Regionalization of the mouse visceral endoderm as the blastocyst transforms into the egg cylinder.

BACKGROUND: Reciprocal interactions between two extra-embryonic tissues, the extra-embryonic ectoderm and the visceral endoderm, and the pluripotent epiblast, are required for the establishment of anterior-posterior polarity in the mouse. After implantation, two visceral endoderm cell types can be distinguished, in the embryonic and extra-embryonic regions of the egg cylinder. In the embryonic region, the specification of the anterior visceral endoderm (AVE) is central to the process of anterior-posterior patterning. Despite recent advances in our understanding of the molecular interactions underlying the differentiation of the visceral endoderm, little is known about how cells colonise the three regions of the tissue. RESULTS: As a first step, we performed morphological observations to understand how the extra-embryonic region of the egg cylinder forms from the blastocyst. Our analysis suggests a new model for the formation of this region involving cell rearrangements such as folding of the extra-embryonic ectoderm at the early egg cylinder stage. To trace visceral endoderm cells, we microinjected mRNAs encoding fluorescent proteins into single surface cells of the inner cell mass of the blastocyst and analysed the distribution of labelled cells at E5.0, E5.5 and E6.5. We found that at E5.0 the embryonic and extra-embryonic regions of the visceral endoderm do not correspond to distinct cellular compartments. Clusters of labelled cells may span the junction between the two regions even after the appearance of histological and molecular differences at E5.5. We show that in the embryonic region cell dispersion increases after the migration of the AVE. At this time, visceral endoderm cell clusters tend to become oriented parallel to the junction between the embryonic and extra-embryonic regions. Finally we investigated the origin of the AVE and demonstrated that this anterior signalling centre arises from more than a single precursor between E3.5 and E5.5. CONCLUSION: We propose a new model for the formation of the extra-embryonic region of the egg cylinder involving a folding of the extra-embryonic ectoderm. Our analyses of the pattern of labelled visceral endoderm cells indicate that distinct cell behaviour in the embryonic and extra-embryonic regions is most apparent upon AVE migration. We also demonstrate the polyclonal origin of the AVE. Taken together, these studies lead to further insights into the formation of the extra-embryonic tissues as they first develop after implantation.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation

Initiation of the development of the anterior–posterior axis in the mouse embryo has been thought to take place only when the anterior visceral endoderm (AVE) emerges and starts its asymmetric migration. However, expression of Lefty1, a marker of the AVE, was recently found to initiate before embryo implantation. This finding has raised two important questions: are the cells that show such early, preimplantation expression of this AVE marker the real precursors of the AVE and, if so, how does this contribute to the establishment of the AVE? Here, we address both of these questions. First, we show that the expression of another AVE marker, Cer1, also commences before implantation and its expression becomes consolidated in the subset of ICM cells that comprise the primitive endoderm. Second, to determine whether the cells showing this early Cer1 expression are true precursors of the AVE, we set up conditions to trace these cells in time-lapse studies from early periimplantation stages until the AVE emerges and becomes asymmetrically displaced. We found that Cer1-expressing cells are asymmetrically located after implantation and, as the embryo grows, they become dispersed into two or three clusters. The expression of Cer1 in the proximal domain is progressively diminished, whilst it is reinforced in the distal–lateral domain. Our time-lapse studies demonstrate that this distal–lateral domain is incorporated into the AVE together with cells in which Cer1 expression begins only after implantation. Thus, the AVE is formed from both part of an ancestral population of Cerl-expressing cells and cells that acquire Cer1 expression later. Finally, we demonstrate that when the AVE shifts asymmetrically to establish the anterior pole, this occurs towards the region where the earlier postimplantation expression of Cer1 was strongest. Together, these results suggest that the orientation of the anterior–posterior axis is already anticipated before AVE migration

Cardiac cell lineages that form the heart.

International audienceMyocardial cells ensure the contractility of the heart, which also depends on other mesodermal cell types for its function. Embryological experiments had identified the sources of cardiac precursor cells. With the advent of genetic engineering, novel tools have been used to reconstruct the lineage tree of cardiac cells that contribute to different parts of the heart, map the development of cardiac regions, and characterize their genetic signature. Such knowledge is of fundamental importance for our understanding of cardiogenesis and also for the diagnosis and treatment of heart malformations

Landmarks and lineages in the developing heart.

Comment on The Tbx2+ primary myocardium of the atrioventricular canal forms the atrioventricular node and the base of the left ventricle. [Circ Res. 2009]International audienceThe primary myocardium of the embryonic heart, including the atrioventricular canal and outflow tract, is essential for septation and valve formation. In the chamber-forming heart, the expression of the T-box transcription factor Tbx2 is restricted to the primary myocardium. To gain insight into the cellular contributions of the Tbx2+ primary myocardium to the components of the definitive heart, genetic lineage tracing was performed using a novel Tbx2Cre allele. These analyses revealed that progeny of Tbx2+ cells provide an unexpectedly large contribution to the Tbx2-negative ventricles. Contrary to common assumption, we found that the embryonic left ventricle only forms the left part of the definitive ventricular septum and the apex. The atrioventricular node, but not the atrioventricular bundle, was found to derive from Tbx2+ cells. The Tbx2+ outflow tract formed the right ventricle and right part of the ventricular septum. In Tbx2-deficient embryos, the left-sided atrioventricular canal was found to prematurely differentiate to chamber myocardium and to proliferate at increased rates similar to those of chamber myocardium. As a result, the atrioventricular junction and base of the left ventricle were malformed. Together, these observations indicate that Tbx2 temporally suppresses differentiation and proliferation of primary myocardial cells. A subset of these Tbx2Cre-marked cells switch off expression of Tbx2, which allows them to differentiate into chamber myocardium, to initiate proliferation, and to provide a large contribution to the ventricles. These findings imply that errors in the development of the early atrioventricular canal may affect a much larger region than previously anticipated, including the ventricular base

Imaging and analyzing primary cilia in cardiac cells.

