FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression

Background  

FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.     

Epiblast and primitive-streak origins of the endoderm in the gastrulating chick embryo

Gastrulation is characterized by the extensive movements of cells. Fate mapping is used to follow such cell movements as they occur over time, and prospective fate maps have been constructed for several stages of the model organisms used in modern studies in developmental biology. In chick embryos, detailed fate maps have been constructed for both prospective mesodermal and ectodermal cells. However, the origin and displacement of the prospective endodermal cells during crucial periods in gastrulation remain unclear. This study had three aims. First, we determined the primitive-streak origin of the endoderm using supravital fluorescent markers, and followed the movement of the prospective endodermal cells as they dispersed to generate the definitive endodermal layer. We show that between stages 3a/b and 4, the intraembryonic definitive endoderm receives contributions mainly from the rostral half of the primitive streak, and that endodermal movements parallel those of ingressing adjacent mesodermal subdivisions. Second, the question of the epiblast origin of the endodermal layer was addressed by precisely labeling epiblast cells in a region known to give rise to prospective somitic cells, and following their movement as they underwent ingression through the primitive streak. We show that the epiblast clearly contributes prospective endodermal cells to the primitive streak, and subsequently to definitive endoderm of the area pellucida. Finally, the relationship between the hypoblast and the definitive endoderm was defined by following labeled rostral primitive-streak cells over a short period of time as they contributed to the definitive endoderm, and combining this with in situ hybridization with a riboprobe for Crescent, a marker of the hypoblast. We show that as the definitive endodermal layer is laid down, there is cell-cell intercalation at its interface with the displaced hypoblast cells. These data were used to construct detailed prospective fate maps of the endoderm in the chick embryo, delineating the origins and migrations of endodermal cells in various rostrocaudal levels of the primitive streak during key periods in early development.     

Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse

The fate of the embryonic endoderm (generally called visceral embryonic endoderm) of prestreak and early primitive streak stages of the mouse embryo was studied in vitro by microinjecting horseradish peroxidase into single axial endoderm cells of 6.7-day-old embryos and tracing the labelled descendants either through gastrulation (1 day of culture) or to early somite stages (2 days of culture). Descendants of endoderm cells from the anterior half of the axis were found at the extreme cranial end of the embryo after 1 day and in the visceral yolk sac endoderm after 2 days, i.e. they were displaced anteriorly and anterolaterally. Descendants of cells originating over and near the anterior end of the early primitive streak, i.e. posterior to the distal tip of the egg cylinder, were found after 1 day over the entire embryonic axis and after 2 days in the embryonic endoderm at the anterior intestinal portal, in the foregut, along the trunk and postnodally, as well as anteriorly and posteriorly in the visceral yolk sac. Endoderm covering the posterior half of the early primitive streak contributed to postnodal endoderm after 1 day (at the late streak stage) and mainly to posterior visceral yolk sac endoderm after 2 days. Clonal descendants of axial endoderm were located after 2 days either over the embryo or in the yolk sac; the few exceptions spanned the caudal end of the embryo and the posterior yolk sac. The clonal analysis also showed that the endoderm layer along the posterior half of the axis of prestreak- and early-streak-stage embryos is heterogeneous in its germ layer fate. Whereas the germ layer location of descendants from anterior sites did not differ after 1 day from that expected from the initial controls (approx. 90% exclusively in endoderm), only 62% of the successfully injected posterior sites resulted in labelled cells exclusively in endoderm; the remainder contributed partially or entirely to ectoderm and mesoderm. This loss from the endoderm layer was compensated by posterior-derived cells that remained in endoderm having more surviving descendants (8.4 h population doubling time) than did anterior-derived cells (10.5 h population doubling time). There was no indication of cell death at the prestreak and early streak stages; at least 93% of the cells were proliferating and more than half of the total axial population were in, or had completed, a third cell cycle after 22 h culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ventral closure, headfold fusion and definitive endoderm migration defects in mouse embryos lacking the fibronectin leucine-rich transmembrane protein FLRT3

