Angiogenesis is the generation of mature vascular networks from pre-existing vessels. Angiogenesis is crucial during the organism' development, for wound healing and for the female reproductive ...cycle. Several murine experimental systems are well suited for studying developmental and pathological angiogenesis. They include the embryonic hindbrain, the post-natal retina and allantois explants. In these systems vascular networks are visualised by appropriate staining procedures followed by microscopical analysis. Nevertheless, quantitative assessment of angiogenesis is hampered by the lack of readily available, standardized metrics and software analysis tools. Non-automated protocols are being used widely and they are, in general, time--and labour intensive, prone to human error and do not permit computation of complex spatial metrics. We have developed a light-weight, user friendly software, AngioTool, which allows for quick, hands-off and reproducible quantification of vascular networks in microscopic images. AngioTool computes several morphological and spatial parameters including the area covered by a vascular network, the number of vessels, vessel length, vascular density and lacunarity. In addition, AngioTool calculates the so-called "branching index" (branch points/unit area), providing a measurement of the sprouting activity of a specimen of interest. We have validated AngioTool using images of embryonic murine hindbrains, post-natal retinas and allantois explants. AngioTool is open source and can be downloaded free of charge.
The allantois-derived umbilical component of the chorio-allantoic placenta shuttles fetal blood to and from the chorion, thereby ensuring fetal-maternal exchange. The progenitor populations that ...establish and supply the fetal-umbilical interface lie, in part, within the base of the allantois, where the germ line is claimed to segregate from the soma. Results of recent studies in the mouse have reported that STELLA (DPPA-3, PGC7) co-localizes with PRDM1 (BLIMP1), the bimolecular signature of putative primordial germ cells (PGCs) throughout the fetal-placental interface. Thus, if PGCs form extragonadally within the posterior region of the mammal, they cannot be distinguished from the soma on the basis of these proteins. We used immunohistochemistry, immunofluorescence, and confocal microscopy of the mouse gastrula to co-localize STELLA with a variety of gene products, including pluripotency factor OCT-3/4, mesendoderm-associated T and MIXl1, mesendoderm- and endoderm-associated FOXa2 and hematopoietic factor Runx1. While a subpopulation of cells localizing OCT-3/4 was always found independently of STELLA, STELLA always co-localized with OCT-3/4. Despite previous reports that T is involved in specification of the germ line, co-localization of STELLA and T was detected only in a small subset of cells in the base of the allantois. Slightly later in the hindgut lip, STELLA+/(OCT-3/4+) co-localized with FOXa2, as well as with RUNX1, indicative of definitive endoderm and hemangioblasts, respectively. STELLA was never found with MIXl1. On the basis of these and previous results, we conclude that STELLA identifies at least five distinct cell subpopulations within the allantois and hindgut, where they may be involved in mesendodermal differentiation and hematopoiesis at the posterior embryonic-extraembryonic interface. These data provide a new point of departure for understanding STELLA's potential roles in building the fetal-placental connection.
•STELLA co-localizes with mesendodermal proteins at the fetal-placental interface.•STELLA only rarely – and transiently – co-localizes with Brachyury.•STELLA is not a specific identifier of germ cell antecedents in the gastrula.
The allantois is the embryonic precursor of the umbilical cord in mammals and is one of several embryonic regions, including the yolk sac and dorsal aorta, that undergoes vasculogenesis, the de novo ...formation of blood vessels. Despite its importance in establishing the chorioallantoic placenta and umbilical circulation, the allantois frequently is overlooked in embryologic studies. Nonetheless, recent studies demonstrate that vasculogenesis, vascular remodeling, and angiogenesis are essential allantois functions in the establishment of the chorioallantoic placenta. Here, we review blood vessel formation in the murine allantois, highlighting the expression of genes and involvement of pathways common to vasculogenesis or angiogenesis in other parts of the embryo. We discuss experimental techniques available for manipulation of the allantois that are unavailable for yolk sac or dorsal aorta, and review how this system has been used as a model system to discover new genes and mechanisms involved in vessel formation. Finally, we discuss the potential of the allantois as a model system to provide insights into disease and therapeutics.
Cdx gene products regulate the extent of axial elongation from the posterior growth zone. These transcription factors sustain the emergence of trunk and tail tissues by providing a suitable niche in ...the axial progenitor zone, via regulation of Wnt signaling. Cdx genes are expressed in and along the complete primitive streak including its posterior part wherefrom the extraembryonic mesoderm of the allantois emerges. Cdx genes are required for the full development of the allantois and its derivatives in the placental labyrinth. The mouse germ cell lineage also originates from the proximo-posterior epiblast of the primitive streak, and is established within the extraembryonic mesoderm that generates the allantois. We asked whether the expression of Cdx genes around the newly specified PGCs is necessary for the maintenance and expansion of this population, as it is for the allantois and axial progenitors. We observed a significantly lower number of PGCs in Cdx2null embryos than in controls. We found that Wnt3a loss of function decreases the PGC population to the same extent as Cdx2 inactivation. Moreover, exogenous Wnt3a corrects the lower PGC number in Cdx2null posterior embryonic tissues cultured in vitro. Cdx2 is not expressed in PGCs themselves, and we propose that the expression of Cdx2 in posterior extraembryonic tissues contributes to the proper niche of the germ cell progenitors by stimulating canonical Wnt signaling. Since PGC residence within the posterior growth zone is a mouse-specific feature, our data suggest that mouse PGCs opportunistically became dependent on the axial progenitor niche.