International audienceThe primary cilium is a small sensory organelle that is required for different aspects of embryonic development, including the formation of the heart. The structure and composition of cilia have been extensively studied, so that several markers of primary cilia have now been identified. However, the role of cilia in specific cell types remains poorly understood. We describe here a series of approaches to image primary cilia in the rodent heart or in primary cultures of cells dissociated from the heart. As the cilium is a marker of cell polarity, we also provide, for quantitative image analysis of cilium orientation, tools which are generally applicable to other types of tissues

Oriented clonal cell growth in the developing mouse myocardium underlies cardiac morphogenesis.

International audienceDuring heart morphogenesis, cardiac chambers arise by differential expansion of regions of the primitive cardiac tube. This process is under the control of specific transcription factors such as Tbx5 and dHAND. To gain insight into the cellular mechanisms that underlie cardiogenesis, we have used a retrospective clonal approach based on the spontaneous recombination of an nlaacZ reporter gene targeted to the murine alpha-cardiac actin locus. We show that clonal growth of myocardial cells is oriented. At embryonic day (E) 10.5, the shape of clones is characteristic of a given cardiac region and reflects its morphology. This is already detectable in the primitive cardiac tube at E8.5, and is maintained after septation at E14.5 with additional modulations. The clonal analysis reveals new subdivisions of the myocardium, including an interventricular boundary region. Our results show that the myocardium, from the time of its formation, is a polarized and regionalized tissue and point to the role of oriented clonal cell growth in cardiac chamber morphogenesis

Smooth muscle of the dorsal aorta shares a common clonal origin with skeletal muscle of the myotome.

We show that cells of the dorsal aorta, an early blood vessel, and of the myotome, the first skeletal muscle to form within the somite, derive from a common progenitor in the mouse embryo. This conclusion is based on a retrospective clonal analysis, using a nlaacZ reporter targeted to the alpha-cardiac actin gene. A rare intragenic recombination event results in a functional nlacZ sequence, giving rise to clones of beta-galactosidase-positive cells. Periendothelial and vascular smooth muscle cells of the dorsal aorta are the main cell types labelled, demonstrating that these are clonally related to the paraxial mesoderm-derived cells of skeletal muscle. Rare endothelial cells are also seen in some clones. In younger clones, arising from a recent recombination event, myotomal labelling is predominantly in the hypaxial somite, adjacent to labelled smooth muscle cells in the aorta. Analysis of Pax3(GFP/+) embryos shows that these cells are Pax3 negative but GFP positive, with fluorescent cells in the intervening region between the aorta and the somite. This is consistent with the direct migration of smooth muscle precursor cells that had expressed Pax3. These results are discussed in terms of the paraxial mesoderm contribution to the aorta and of the mesoangioblast stem cells that derive from it

Right ventricular myocardium derives from the anterior heart field.

International audienceThe mammalian heart develops from a primary heart tube, which is formed by fusion of bilateral cardiac territories in which myocardial and endothelial cells have already begun to differentiate from splanchnic mesoderm. A population of myocardial precursors has been identified in pharyngeal mesoderm, anterior to the early heart tube. Cell labeling studies have indicated that this novel territory, called the anterior heart field (AHF), gives rise to the myocardial wall of the outflow tract. We now report that not only the myocardium of the outflow tract but also myocardial cells of the embryonic right ventricle are derived from this source. Explants of pharyngeal mesoderm or of the early heart tube were cultured from transgenic mice in which transgene expression marks different regions of the heart. Pharyngeal mesoderm from 5 to 7 somite embryos gives rise to cardiomyocytes with right ventricular and outflow tract identities, whereas the heart tube as this stage has an essentially left ventricular identity. DiI labeling confirms that the early heart tube is destined to contribute to the embryonic left ventricle and indicates that right ventricular myocardium is added from extracardiac mesoderm. Retrospective clonal analysis of the heart at embryonic day (E) 10.5 reveals the existence of a clonal boundary in the interventricular region, which appears during ventricular septation, underlining different origins of the two ventricular compartments. This study demonstrates the differences in the embryological origin of right and left ventricular myocardium, which has important implications for congenital heart disease

A retrospective clonal analysis of the myocardium reveals two phases of clonal growth in the developing mouse heart.

International audienceKey molecules which regulate the formation of the heart have been identified; however, the mechanism of cardiac morphogenesis remains poorly understood at the cellular level. We have adopted a genetic approach, which permits retrospective clonal analysis of myocardial cells in the mouse embryo, based on the targeting of an nlaacZ reporter to the alpha-cardiac actin gene. A rare intragenic recombination event leads to a clone of beta-galactosidase-positive myocardial cells. Analysis of clones at different developmental stages demonstrates that myocardial cells and their precursors follow a proliferative mode of growth, rather than a stem cell mode, with an initial dispersive phase, followed by coherent cell growth. Clusters of cells are dispersed along the venous-arterial axis of the heart tube. Coherent growth is oriented locally, with a main axis, which corresponds to the elongation of the cluster, and rows of cells, which form secondary axes. The angle between the primary and secondary axes varies, indicating independent events of growth orientation. At later stages, as the ventricular wall thickens, wedge shaped clusters traverse the wall and contain rows of cells at a progressive angle to each other. The cellular organisation of the myocardium appears to prefigure myofibre architecture. We discuss how the characteristics of myocardial cell growth, which we describe, underlie the formation of the heart tube and its subsequent regionalised expansion