Cessation of gastrulation is mediated by suppression of epithelial-mesenchymal transition at the ventral ectodermal ridge

In the gastrula stage embryo, the epiblast migrates toward the primitive streak and ingresses through the primitive groove. Subsequently, the ingressing epiblast cells undergo epithelial-mesenchymal transition (EMT) and differentiate into the definitive endoderm and mesoderm during gastrulation. However, the developmental mechanisms at the end of gastrulation have not yet been elucidated. Histological and genetic analyses of the ventral ectodermal ridge (VER), a derivative of the primitive streak, were performed using chick and mouse embryos. The analyses showed a continued cell movement resembling gastrulation associated with EMT during the early tailbud stage of both embryos. Such gastrulation-like cell movement was gradually attenuated by the absence of EMT during tail development. The kinetics of the expression pattern of noggin (Nog) and basal membrane degradation adjacent to the chick and the mouse VER indicated a correlation between the temporal and/or spatial expression of Nog and the presence of EMT in the VER. Furthermore, Nog overexpression suppressed EMT and arrested ingressive cell movement in the chick VER. Mice mutant in noggin displayed dysregulation of EMT with continued ingressive cell movement. These indicate that the inhibition of Bmp signaling by temporal and/or spatial Nog expression suppresses EMT and leads to the cessation of the ingressive cell movement from the VER at the end of gastrulation.     

Developmental abnormalities of NT mouse embryos appear early after implantation

In mammals, cloning by nuclear transfer (NT) into an enucleated oocyte is a very inefficient process, even if it can generate healthy adults. We show that blastocysts derived from embryonic stem (ES) donor cells develop at a high rate, correctly express the pluripotential marker gene Oct4 in ICM cells and display normal growth in vitro. Moreover, the majority of them implant in the uterus of recipient females. We combine embryological studies, gene expression analysis during gastrulation and generation of chimaeric embryos to identify the developmental origin (stage and tissue affected) of NT embryo mortality. The majority died before mid-gestation from defects arising early, either at peri-implantation stages or during the gastrulation period. The first type of defect is a non-cell autonomous defect of the epiblast cells and is rescued by complementation of NT blastocysts with normal ES or ICM cells. The second type of defect affects growth regulation and the shape of the embryo but does not directly impair the initial establishment of the patterning of the embryo. Only chimaeras formed by the aggregation of NT and tetraploid embryos reveal no growth abnormalities at gastrulation. These studies indicate that the trophoblast cell lineage is the primary source of these defects. These embryological studies provide a solid basis for understanding reprogramming errors in NT embryos. In addition, they unveil new aspects of growth regulation while increasing our knowledge on the role of crosstalk between the extra-embryonic and the embryonic regions of the conceptus in the control of growth and morphogenesis.     

Mesoderm-inducing factors: a small class of molecules

Mesoderm-inducing factors (MIF's) from chick embryos, XTC cells and WEHI-3 cells were studied using various procedures. The object was to find whether they are similar to heparin-binding growth factors (HBGFs-the only known pure mesoderm-inducing substances) and, if not, whether they are similar to each other. The major active components from all three MIF sources behave as somewhat hydrophobic, acid-stable molecules and do not bind to heparin. They all have relative molecular masses of about 13,000 measured by HPLC size exclusion chromatography. The isoelectric points measured by chromatofocusing were 6.7 (WEHI) and 7.7 (XTC). The chick MIF seemed somewhat heterogeneous by chromatofocusing and a portion of its activity bound to heparin sepharose. All three MIFs have similar effects on explants of Xenopus blastula ectoderm to the heparin-binding growth factors, causing an elongation at the time of gastrulation followed by the development of mesenchyme, mesothelium and muscle cells, the proportion of muscle increasing with dose. Unlike the HBGFs they all also induce notochord if sufficiently high concentrations are used. Our study shows that the MIFs examined here form a small group of potent agents distinct from the HBGFs and from other known growth and differentiations factors. Their occurrence in various tissues and cell lines suggests that they have functions in the adult organism as well as during early development.     

Functions of the IGFs in early mammalian development

The identification of growth factors and/or receptors produced by mammalian embryos or present in the maternal reproductive tract is of basic interest, as well as having practical application. Early studies established that receptors binding insulin and the insulin-like growth factors (IGFs) are expressed by preimplantation mouse embryos. These studies have been confirmed at the molecular level using RT-PCR techniques. In addition, high resolution electron microscopy has shown that insulin is internalized by the cells of the blastocyst stage mouse embryo, and that immunologically intact insulin is detectable in the cells of the trophectoderm and inner cell mass. Similar studies with gold labelled IGF-I have shown that this ligand is also bound and internalized by mouse blastocysts. However, although all blastocysts express receptors that bind IGF-I on the basolateral cell surface of the trophectoderm, only 30% exhibit apically located receptors. In order to elucidate the functions of IGFs in early mouse development, we are in the process of constructing protein databases for embryos at the eight-cell and blastocyst stage. By the use of the database, it should prove possible to elucidate targets of growth factor action. © 1993 Wiley-Liss, Inc.     

