryos, the emergence and growth of the allantois is critical for the establishment of the chorioallantoic placenta. The allantoic membrane contributes to all the vasculature that perfuses the placental tissues and the fetal membranes. Using suppressive subtractive hybridization to compare mRNA from Day 13 ovine preimplantation conceptuses (prior to allantoic emergence) with Day 17 allantoic membrane, we identified nine genes whose expression was associated with the emergence of the allantoic sac. Collagen alpha 1 type XII, collagen alpha 2 type I, collagen alpha 2 type V, epsilon 4 beta-globin, osteonectin, and uroplakin were expressed at significantly greater levels in ovine Day 17 allantois compared to Day 13 conceptuses. These genes are associated with the extracellular matrix and most likely are involved in establishing and strengthening the structural integrity of the allantoic sac and in the development of the blood vessels. RalB expression increased with development although at significantly greater levels in the allantois only at Day 19. Hoxa-10 and RhoA showed no differential expression during this period. All these genes showed a similar temporal pattern of expression in bovine conceptuses at equivalent stages of development with significantly greater expression of all these genes, except for Hoxa-10, found in Day 24 allantois compared to Day 14 conceptuses. This suggests that the role they play in allantoic emergence, growth and function is conserved in both ruminant species and that their expression is regulated in a similar manner. The interactions and regulation of this process remains to be fully explained.     

血管内皮细胞发育及分子机制

心血管系统是胚胎发育中最先形成的器官之一, 为机体提供营养成分和氧气。血管发育包括两部分, 一是内皮祖细胞(Angioblast)聚集形成血管原基(Vasculogenesis), 二是从已有血管形成新的血管分支(Angiogenesis)。此后由初级内皮细胞管召集平滑肌细胞形成功能性血管(Vessel maturation)。内皮祖细胞起源途径包括：由Flk1阳性中胚层细胞到成血成血管细胞(Hemangioblast)到血管内皮祖细胞; 或由Flk1阳性中胚层细胞直接到血管内皮祖细胞。Flk1阳性中胚层细胞受到vegf、flk1、cloche、lycat、etsrp等关键基因或信号通路的调节, 其中核心问题是原肠期中胚层如何形成Flk1阳性中胚层细胞及进一步分化成血管内皮祖细胞和成血血管细胞。文章集中评述内皮祖细胞发育、分化及其分子遗传调控机制, 并展望本领域未来发展方向。     

Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation

T-box gene family members have important roles during murine embryogenesis, gastrulation, and organogenesis. Although relatively little is known about how T-box genes are regulated, published gene expression studies have revealed dynamic and specific patterns in both embryonic and extraembryonic tissues of the mouse conceptus. Mutant alleles of the T-box gene Brachyury (T) have identified roles in formation of mesoderm and its derivatives, such as somites and the allantois. However, given the cell autonomous nature of T gene activity and conflicting results of gene expression studies, it has been difficult to attribute a primary function to T in normal allantoic development. We report localization of T protein by sectional immunohistochemistry in both embryonic and extraembryonic tissues during mouse gastrulation, emphasizing T localization within the allantois. T was detected in all previously reported sites within the conceptus, including the primitive streak and its derivatives, nascent embryonic mesoderm, the node and notochord, as well as notochord-associated endoderm and posterior neurectoderm. In addition, we have clarified T within the allantois, where it was first detected in the proximal midline of the late allantoic bud (approximately 7.5 days postcoitum, dpc) and persisted within an expanded midline domain until 6-somite pairs (s; approximately 8.5 dpc). Lastly, we have discovered several novel T sites, including the developing heart, visceral endoderm, extraembryonic ectoderm, and its derivative, chorionic ectoderm. Together, these data provide a unified picture of T in the mammalian conceptus, and demonstrate T's presence in unrelated cell types and tissues in highly dynamic spatiotemporal patterns in both embryonic and extraembryonic tissues.