The three fibronectin leucine-rich repeat transmembrane (FLRT) proteins contain 10 leucine-rich repeats (LRR), a type III fibronectin (FN) domain, followed by the transmembrane region, and a short cytoplasmic tail. XFLRT3, a Nodal/TGFβ target, regulates cell adhesion and modulates FGF signalling during Xenopus gastrulation. The present study describes the onset and pattern of FLRT1-3 expression in the early mouse embryo. FLRT3 expression is activated in the anterior visceral endoderm (AVE), and during gastrulation appears in anterior streak derivatives namely the node, notochord and the emerging definitive endoderm. To explore FLRT3 function we generated a null allele via gene targeting. Early Nodal activities required for anterior-posterior (A-P) patterning, primitive streak formation and left-right (L-R) axis determination were unperturbed. However, FLRT3 mutant embryos display defects in headfold fusion, definitive endoderm migration and a failure of the lateral edges of the ventral body wall to fuse, leading to cardia bifida. Surprisingly, the mutation has no effect on FGF signalling. Collectively these experiments demonstrate that FLRT3 plays a key role in controlling cell adhesion and tissue morphogenesis in the developing mouse embryo.     

Distinct roles for visceral endoderm during embryonic mouse development.

The murine visceral endoderm is an extraembryonic cell layer that appears prior to gastrulation and performs critical functions during embryogenesis. The traditional role ascribed to the visceral endoderm entails nutrient uptake and transport. Besides synthesizing a number of specialized proteins that facilitate uptake, digestion, and secretion of nutrients, the extraembryonic visceral endoderm coordinates blood cell differentiation and vessel formation in the adjoining mesoderm, thereby facilitating efficient exchange of nutrients and gases between the mother and embryo. Recent studies suggest that in addition to this nutrient exchange function the visceral endoderm overlying the egg cylinder stage embryo plays an active role in guiding early development. Cells in the anterior visceral endoderm function as an early organizer. Prior to formation of the primitive streak, these cells express specific gene products that specify the fate of underlying embryonic tissues. In this review we highlight recent investigations demonstrating this dual role for visceral endoderm as a provider of both nutrients and developmental cues for the early embryo.     

Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer

The fate of the embryonic endoderm (generally called visceral embryonic endoderm) of midstreak to neural plate stages of the mouse embryo was studied by microinjecting horseradish peroxidase (HRP) into single axial endoderm cells in situ, and tracing the labeled descendants to early somite stages in vitro. Axial endoderm cells along the anterior fifth of the late streak/neural plate stage embryo contributed descendants either to the yolk sac endoderm or to the anterior intestinal portal. Cells of the exposed head process contributed to the trunk endoderm and notochord; neighboring endoderm cells contributed to the dorsal foregut. Contributions to the ventral foregut came from endoderm at, and anterior to, the distal tip of the younger, midstreak embryo (in which the head process was not yet exposed). Endoderm over the primitive streak contributed to the postsomite endoderm. We argue from these results and those in the literature that during gastrulation the axial embryonic endoderm is of mixed lineage: (1) an anterior population of cells is derived from primitive endoderm and contributes to the yolk sac endoderm; (2) a population at, and anterior to, the distal tip of the midstreak embryo, extending more anteriorly at late streak/neural plate stages, is presumed to emerge from primitive ectoderm at the beginning of gastrulation and contributes to the foregut and anterior intestinal portal; (3) the axial portion of the head process that begins to incorporate into the ventral surface at the late streak stage contributes to notochord and trunk endoderm. Cells or their descendants that were destined to die within 24 hr were evident at the midstreak stage. There was a linear trend in the incidence of cell death among labeled cells at the late streak/neural plate stages, ranging from 27% caudal to the node to 57% in the anterior fifth of the embryo. The surviving axial endoderm cells divided sufficiently fast to double the population in 24 hr.     

A systematic screen for genes expressed in definitive endoderm by Serial Analysis of Gene Expression (SAGE)

Background  

The embryonic definitive endoderm (DE) gives rise to organs of the gastrointestinal and respiratory tract including the liver, pancreas and epithelia of the lung and colon. Understanding how DE progenitor cells generate these tissues is critical to understanding the cause of visceral organ disorders and cancers, and will ultimately lead to novel therapies including tissue and organ regeneration. However, investigation into the molecular mechanisms of DE differentiation has been hindered by the lack of early DE-specific markers.     

