Endoderm development in vertebrates: fate mapping, induction and regional specification

The formation of the vertebrate body plan begins with the differentiation of cells into three germ layers: ectoderm, mesoderm and endoderm. Cells in the endoderm give rise to the epithelial lining of the digestive tract, associated glands and respiratory system. One of the fundamental problems in developmental biology is to elucidate how these three primary germ layers are established from the homologous population of cells in the early blastomere. To address this question, ectoderm and mesoderm development have been extensively analyzed, but study of endoderm development has only begun relatively recently. In this review, we focus on the 'where', 'when' and 'how' of endoderm development in four vertebrate model organisms: the zebrafish, Xenopus, chick and mouse. We discuss the classical fate mapping of the endoderm and the more recent progress in characterizing its induction, segregation and regional specification.     

Cell movements during gastrulation: come in and be induced

The conversion of an epithelial monolayer into a multilayered structure consisting of the three germ layers, ectoderm, mesoderm and endoderm, constitutes a conserved theme in the early development of animals. This is accomplished by morphogenetic movements that occur during gastrulation and serve not only to generate shape but also to ensure that cells receive the right signals at the right time. Recent evidence of the role of molecular interactions facilitated by cell movements in continuously defining the chick 'organizer' during gastrulation challenges the notion that it is a fixed cell population derived from an exclusive cell lineage.     

Changing patterns of cytokeratins and vimentin in the early chick embryo

The distribution of cytokeratins and vimentin intermediate filaments in the first 48 h of chick development has been determined using immunofluorescent labelling. During formation of the germ layers, cytokeratin expression is associated with the appearance of an integral epithelium (ectoderm), whereas vimentin expression is associated with cells that detach and migrate from this epithelium to form endoderm and mesoderm. Subsequently, vimentin persists in the endoderm and mesoderm and the tissues derived therefrom, such as the somites and developing heart, throughout the period of study. The appearance of cytokeratins at later stages of development occurs in some epithelia such as the ectoderm, endoderm, lateral plate and epimyocardium but not others including the neural plate, neural tube and somites. Expression of cytokeratins in endoderm and mesenchymal tissues occurs in tandem with vimentin. In conclusion, vimentin expression is related to its distribution in the epiblast before germ layer formation. Its initial appearance may be related to the motile behaviour of cells about to ingress through the primitive streak. The appearance of cytokeratin filaments, however, does not reflect germ layer derivation but rather the need for an epithelial sheet.     

Phospholipid and nucleic acid gradients in the developing amphibian embryo

Rana pipiens embryos at the end of the blastula stage were dissociated and the cell suspension was separated into presumptive ectoderm, mesoderm, light endoderm, and heavy endoderm cells by a discontinuous density gradient centrifugation technique. The isolated germ layers were analyzed for total lipid, lipid phosphorus, plasmalogen, RNA, and DNA. Per gram dry weight, DNA showed a threefold decrease from ectoderm to heavy endoderm. On the same basis, the RNA content of the mesoderm was 34 per cent higher than that of ectoderm, and 320 and 570 per cent higher than that of light and heavy endoderm, respectively. In addition to the RNA and DNA gradients, there were at least two superimposed lipid gradients: a neutral lipid gradient decreasing from ectoderm to endoderm, and a total phospholipid gradient increasing from ectoderm to endoderm. In contrast to total phospholipid, a specific phospholipid class, ethanolamine plasmalogen, decreased from ectoderm to endoderm. The total lipid content per gram dry weight was the same in all the germ layers. Total phospholipids were analyzed quantitatively by thin layer chromatography. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and inositol phospholipid constituted 34, 13, 12, and 34 per cent, respectively, of the total lipid phosphorus. The phospholipid composition was different in each germ layer. The possible role of specific lipids in embryonic induction and differentiation is discussed.     

Expression of the cell surface-associated glycoprotein, fibronectin, in the early mouse embryo.

