Changes in protein glycosylation during chick embryo development

To investigate the molecular changes in cell-surface glycoproteins during chick embryo development, fibroblasts from 8- and 16-day embryos were extensively digested by pronase after (i) metabolic labeling with radioactive precursors and (ii) external labeling. Two main classes of glycopeptide pronase digestion product were distinguished by Sephadex G-50 column chromatography. The large material excluded was mostly composed of glycosaminoglycans. The small retarded glycopeptides underwent age-related modifications. Those in the 8-day cells were mainly N-linked, whereas 16-day cells contained both O- and N-linked glycopeptides. The evolution of high-mannose chains in younger cells to complex-type chains in the older cells is suggested by (i) the decrease in the mannose-to-galactose and mannose-to-N-acetylglucosamine ratio with embryo development, and (ii) the fact that endo-β-N-acetylglucosaminidase H treatment released more oligomannosyls from younger than from older embryo cell glycopeptides. Small glycopeptides were also more highly sialylated in 16-day cells than in 8-day cells. The present results provide the first biochemical evidence that both quantitative and qualitative modifications occur in cell-surface glycoconjugates during the late stages of chick embryo development.     

Essential function of PTP-PEST during mouse embryonic vascularization, mesenchyme formation, neurogenesis and early liver development

PTP (protein-tyrosine phosphatase)-PEST is a ubiquitously expressed cellular regulator of integrin signalling. It has been shown to bind several molecules such as Shc, paxillin and Grb2, that are involved downstream of FAK (focal adhesion kinase) pathway. Through its specific association to p130cas and further dephosphorylation, PTP-PEST plays a critical role in cell-matrix interactions, which are essential during embryogenesis. We report here that ablation of the gene leads to early embryonic lethality, correlating well with the high expression of the protein during embryonic development. We observed an increased level of tyrosine phosphorylation of p130cas protein in E9.5 PTP-PEST(-/-) embryos, a first evidence of biochemical defect leading to abnormal growth and development. Analysis of null mutant embryos revealed that they reach gastrulation, initiate yolk sac formation, but fail to progress through normal subsequent developmental events. E9.5-10.5 PTP-PEST(-/-) embryos had morphological abnormalities such as defective embryo turning, improper somitogenesis and vasculogenesis, impaired liver development, accompanied by degeneration in both neuroepithelium and somatic epithelia. Moreover, in embryos surviving until E10.5, the caudal region was truncated, with severe mesenchyme deficiency and no successful liver formation. Defects in embryonic mesenchyme as well as subsequent failure of proper vascularization, liver development and somatogenesis, seemed likely to induce lethality at this stage of development, and these results confirm that PTP-PEST plays an essential function in early embryogenesis.     

The translational functions of embryonic poly(A)‐binding protein during gametogenesis and early embryo development

Embryonic poly(A)‐binding protein (EPAB) is an RNA‐binding protein that binds to the poly(A) tails and AU‐rich element at the 3′ ends of messenger RNA (mRNAs). The main functions of EPAB are to protect stored mRNAs from undergoing deadenylation and subsequent degradation and to be involved in their translational regulation during spermatogenesis, oogenesis, and early embryogenesis. Following the first characterization of Epab in the Xenopus oocytes and early embryos, spatial and temporal expression and potential roles of the Epab gene have been determined in the vertebrate germ cells and early embryos. In this review, we have comprehensively evaluated all studies in this field and discussed the particular functions of EPAB in the spermatogenic cells, oocytes, early embryos, and somatic cells in vertebrates.     

Measurement of intra-embryonic pH during the early stages of development in the chick embryo

Summary Measurements have been made of the pH in the extracellular space, adjacent to the neural tube, in 73 isolated chick embryos in vitro at stages from 4–22 somites. A pH of 7.8–8.4 was observed in the segmented region, while caudally, in the segmental plate, the pH was consistently lower falling by as much as 0.5 pH units at the regressing primitive streak. Variations were noted in the pH of embryos of the same age but the regional variation in pH was a consistent finding in all of the embryos examined. The buffering capacity of the extracellular space was found to be 12.9 mequiv/pH unit/1 in the segmented region and 13.9 mequiv/pH unit/1 in the segmental plate. Thus it is unlikely that the regional variations in pH result from local variations in the buffering power of the extracellular space. Varying the K⁺, Cl^-, Mg²⁺ or HCO ₃ ^- ion concentrations in the bathing medium caused little change in the intra-embryonic pH, while reducing the concentrations of Na⁺ or Ca²⁺ caused a small acidification. This suggests that the ectoderm and endoderm form an effective barrier between the embryo and the external environment. Exposure of the embryo to KCN reduced the intra-embryonic pH suggesting that the alkaline environment is maintained by active processes.     