Cripto is required for mesoderm and endoderm cell allocation during mouse gastrulation

During mouse gastrulation, cells in the primitive streak undergo epithelial–mesenchymal transformation and the resulting mesenchymal cells migrate out laterally to form mesoderm and definitive endoderm across the entire embryonic cylinder. The mechanisms underlying mesoderm and endoderm specification, migration, and allocation are poorly understood. In this study, we focused on the function of mouse Cripto, a member of the EGF-CFC gene family that is highly expressed in the primitive streak and migrating mesoderm cells on embryonic day 6.5. Conditional inactivation of Cripto during gastrulation leads to varied defects in mesoderm and endoderm development. Mutant embryos display accumulation of mesenchymal cells around the shortened primitive streak indicating a functional requirement of Cripto during the formation of mesoderm layer in gastrulation. In addition, some mutant embryos showed poor formation and abnormal allocation of definitive endoderm cells on embryonic day 7.5. Consistently, many mutant embryos that survived to embryonic day 8.5 displayed defects in ventral closure of the gut endoderm causing cardia bifida. Detailed analyses revealed that both the Fgf8–Fgfr1 pathway and p38 MAP kinase activation are partially affected by the loss of Cripto function. These results demonstrate a critical role for Cripto during mouse gastrulation, especially in mesoderm and endoderm formation and allocation.     

Transitions between epithelial and mesenchymal states and the morphogenesis of the early mouse embryo

Multicellular organisms arise from the generation of different cell types and the organization of cells into tissues and organs. Cells of metazoa display two main phenotypes, the ancestral epithelial state and the recent mesenchymal derivative. Epithelial cells are usually stationary and reside in twodimensional sheets. By contrast mesenchymal cells are loosely packed and can move to new positions, thereby providing a vehicle for cell rearrangement, dispersal and novel cell-cell interactions. Transitions between epithelial and mesenchymal states drive key morphogenetic events in the early vertebrate embryo, including gastrulation, germ layer formation and somitogenesis. The cell behaviors and molecular mechanisms promoting transitions between these two states in the early mouse embryo are discussed in this review.Key words: mouse embryo, EMT, MET, morphogenesis, gastrulation, somitogenesis, epiblast, mesoderm, endoderm, primitive streak, paraxial mesoderm     

Visceral endoderm induces specification of cardiomyocytes in mice

The endoderm plays an inductive role in the formation of cardiomyocytes in many vertebrates. Here, we provide further evidence for this in the mouse and demonstrate enhanced cardiomyogenesis in mouse embryonic stem cells cultured in the presence of native visceral endoderm. Isolated mesoderm from late-primitive streak stage mouse embryos that still have an open proamniotic canal had a reduced capacity to form cardiomyocytes after 4 days in culture compared with mesoderm isolated from later stages but prior to cardiomyogenesis. Moreover, removal of the visceral endoderm but not the primitive streak reduced the formation of beating areas in embryo explants in culture. Coculture with the END2 cell line, which has visceral endoderm-like properties, restored the formation of beating areas. Immunohistochemical analysis showed that the expected candidate signaling pathways downstream of Wnts and bone morphogenetic proteins (BMPs) were active in the embryo at the appropriate time and place to be involved. Overall, the results show that, as in other vertebrates, the (visceral) endoderm plays an important role in the early events of mouse cardiomyogenesis.     

Pre-gastrula expression of zebrafish extraembryonic genes

Background  

Many species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL).     

Control of early anterior-posterior patterning in the mouse embryo by TGF-beta signalling