The expression of the cell surface-associated glycoprotein fibronectin was studied by indirect immunofluorescence in the early stages of mouse embryogenesis. Fibronectin was not detectable in early preimplantation embryos. Trace amounts of the protein were first found between the cells of the inner cell mass of late blastocysts. In implanted early egg cylinders, fibronectin was deposited between the ectoderm and endoderm of the inner cell mass and in the nascent Reichert's membrane. With development, the visceral and the parietal endoderm cells became positive for the protein, but no fibronectin was detected in ectoderm cells. During segregation of mesoderm from ectoderm, fibronectin appeared in mesoderm cells and as a band between the two germ layers. In the developing amnion and chorion, the protein was localized between the ectodermal and mesodermal cell layers. The results indicate that fibronectin is an early differentiation market for the stage of endoderm formation in the inner cell mass of the mouse blastocyst. It is also a marker of mesoderm appearance and seems to be associated with the accumulating extracellular matrix material in the developing embryo.     

Generation of the germ layers along the animal-vegetal axis in Xenopus laevis

After completion of gastrulation, typical vertebrate embryos consist of three cell sheets, called germ layers. The outer layer, the ectoderm, which produces the cells of the epidermis and the nervous system; the inner layer, the endoderm, producing the lining of the digestive tube and its associated organs (pancreas, liver, lungs etc.) and the middle layer, the mesoderm, which gives rise to several organs (heart, kidney, gonads), connective tissues (bone, muscles, tendons, blood vessels), and blood cells. The formation of the germ layers is one of the earliest embryonic events to subdivide multicellular embryos into a few compartments. In Xenopus laevis, the spatial domains of three germ layers are largely separated along the animal-vegetal axis even before gastrulation; ectoderm in the animal pole region; mesoderm in the equatorial region and endoderm in the vegetal pole region. In this review, we summarise the recent advances in our understanding of the formation of the germ layers in Xenopus laevis.     

Formation of cytoskeletal elements during mouse embryogenesis

Abstract. The ultrastructure of the day 8.5 mouse embryo has been studied by transmission electron microscopy, with special emphasis on the primary mesenchymal cells and their interaction with cells of the embryonic ectoderm and the proximal endoderm. The organization of the two polar epithelial cell layers (embryonic ectoderm and proximal endoderm), the isolated cells of the distal endoderm and the primary mesenchymal cells is described. Primary mesenchymal cells are different from embryonic ectoderm cells, from which they are derived, not only by the absence of desmosomes and intermediate-sized filaments of the cytokeratin type but also by their variable morphology not exhibiting stable polar architecture, and their numerous cytoplasmic processes which make contacts with the basal lamina of the ectoderm, the basal cell surface of the proximal endoderm, and other mesenchymal cells. Over most of the embryo the embryonic ectoderm is covered by a typical basal lamina, except for certain regions that are frequently characterized by cytoplasmic projections ('blebs') from the basal cell surface membrane. In contrast, the basal surface of the proximal endoderm is not covered by a continuous basal lamina and reveals mushroom-like protrusions of the cortical cytoplasm. Junctions between primary mesenchymal cells are numerous and include adhaerens-type formations of various sizes as well as gap junctions. Occasionally, a special type of junction between mesenchymal cells and embryonic ectoderm has been found, resulting in local interruptions of the basal lamina. The observations are discussed in relation to possible mechanisms of mesoderm formation and the drastic changes of cell character that accompany this process, including cytoskeletal changes such as the disappearance of cytokeratin filaments and the expression of vimentin.     

胚胎干细胞的研究进展

胚胎干细胞是来源于早期胚胎内细胞团的、具有多向分化潜能的细胞,已经广泛用于生命科学的许多领域,它在医学方面的应用成为医学领域的研究热点.本文对胚胎干细胞在诱导分化、基础研究和临床应用上的研究进展进行了综述,对今后的研究方向进行了展望.     

