MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.     

Vimentin downregulation is an inherent feature of murine erythropoiesis and occurs independently of lineage.

In mammalian erythropoiesis, the mature cells of the primitive lineage remain nucleated while those of the definitive lineage are anuclear. One of the molecular and structural changes that precedes enucleation in cells of the definitive lineage is the cessation in the expression of the gene for the intermediate filament (IF) protein vimentin and the removal of all vimentin filaments from the cytoplasm. We show here that in immature primitive cells vimentin is synthesized and forms a cytoplasmic network of IFs. As differentiation proceeds in vivo, vimentin gene expression is downregulated in these cells; this is accompanied by the loss of vimentin filaments from the cytoplasm. This loss temporally coincides with the nucleus becoming freely mobile within the cytoplasm, suggesting that, while IF removal is not directly linked to the physical process of enucleation, it may be a prerequisite for the initiation of nuclear mobility in both lineages. These changes are also observed in early primitive cells cultured in vitro, suggesting that they constitute an intrinsic part of the murine erythroid differentiation program independent of lineage and hematopoietic microenvironment.     

The inactivation of the π gene in chicken erythroblasts of adult lineage is not mediated by packaging of the embryonic part of the α-globin gene domain into a repressive heterochromatin-like structure

The developmental switch of globin gene expression is a characteristic feature of vertebrate organisms. The switch of β-globin expression is believed to depend on reconfiguration of the active chromatin hub, which contains transcribed genes and regulatory elements. Mechanisms controlling the switch of α-globin gene expression are less clear. Here, we studied the mode of chromatin packaging of the chicken α-globin gene domain in red blood cells (RBCs) of primitive and definite lineages and the spatial configuration of this domain in RBCs of primitive lineage. It has been demonstrated that RBCs of primitive lineage already contain the adult-type active chromatin hub but the embryonal α-type globin π gene is not recruited to this hub. Distribution of active and repressive histone modifications over the α-globin gene domain in RBCs of definite and primitive lineages does not corroborate the hypothesis that inactivation of the π gene in RBCs of adult lineage is mediated via formation of a local repressed chromatin domain. This conclusion is supported by the demonstration that in chicken erythroblasts of adult lineage, the embryonal and adult segments of the α-globin gene domain show similar elevated sensitivities to DNase I.     

The inactivation of the π gene in chicken erythroblasts of adult lineage is not mediated by packaging of the embryonic part of the α-globin gene domain into a repressive heterochromatin-like structure

The developmental switch of globin gene expression is a characteristic feature of vertebrate organisms. The switch of β-globin expression is believed to depend on reconfiguration of the active chromatin hub, which contains transcribed genes and regulatory elements. Mechanisms controlling the switch of α-globin gene expression are less clear. Here, we studied the mode of chromatin packaging of the chicken α-globin gene domain in red blood cells (RBCs) of primitive and definite lineages and the spatial configuration of this domain in RBCs of primitive lineage. It has been demonstrated that RBCs of primitive lineage already contain the adult-type active chromatin hub but the embryonal α-type globin π gene is not recruited to this hub. Distribution of active and repressive histone modifications over the α-globin gene domain in RBCs of definite and primitive lineages does not corroborate the hypothesis that inactivation of the π gene in RBCs of adult lineage is mediated via formation of a local repressed chromatin domain. This conclusion is supported by the demonstration that in chicken erythroblasts of adult lineage, the embryonal and adult segments of the α-globin gene domain show similar elevated sensitivities to DNase I.     

Manipulation of Cell:Cell Contacts and Mesoderm Suppressing Activity Direct Lineage Choice from Pluripotent Primitive Ectoderm-Like Cells in Culture

In the mammal, the pluripotent cells of embryo differentiate and commit to either the mesoderm/endoderm lineages or the ectoderm lineage during gastrulation. In culture, the ability to direct lineage choice from pluripotent cells into the mesoderm/endoderm or ectoderm lineages will enable the development of technologies for the formation of highly enriched or homogenous populations of cells. Here we show that manipulation of cell:cell contact and a mesoderm suppressing activity in culture affects the outcome of pluripotent cell differentiation and when both variables are manipulated appropriately they can direct differentiation to either the mesoderm or ectoderm lineage. The disruption of cell:cell contacts and removal of a mesoderm suppressor activity results in the differentiation of pluripotent, primitive ectoderm-like cells to the mesoderm lineage, while maintenance of cell:cell contacts and inclusion, within the culture medium, of a mesoderm suppressing activity results in the formation of near homogenous populations of ectoderm. Understanding the contribution of these variables in lineage choice provides a framework for the development of directed differentiation protocols that result in the formation of specific cell populations from pluripotent cells in culture.     