Prior to gastrulation the mouse embryo exists as a symmetrical cylinder consisting of three tissue layers. Positioning of the future anterior-posterior axis of the embryo occurs through coordinated cell movements that rotate a pre-existing proximal-distal (P-D) axis. Overt axis formation becomes evident when a discrete population of proximal epiblast cells become induced to form mesoderm, initiating primitive streak formation and marking the posterior side of the embryo. Over the next 12-24 h the primitive streak gradually elongates along the posterior side of the epiblast to reach the distal tip. The most anterior streak cells comprise the 'organizer' region and include the precursors of the so-called 'axial mesendoderm', namely the anterior definitive endoderm and prechordal plate mesoderm, as well as those cells that give rise to the morphologically patent node. Signalling pathways controlled by the transforming growth factor-beta ligand nodal are involved in orchestrating the process of axis formation. Embryos lacking nodal activity arrest development before gastrulation, reflecting an essential role for nodal in establishing P-D polarity by generating and maintaining the molecular pattern within the epiblast, extraembryonic ectoderm and the visceral endoderm. Using a genetic strategy to manipulate temporal and spatial domains of nodal expression reveals that the nodal pathway is also instrumental in controlling both the morphogenetic movements required for orientation of the final axis and for specification of the axial mesendoderm progenitors.     

Wnt3 signaling in the epiblast is required for proper orientation of the anteroposterior axis

The establishment of anteroposterior (AP) polarity in the early mouse epiblast is crucial for the initiation of gastrulation and the subsequent formation of the embryonic (head to tail) axis. The localization of anterior and posterior determining genes to the appropriate region of the embryo is a dynamic process that underlies this early polarity. Several studies indicate that morphological and molecular markers which define the early AP axis are first aligned along the short axis of the elliptical egg cylinder. Subsequently, just prior to the time of primitive streak formation, a conformational change in the embryo realigns these markers with the long axis. We demonstrate that embryos lacking the signaling factor Wnt3 exhibit defects in this axial realignment. In addition, chimeric analyses and conditional removal of Wnt3 activity reveal that Wnt3 expression in the epiblast is required for induction of the primitive streak and mesoderm whereas activity in the posterior visceral endoderm is dispensable.     

Foxh1 and Foxa2 are not required for formation of the midgut and hindgut definitive endoderm

The definitive endoderm forms during gastrulation and is rapidly transformed into the gut tube which is divided along the anterior-posterior axis into the foregut, midgut, and hindgut. Lineage tracing and genetic analysis have examined the origin of the definitive endoderm during gastrulation and demonstrated that the majority of definitive endoderm arises at the anterior end of the primitive streak (APS). Foxh1 and Foxa2 have been shown to play a role in specification of the APS and definitive endoderm. However, prior studies have focused on the role of these factors in specification of foregut definitive endoderm, while their role in the specification of midgut and hindgut definitive endoderm is less understood. Furthermore, previous analyses of these mutants have utilized definitive endoderm markers that are restricted to the anterior endoderm, expressed in extraembryonic endoderm, or present in other germ layers. Here, we characterized the expression of several novel definitive and visceral endoderm markers in Foxh1 and Foxa2 null embryos. In accordance with previous studies, we observed a deficiency of foregut definitive endoderm resulting in incorporation of visceral endoderm into the foregut. Interestingly, this analysis revealed that formation of midgut and hindgut definitive endoderm is unaffected by loss of Foxh1 or Foxa2. This finding represents a significant insight into specification and regionalization of mouse definitive endoderm.     

IGF-1/IGFBP-1 increases blastocyst formation and total blastocyst cell number in mouse embryo culture and facilitates the establishment of a stem-cell line

Background  

Apoptosis occurs frequently for blastocysts cultured in vitro, where conditions are suboptimal to those found in the natural environment. Insulin-like growth factor-1 (IGF-1) plays an important role in preventing apoptosis in the early development of the embryo, as well as in the progressive regulation of organ development. We hypothesize that IGF-1 and its dephosphorylated binding protein (IGFBP-1) may be able to improve embryo culture with an associated reduced cell death, and that the resultant increase in the total cell number of the embryo could increase the chances of establishing an embryonic stem-cell line.     

An expression profile analysis of ES cell-derived definitive endodermal cells and Pdx1-expressing cells

Background  

We developed an efficient in vitro method to differentiate mouse ES cells into the definitive endoderm (DE) and then Pdx1-expressing pancreatic lineages using mesodermal-derived supporting cells, M15. Using this method, resulting ES cell-derived DE and Pdx1-expressing cells were isolated by cell sorting, and their gene expression profiles were investigated with DNA microarray. Genes that were specifically expressed in DE and/or in Pdx1-expressing cells were extracted and their expression patterns in normal embryonic development were studied.