Establishment and therapeutic use of human embryonic stem cell lines

Embryonic stem (ES) cell lines, which are derived from the inner cell mass of blastocysts, proliferate indefinitely in vitro, retaining their potency to differentiate into various cell types derived from all of the three embryonic germ layers: the ectoderm, mesoderm and endoderm. Establishment of human ES cell lines in 1998 has indicated the great potential of ES cells for applications in medical research and other purposes such as cell transplantation therapy. Careful assessment of safety and effectiveness using proper animal models is required before such therapies can be attempted on human patients. Monkey ES cell lines provide valuable models for such research.     

Formation of cytoskeletal elements during mouse embryogenesis. IV. Ultrastructure of primary mesenchymal cells and their cell-cell interactions

The ultrastructure of the day 8.5 mouse embryo has been studied by transmission electron microscopy, with special emphasis on the primary mesenchymal cells and their interaction with cells of the embryonic ectoderm and the proximal endoderm. The organization of the two polar epithelial cell layers (embryonic ectoderm and proximal endoderm), the isolated cells of the distal endoderm and the primary mesenchymal cells is described. Primary mesenchymal cells are different from embryonic ectoderm cells, from which they are derived, not only by the absence of desmosomes and intermediate-sized filaments of the cytokeratin type but also by their variable morphology not exhibiting stable polar architecture, and their numerous cytoplasmic processes which make contacts with the basal lamina of the ectoderm, the basal cell surface of the proximal endoderm, and other mesenchymal cells. Over most of the embryo the embryonic ectoderm is covered by a typical basal lamina, except for certain regions that are frequently characterized by cytoplasmic projections ("blebs') from the basal cell surface membrane. In contrast, the basal surface of the proximal endoderm is not covered by a continuous basal lamina and reveals mushroom-like protrusions of the cortical cytoplasm. Junctions between primary mesenchymal cells are numerous and include adhaerens-type formations of various sizes as well as gap junctions. Occasionally, a special type of junction between mesenchymal cells and embryonic ectoderm has been found, resulting in local interruptions of the basal lamina. The observations are discussed in relation to possible mechanisms of mesoderm formation and the drastic changes of cell character that accompany this process, including cytoskeletal changes such as the disappearance of cytokeratin filaments and the expression of vimentin.     

The problem of germ layers in sponges (Porifera) and some issues concerning early metazoan evolution

Nowadays the formation of germ layers (endoderm and mesoderm) is associated with gastrulation. The question of whether the cell movements during early embryonic development in sponges (Porifera) are gastrulation as in eumetazoans remains in dispute. Recent data on the histological organization, digestion and embryonic morphogenesis in sponges are analyzed here in an attempt to answer this question. Unique features of these basal Metazoa are the lack of intestinal epithelium, digestive parenchyma or any cell population specialized in digestion. Food particles are captured by cells of almost all types. These data show that sponges have no embryonic layers such as ectoderm or endoderm, characteristic to eumetazoans, and, consequently, no gastrulation. We make an assumption that the formation of germ layers cannot be considered as a recapitulation of events that took place in the common ancestor of Porifera and Eumetazoa. The unity of Metazoa is expressed not in the presence of gastrulation processes per se, but in the universal nature of cell movement mechanisms ensuring various types of morphogenesis, including those underlying gastrulation. It is concluded that metazoan mechanisms of morphogenetic movements must have emerged in the course of evolution prior to the separation of the germ layers like endoderm and ectoderm.     

Development of separated germ layers of rodent embryos on ectopic sites: a reappraisal.