Patterning of the embryo: the first spatial decisions in the life of a mouse

Although in most species the polarity of the embryo takes its roots from the spatial patterning of the egg, mammals were viewed as an exception. This was because the anteroposterior polarity of the mouse embryo could not be seen until gastrulation, and no developmental cues were known that could define polarity at earlier stages. Why should we now re-consider this view? While mechanisms of axis formation in mammals could, in principle, be unique, the evolutionary conservation of numerous other developmental processes raises the question of why mammals would have evolved a different way or timing of organising their embryonic polarity. Indeed, recent evidence shows that well before the onset of gastrulation, the mouse embryo initiates asymmetric patterns of gene expression in its visceral endoderm. Although this extra-embryonic tissue does not contribute to the body itself, it is involved in axis formation. Other recent work has revealed that spatial distribution of cells in the visceral endoderm can be traced back to polarity present at the blastocyst stage. These insights have raised the possibility that embryonic polarity might also originate early during development of mammalian embryos. Indeed it now appears that there are at least two spatial cues that operate in the mouse egg to shape polarity of the blastocyst. One of these is at the animal pole, which is defined by the site of female meiosis, and another is associated with the position of sperm entry. In this review I discuss these recent findings, which have led to the recognition that mouse embryos initiate development of their polarity at the earliest stages of their life. This novel perspective raises questions about the nature of cellular and molecular mechanisms that could convert developmental cues in the zygote to axes of the blastocyst, and hence into polarity of the post-implantation embryo. It also brings to light the need to understand how such mechanisms could enable early mouse development to be so regulative.     

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.     

Presence of serotonin in early chick embryos

With biochemical analysis and with autoradiography based on injection of 5-[3H]hydroxytryptophan, it was possible to demonstrate the presence of serotonin (5-hydroxytryptamine) in early chick embryos as early as the pre-streak stage. The biochemical analysis which covered the early developmental period (0.5-6 days of incubation) revealed an elevated concentration of serotonin at gastrulation; from then it stayed at a lower and fairly even level. Autoradiographs of embryos at the pre-streak stage, the primitive streak stage, the head fold stage and the 4-6 somites stage indicated the presence of serotonin in intracellular yolk granules and in cell nuclei. Moreover, the amine appeared associated with microfilaments and microtubules, particularly in developing neural cells. Notably the elevated concentration of serotonin at gastrulation, but also the intracellular distribution of the amine during early organogenesis, indicates a prominent role for it in cell-shape changes and morphogenesis in the early chick embryo.     

Segregation of the embryonic vascular and hemopoietic systems.

The origin of endothelial cells and their subsequent assembly into the primary vascular system have been mostly analyzed in the avian embryo. Following the discovery of specific growth factors and their cognate receptors, the molecular mechanisms underlying these processes have been unraveled in both birds and mammals. In particular, experimental studies of the angiogenic vascular endothelial growth factor (VEGF) and its receptors, carried out in both vertebrate classes, have provided significant insight into the developmental biology of endothelial cells. The VEGF receptor VEGFR2 is the earliest marker known to be expressed by endothelial precursor cells of avian and mouse embryos. Based on the localization of VEGFR2+ cells in the avian embryo and on clonal culture experiments, two types of endothelial precursor cells can be distinguished from gastrulation stages onward: posterior mesodermal VEGFR2+ hemangioblasts, which have the capacity to differentiate into endothelial and hemopoietic cells, and anterior VEGFR2+ angioblasts, which can only give rise to endothelial cells.     

Chimeras between parthenogenetic or androgenetic blastomeres and normal embryos: allocation to the inner cell mass and trophectoderm

A series of chimeras was generated by injecting single normal, parthenogenetic, or androgenetic blastomeres carrying transgenic markers under the zona pellucida of nontransgenic eight-cell embryos. These chimeras were cultured to the blastocyst stage and sectioned, and the transgenic component was detected by in situ hybridization. No statistically significant difference was found among the normal, parthenogenetic, and androgenetic chimeras in the number of chimeric blastocysts with a transgenic contribution to the inner cell mass (ICM), the trophectoderm, or both the ICM and trophectoderm. Since androgenetic and parthenogenetic cells were present in chimeras at a high frequency in both the ICM and trophectoderm at the blastocyst stage, but not in similar chimeras at late gastrulation, these cells must not respond normally to developmental signals subsequent to blastocyst formation and prior to late gastrulation.     