► The Cdx2null mutation affects derivatives of the posterior extraembryonic mesoderm. ► The specified PGC population does not expand in Cdx2null mutant embryos. ► Wnt3anull embryos have a reduced PGC population. ► The PGC population of Cdx2null posterior tissues is corrected to wild type level in the presence of added Wnt3a.
•Hypoblast/visceral endoderm of Placentalia may be hematopoietic.•Putative blood cells have been observed in the hypoblast layer of the yolk sac.•Mouse allantois-associated visceral endoderm yields ...putative blood cells.
The extra-embryonic hypoblast/visceral endoderm of Placentalia carries out a variety of functions during gestation, including hematopoietic induction. Results of decades-old and recent experiments have provided compelling evidence that, in addition to its inducing properties, hypoblast/visceral endoderm itself is a source of placental blood cells. Those observations that highlight extra-embryonic endoderm's role as an overlooked source of placental blood cells across species are briefly discussed here, with suggestions for future exploration.
The embryonic-maternal interface of the placental labyrinth, allantois, and yolk sac are vital during embryogenesis; however, the precise mechanism underlying the vascularization of these structures ...remains unknown. Herein we focus on the role of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), which are intracellular Ca2+ release channels, in placentation. Double knockout (DKO) of type 1 and 3 IP3Rs (IP3R1 and IP3R3, respectively) in mice resulted in embryonic lethality around embryonic day (E) 11.5. Because IP3R1 and IP3R3 were co-expressed in endothelial cells in the labyrinth, allantois, and yolk sac, we investigated extra-embryonic vascular development in IP3R1- and IP3R3-DKO mice. The formation of chorionic plates and yolk sac vessels seemed dysregulated around the timing of the chorio-allantoic attachment, immediately followed by the disorganization of allantoic vessels, the decreased expression of the spongiotrophoblast cell marker Tpbpa and the growth retardation of the embryos in DKO mice. Fluorescent immunohistochemistry demonstrated downregulation of a vascular endothelial marker, CD31, in labyrinth embryonic vessels and poor elongation of extra-embryonic mesoderm into the labyrinth layer in DKO placenta, whereas the branching of the DKO chorionic trophoblast was initiated. In addition, allantoic and yolk sac vessels in extra-embryonic tissues were less remodeled in DKO mice. In vitro endothelial cord formation and migration activities of cultured vascular endothelial cells derived from human umbilical vein were downregulated under the inhibition of IP3R. Our results suggest that IP3R1 and IP3R3 are required for extra-embryonic vascularization in the placenta, allantois, and yolk sac. This is the first demonstration of the essential role of IP3/IP3Rs signaling in the development of the vasculature at the embryonic-maternal interface.
•IP3R type 1 (IP3R1) and type 3 (IP3R3) were expressed in extra-embryonic tissues.•IP3R1 and IP3R3 were expressed in vascular endothelium.•IP3R1−/−IP3R3−/− mutant embryos were embryonic lethal.•Poor elongation of extra-embryonic mesoderm into the labyrinth of IP3R1−/−IP3R3−/− mutants.•The vessels in IP3R1−/−IP3R3−/− allantois and yolk sac were disorganized.
Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro
, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, ...including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells
. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.
Steel factor is an essential survival and proliferation factor for primordial germ cells (PGCs) during their migration in the early mouse embryo. PGCs arise during gastrulation, and migrate into the ...posterior endoderm that becomes the hindgut. Previous reports have suggested that PGCs become dependent on Steel factor when they colonize the hindgut. However, in the absence of a good marker for living PGCs, their behavior before hindgut colonization has not been previously studied. We report here the normal behavior of PGCs in live embryos before hindgut colonization, and the roles of Steel factor, using a reporter line in which GFP is driven by the promoter of the Stella gene, whose activation accompanies the initial specification of PGCs. We show first that PGCs are surrounded by Steel factor-expressing cells from their first appearance in the allantois to the time they enter the genital ridges. Second, fewer PGCs are found in the allantois in Steel-null embryos, but this is not due to a failure of PGC specification. Third, the analysis of cultured Steel-null early embryos shows that Steel factor is required for normal PGC motility, both in the allantois and in the hindgut. Germ cells migrate actively in the allantois, and move directionally from the allantois into the proximal epiblast. In the absence of Steel factor, caused by either null mutation or antibody blockade, PGC motility is dramatically decreased, but directionality is maintained, demonstrating a primary role for Steel factor in PGC motility. This was found both before and after colonization of the hindgut. These data, together with previously published data, show that PGCs are Steel factor dependent from their initial specification until they colonize the genital ridges, and suggest the existence of a ;spatio-temporal niche' that travels with this important pluripotential cell population in the embryo.
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