The method of separation of germ layers of rodent embryos by treating the embryonic shields with proteolytic enzymes and by microsurgery with the subsequent transplantation to ectopic sites has helped to gain a more detailed insight into what is going on during gastrulation in mammals. The space under the kidney capsule of adult animals seems to be the most appropriate ectopic site for transplantation of early postimplantation rat embryos or separated germ layers. After transplantation the grafts develop into teratomas whose complex histological structure reflects the initial developmental capacities of the graft. At the pre-primitive streak and the early primitive streak stages the primitive ectoderm differentiates into tissue derivatives of all three definitive germ layers, often in complex organotypic combinations. This is indirect evidence that all cells of the embryonic body originate from the primitive embryonic ectoderm. Halves of the primitive ectoderm obtained by a longitudinal or transverse cut through the egg cylinder give the same result. At the head fold stage the capacity for differentiation of the ectoderm is restricted to ectodermal and mesodermal derivatives. One day before gastrulation the isolated primitive ectoderm is not able to differentiate as renal isograft. The mesoderm isolated at the head fold stage and at later stages when its segmentation occurs, differentiates almost exclusively into the brown adipose tissue. The embryonic endoderm differentiates only in combination with the mesoderm. After transplantation the embryonic ectoderm loses its epithelial organization and breaks up into a mass of mesenchyme-like cells in which epithelial structures subsequently appear and differentiate in a way reminiscent of the reaggregation of cells in mixed cell suspension in vitro.     

Conserved patterns of cell movements during vertebrate gastrulation

Vertebrate embryogenesis entails an exquisitely coordinated combination of cell proliferation, fate specification and movement. After induction of the germ layers, the blastula is transformed by gastrulation movements into a multilayered embryo with head, trunk and tail rudiments. Gastrulation is heralded by formation of a blastopore, an opening in the blastula. The axial side of the blastopore is marked by the organizer, a signaling center that patterns the germ layers and regulates gastrulation movements. During internalization, endoderm and mesoderm cells move via the blastopore beneath the ectoderm. Epiboly movements expand and thin the nascent germ layers. Convergence movements narrow the germ layers from lateral to medial while extension movements elongate them from head to tail. Despite different morphology, parallels emerge with respect to the cellular and genetic mechanisms of gastrulation in different vertebrate groups. Patterns of gastrulation cell movements relative to the blastopore and the organizer are similar from fish to mammals, and conserved molecular pathways mediate gastrulation movements.     

Fibroblast growth factor signalling controls successive cell behaviours during mesoderm layer formation in Drosophila

Fibroblast growth factor (FGF)-dependent epithelial-mesenchymal transitions and cell migration contribute to the establishment of germ layers in vertebrates and other animals, but a comprehensive demonstration of the cellular activities that FGF controls to mediate these events has not been provided for any system. The establishment of the Drosophila mesoderm layer from an epithelial primordium involves a transition to a mesenchymal state and the dispersal of cells away from the site of internalisation in a FGF-dependent fashion. We show here that FGF plays multiple roles at successive stages of mesoderm morphogenesis in Drosophila. It is first required for the mesoderm primordium to lose its epithelial polarity. An intimate, FGF-dependent contact is established and maintained between the germ layers through mesoderm cell protrusions. These protrusions extend deep into the underlying ectoderm epithelium and are associated with high levels of E-cadherin at the germ layer interface. Finally, FGF directs distinct hitherto unrecognised and partially redundant protrusive behaviours during later mesoderm spreading. Cells first move radially towards the ectoderm, and then switch to a dorsally directed movement across its surface. We show that both movements are important for layer formation and present evidence suggesting that they are controlled by genetically distinct mechanisms.     

高精度时空转录组揭示小鼠早期胚胎三胚层细胞谱系发生过程

从受精卵发育成具有不同细胞类型个体的过程中,细胞命运受到多个层次的调控。在哺乳动物的早期胚胎发育过程中,原肠运动是外、中、内三个胚层的建立过程,为后续的器官发生和形态建成提供了发育蓝图。然而目前对于三胚层命运建立的分子机制认识并不清晰。该文通过对小鼠早期胚胎的时空转录组分析,从分子层面揭示了外、中、内三胚层谱系发生的整个过程。     

Definition of germ layer cell lineage alternative splicing programs reveals a critical role for Quaking in specifying cardiac cell fate

Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.     