Comparative gene expression analysis and fate mapping studies suggest an early segregation of cardiogenic lineages in Xenopus laevis

Retrospective clonal analysis in mice suggested that the vertebrate heart develops from two sources of cells called first and second lineages, respectively. Cells of the first lineage enter the linear heart tube and initiate terminal differentiation earlier than cells of the second lineage. It is thought that both heart lineages arise from a common progenitor cell population prior to the cardiac crescent stage (E7.5 of mouse development). The timing of segregation of different lineages as well as the molecular mechanisms underlying this process is not yet known. Furthermore, gene expression data for those lineages are very limited. Here we provide the first comparative study of cardiac marker gene expression during Xenopus laevis embryogenesis complemented by single cell RT-PCR analysis. In addition we provide fate mapping data of cardiac progenitor cells at different stages of development. Our analysis indicates an early segregation of cardiac lineages and a fairly complex heterogeneity of gene expression in the cardiac progenitor cells. Furthermore, this study sets a reference for all further studies analyzing cardiac development in X. laevis.     

Development of erythropoiesis in the mouse

Hematopoiesis consists of a small number of stem cells which rise to a progeny of several distinct lineages. Among these, cells of the erythroid lineage emerge early in the mammalian embryo and provide indispensable functions throughout gestation and postnatal life. Knowledge of the existence of two distinct erythroid lineages, traditionally known as primitive and definitive, has more than a century. In the last decade, it has become evident that the ontogeny of erythropoiesis is more complex than a simple two-steps model. This review summarizes the development and differentiation of the erythroid lineage cells during gestation, and also the characteristics of erythropoiesis during the infant stage, discussing which issues remain open and which need further research.     

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile.     

Intercellular Interactions,Position, and Polarity in Establishing Blastocyst Cell Lineages and Embryonic Axes

The formation of the three lineages of the mouse blastocyst provides a powerful model system to study interactions among cell behavior, cell signaling, and lineage development. Hippo signaling differences between the inner and outer cells of the early cleavage stages, combined with establishment of a stably polarized outer epithelium, lead to the establishment of the inner cell mass and the trophectoderm, whereas FGF signaling differences among the individual cells of the ICM lead to gradual separation and segregation of the epiblast and primitive endoderm lineages. Events in the late blastocyst lead to the formation of a special subset of cells from the primitive endoderm that are key sources for the signals that establish the subsequent body axis. The slow pace of mouse early development, the ability to culture embryos over this time period, the increasing availability of live cell imaging tools, and the ability to modify gene expression at will are providing increasing insights into the cell biology of early cell fate decisions.     

A system to enrich for primitive streak-derivatives, definitive endoderm and mesoderm, from pluripotent cells in culture

Two lineages of endoderm develop during mammalian embryogenesis, the primitive endoderm in the pre-implantation blastocyst and the definitive endoderm at gastrulation. This complexity of endoderm cell populations is mirrored during pluripotent cell differentiation in vitro and has hindered the identification and purification of the definitive endoderm for use as a substrate for further differentiation. The aggregation and differentiation of early primitive ectoderm-like (EPL) cells, resulting in the formation of EPL-cell derived embryoid bodies (EPLEBs), is a model of gastrulation that progresses through the sequential formation of primitive streak-like intermediates to nascent mesoderm and more differentiated mesoderm populations. EPL cell-derived EBs have been further analysed for the formation of definitive endoderm by detailed morphological studies, gene expression and a protein uptake assay. In comparison to embryoid bodies derived from ES cells, which form primitive and definitive endoderm, the endoderm compartment of embryoid bodies formed from EPL cells was comprised almost exclusively of definitive endoderm. Definitive endoderm was defined as a population of squamous cells that expressed Sox17, CXCR4 and Trh, which formed without the prior formation of primitive endoderm and was unable to endocytose horseradish peroxidase from the medium. Definitive endoderm formed in EPLEBs provides a substrate for further differentiation into specific endoderm lineages; these lineages can be used as research tools for understanding the mechanisms controlling lineage establishment and the nature of the transient intermediates formed. The similarity between mouse EPL cells and human ES cells suggests EPLEBs can be used as a model system for the development of technologies to enrich for the formation of human ES cell-derived definitive endoderm in the future.     