Implication of Cytoplasmic Factors in the Lethality of Bufonidae Nucleocytoplasmic Hybrids

The nucleocytoplasmic hybrid combination performed by nuclear graft between B. bufo nucleus and B. calamita egg is 95% lethal during gastrulation mostly at the young gastrula stage (75%) and to a lesser extent at the yolk plug stage (20%). The heterologous B. calamita cytoplasm was treated or fractioned by different techniques including centrifugation, nucleic acid extraction, ammonium sulfate treatment and heating.
The treated cytoplasmic fractions were injected in B. bufo fertilized eggs in order to check their influence on morphogenetic capacities of the B. bufo nucleus. Injection of hyaline cytoplasmic fraction obtained from B. calamita centrifuged eggs blocks the B. Bufo fertilized egg gastrulation in similar rates as in nucleocytoplasmic association performed by interspecific nuclear grafting. Gastrulation, however, is not affected by injection of total nucleic acids extracted from this hyaline fraction. The soluble cytoplasmic fraction obtained after 60% ammonium sulfate precipitation blocks the development at the beginning of gastrulation, this property being inactivated by heating for 5 min at 60°C. Supernatant from 70% ammonium sulfate precipitation blocks the development at the yolk plug stage and is inactivated by heating for 5 min at 80°C.
These experiments suggest that two distinct factors in B. calamita eggs are involved in developmental arrest of injected B. bufo fertilized eggs, these two factors respectively acting on the beginning and on the end of gastrulation.     

Contributions of blastocyst micromanipulation to the study of mammalian development

This is a personal account of why the author chose to focus on devising techniques for micromanipulating the blastocyst stage conceptus as a way of investigating early development in mammals. Its aim is to provide insight into what such technical innovations entailed and how they have contributed to present understanding of both embryology and the analysis of gene function in mammals. The ability to dissect and reconstitute mouse blastocysts, and to inject cells or tissue into them, enabled genes to be harnessed as markers for elucidating the lineage of cells and interactions between tissues from the stage when differentiation is first evident. Most importantly, it made it possible to apply clonal analysis to the study of cell fate in mammals. The scope of blastocyst micromanipulation was further enhanced when embryonal carcinoma (EC) cells and, particularly, embryonic stem (ES) cells were found to be able to participate in normal development and contribute to the germ line following injection into the blastocyst. BioEssays 20:168–180, 1998. © 1998 John Wiley & Sons, Inc.     

Experimental analysis of the extension of the dorsal marginal zone in Pleurodeles waltl gastrulae

The capacity for extension of the dorsal marginal zone (DMZ) in Pleurodeles waltl gastrulae was studied by scanning electron microscopy and grafting experiments. At the onset of gastrulation, the cells of the animal pole (AP) undergo important changes in shape and form a single layer. As gastrulation proceeds, the arrangement of cells also changes in the noninvoluted DMZ: radial intercalation leads to a single layer of cells. Grafting experiments involving either AP or DMZ explants were performed using a cell lineage tracer. When rotated 90 degrees or 180 degrees, grafted DMZ explants were able to involute normally and there was extension according to the animal-vegetal axis of the host. In contrast, neither single nor bilayered explants from AP involutes completely, and neither extends when grafted in place of the DMZ. Furthermore, when inside of the host, these AP grafts curl up and inhibit the closure of the blastopore. Once transplanted to the AP region, the DMZ showed no obvious autonomous extension. DMZs cultured in vitro showed little extension and this only from the late gastrula stage onward. Removal of blastocoel roof blocked involution to a varied extent, depending on the developmental stage of the embryos. From these results, it is argued that differences could well exist in the mechanism of gastrulation between anuran and urodele embryos. That migrating mesodermal cells play a major role in urodele gastrulation is discussed.     

The development of chick spinal cord in tissue culture. I. Fragment cultures from embryos of various developmental stages

Explants from neural tube and spinal cord of chick embryos at developmental stages 8 through 36 were cultured on collagen-coated cover glasses for 21 days. The cultures of neural tube at stages 10 to 14 contained many neuronal precursor cells which gave rise to mature neurons. This was verified by cumulative labeling of cultures with tritiated thymidine. Explants from spinal cords of stages 26 and 27 contained fewer precursor cells, and at stage 36, only 7% of mature neurons were labeled. Regardless of the stage of development at which explants were made (stages 8 through 36), all cultures had a similar appearance after 21 days, indicating that cells from explants taken from earlier developmental stages (before neurons were formed) "caught up" with the explants from later developmental stages, which already had formed neurons at the time of explantation.     

Epithelial type, ingression, blastopore architecture and the evolution of chordate mesoderm morphogenesis

Chordate embryos show an evolutionary trend in the mechanisms they use to internalize presumptive mesoderm, relying predominantly on invagination in the basal chordates, varying combinations of involution and ingression in the anamniote vertebrates and reptiles, and predominantly on ingression in birds and mammals. This trend is associated with variations in epithelial type and changes in embryonic architecture as well as variations in the type of blastopore formed by an embryo. We also note the surprising conservation of the involution, during gastrulation, of at least a subset of the notochordal cells throughout the chordates, and suggest that this indicates a constraint on morphogenic evolution based on a functional linkage between architecture and patterning. Finally, we propose a model for the evolutionary transitions from gastrulation through a urodele amphibian-type blastopore to gastrulation through a primitive streak, as in chick or mouse.     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+