Changes in states of commitment of single animal pole blastomeres of Xenopus laevis

The experiments described in this paper were designed to compare the normal fates of animal pole blastomeres of Xenopus laevis with their state of commitment. Single animal pole blastomeres were labeled with a lineage marker and transplanted into the blastocoels of host embryos of different stages. The distribution of labeled daughter cells in the tadpole reflects the state of commitment of the parent cell at the time of transplantation. It is known that cells from the animal pole of the early blastula normally contribute predominantly to ectoderm with a small, but significant, contribution to the mesoderm. We show that on transplantation to the blastocoels of late blastula host embryos these blastomeres are pluripotent, contributing to all three germ layers. At later stages the normal fate of these cells becomes restricted solely to ectoderm and concomitantly the proportion of pluripotent cells is reduced, although the results depend upon the stage of the host embryo. Blastomeres from late blastula donors transplanted to mid gastrulae contribute solely to ectoderm in 34% of cases; however, in earlier hosts, when the vegetal hemisphere cells have "mesoderm inducing" or "vegetalizing" activity, late blastula animal pole blastomeres contribute to mesoderm and endoderm rather than ectoderm. Thus during the blastula stage animal pole cells pass from pluripotency to a labile state of commitment to ectoderm.     

The structure and activities of echinonectin: a developmentally regulated cell adhesion glycoprotein with galactose-specific lectin activity.

The extracellular matrix of the sea urchin embryo contains a 230 kD homodimeric glycoprotein known as echinonectin (EN). EN contains a cell attachment domain as well as a galactose-specific lectin activity. Cell attachment to EN is differentially regulated in the three primary germ layers, endoderm, ectoderm and mesoderm. Prior to gastrulation all embryonic cells adhere equally to EN-coated substrates, but during gastrulation primary mesenchyme cells lose affinity for EN, ectoderm cells increase their binding to the molecule, and cells of the endoderm maintain a similar or slightly lowered level of binding. The mechanisms governing these adhesive changes and the specific functions they serve in development are not currently understood. They are timed to coincide with distinct morphogenetic events such as primary mesenchyme cell ingression and archenteron formation, suggesting that regulated adhesion to EN plays at least a permissive role in early morphogenesis.     

Expression of cell adhesion molecule E-cadherin in Xenopus embryos begins at gastrulation and predominates in the ectoderm

The expression of the Ca2+-dependent epithelial cell adhesion molecule E-cadherin (also known as uvomorulin and L-CAM) in the early stages of embryonic development of Xenopus laevis was examined. E-Cadherin was identified in the Xenopus A6 epithelial cell line by antibody cross-reactivity and several biochemical characteristics. Four independent mAbs were generated against purified Xenopus E-cadherin. All four mAbs recognized the same polypeptides in A6 cells, adult epithelial tissues, and embryos. These mAbs inhibited the formation of cell contacts between A6 cells and stained the basolateral plasma membranes of A6 cells, hepatocytes, and alveolar epithelial cells. The time of E-cadherin expression in early Xenopus embryos was determined by immunoblotting. Unlike its expression in early mouse embryos, E-cadherin was not present in the eggs or early blastula of Xenopus laevis. These findings indicate that a different Ca2+-dependent cell adhesion molecule, perhaps another member of the cadherin gene family, is responsible for the Ca2+-dependent adhesion between cleavage stage Xenopus blastomeres. Detectable accumulation of E-cadherin started just before gastrulation at stage 9 1/2 and increased rapidly up to the end of gastrulation at stage 15. In stage 15 embryos, specific immunofluorescence staining of E-cadherin was discernible only in ectoderm, but not in mesoderm and endoderm. The ectoderm at this stage consists of two cell layers. The outer cell layer of ectoderm was stained intensely, and staining was localized to the basolateral plasma membrane of these cells. Lower levels of staining were observed in the inner cell layer of ectoderm. The coincidence of E-cadherin expression with the process of gastrulation and its restriction to the ectoderm indicate that it may play a role in the morphogenetic movements of gastrulation and resulting segregation of embryonic germ layers.