The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.     

A chimaeric animal model for confined placental mosaicism

The reason for chromosome mosaicism being sometimes confined to only part of the conceptus is unknown. To address this problem, we produced tetraploid diploid chimaeric mouse conceptuses. At 12 1/2 days, no tetraploid cells were detected in the fetus. They rarely contributed to other derivatives of the primitive ectoderm lineage but were commonly found in the primitive endoderm and trophectoderm lineages. This provides a useful animal model of human confined placental mosaicism and suggests that the primitive endoderm (hypoblast) lineage should be included in future studies of human mosaic conceptuses.     

Influence of chromosomal determinants on development of androgenetic and parthenogenetic cells

We have examined the role of germline-specific chromosomal determinants of development in the mouse. Studies were carried out using aggregation chimaeras between androgenetic----fertilized embryos and compared with similar parthenogenetic----fertilized chimaeras. Several adult chimaeras were found with parthenogenetic cells but none were found with androgenetic cells. Analysis of chimaeras at mid-gestation showed that parthenogenetic cells were detected in the embryo and yolk sac but that androgenetic cells were found only in the trophoblast and yolk sac and not in the embryo. The contribution of parthenogenetic cells to the embryo and yolk sac was increased by aggregating 2-cell parthenogenetic and 4-cell fertilized embryos but the contribution of parthenogenetic cells in extraembryonic tissues remained negligible even after aggregation of 4-cell parthenogenetic and 2-cell fertilized embryos. Furthermore, parthenogenetic cells were primarily found in the yolk sac mesoderm and not in the yolk sac endoderm. These results suggest that maternal chromosomes in parthenogenetic cells permit their participation in the primitive ectoderm lineage but these cells are presumably eliminated by selective pressure or autonomous cell lethality from the primitive endoderm and trophectoderm lineages. Conversely paternal chromosomes in androgenetic cells confer opposite properties since the embryonic cells can be detected in the trophoblast and the yolk sac but not in the embryos, presumably because they are eliminated from the primitive ectoderm lineage. The spatial distribution of cells with different parental chromosomes may occur partly because of differential expression of some genes, such as proto-oncogenes, and partly due to their ability to respond to a variety of diffusible growth factors.     

Islet 1 is expressed in distinct cardiovascular lineages, including pacemaker and coronary vascular cells

Islet1 (Isl1) is a LIM homedomain protein that plays a pivotal role in cardiac progenitors of the second heart field. Here, lineage studies with an inducible isl1-cre demonstrated that most Isl1 progenitors have migrated into the heart by E9. Although Isl1 expression is downregulated in most cardiac progenitors as they differentiate, analysis of an isl1-nlacZ mouse and coimmunostaining for Isl1 and lineage markers demonstrated that Isl1 is expressed in distinct subdomains of the heart, and in diverse cardiovascular lineages. Isl1 expression was observed in myocardial lineages of the distal outflow tract, atrial septum, and in sinoatrial and atrioventricular node. The myocardialized septum of the outflow tract was found to derive from Isl1 expressing cells. Isl1 expressing cells also contribute to endothelial and vascular smooth muscle lineages including smooth muscle of the coronary vessels. Our data indicate that Isl1 is a specific marker for a subset of pacemaker cells at developmental stages examined, and suggest genetic heterogeneity within the central conduction system and coronary smooth muscle. Our studies suggest a role for Isl1 in these distinct domains of expression within the heart.