Simultaneous cell death and desquamation of the embryonic diffusion barrier during epidermal development

The periderm is an epithelial layer covering the emerging epidermis in early embryogenesis of vertebrates. In the chicken embryo, an additional cellular layer, the subperiderm, occurs at later embryonic stages underneath the periderm. The questions arose what is the function of both epithelial layers and, as they are transitory structures, by which mechanism are they removed. By immunocytochemistry, the tight junction (TJ) proteins occludin and claudin-1 were localized in the periderm and in the subperiderm, and sites of close contact between adjacent cells were detected by electron microscopy. Using horseradish peroxidase (HRP) as tracer, these contacts were identified as tight junctions involved in the formation of the embryonic diffusion barrier. This barrier was lost by desquamation at the end of the embryonic period, when the cornified envelope of the emerging epidermis was formed. By TUNEL and DNA ladder assays, we detected simultaneous cell death in the periderm and the subperiderm shortly before hatching. The absence of caspases-3, -6, and -7 activity, key enzymes of apoptosis, and the lack of typical morphological criteria of apoptosis such as cell fragmentation or membrane blebbing point to a special form of programmed cell death (PCD) leading to the desquamation of the embryonic diffusion barrier.     

Effect of gonadotropins,thyrotropin, and adrenocorticotropin on the development of 12-day chick embryonic gonads

Summary Gonadotropins (FSH+LH) and thyrotropin (TSH) stimulated the development of 12-day embryonic gonads, whereas adrenocorticotropin (ACTH) did not show any significant effect. It is concluded that the gonads of 12-day chick embryos have the capacity to respond to both gonadotropic and thyrotropic stimulation. This suggests that at the time when the hypothalamo-pituitary-thyroid axis begins to develop, the capacity of gonadal receptors to distinguish between gonadotropins and thyrotropin has not been established.     

Two independent forms of endocytosis maintain embryonic cell surface homeostasis during early development

Eukaryotic cells have multiple forms of endocytosis which maintain cell surface homeostasis. One explanation for this apparent redundancy is to allow independent retrieval of surface membranes derived from different types of vesicles. Consistent with this hypothesis we find that sea urchin eggs have at least two types of compensatory endocytosis. One is associated with retrieving cortical vesicle membranes, and formed large endosomes by a mechanism that was inhibited by agatoxin, cadmium, staurosporine and FK506. The second type is thought to compensate for constitutive exocytosis, and formed small endosomes using a mechanism that was insensitive to the above mentioned reagents, but was inhibited by phenylarsine oxide (PAO), and by microinjection of mRNA encoding Src kinase. Both mechanisms could act concurrently, and account for all of the endocytosis occurring during early development. Inhibition of either form did not trigger compensation by the other form, and phorbol ester treatment rescued the endocytotic activity blocked by agatoxin, but not the retrieval blocked by PAO.     

Specific regulation of cyclins D1 and D2 by FGF and Shh signaling coordinates cell cycle progression, patterning, and differentiation during early steps of spinal cord development

In the vertebrate embryo, spinal cord elongation requires FGF signaling that promotes the continuous development of the posterior nervous system by maintaining a stem zone of proliferating neural progenitors. Those escaping the caudal neural stem zone, which is expressed to Shh signal, initiate ventral patterning in the neural groove before starting neuronal differentiation in the neural tube. Here we investigated the integration of D-type cyclins, known to govern cell cycle progression under the control of extracellular signals, in the program of spinal cord maturation. In chicken embryo, we find that cyclin D2 is preferentially expressed in the posterior neural plate, whereas cyclin D1 appears in the neural groove. We demonstrated by loss- and gain-of-function experiments that FGF signaling maintains cyclin D2 in the immature caudal neural epithelium, while Shh activates cyclin D1 in the neural groove. Moreover, forced maintenance of cyclin D1 or D2 in the neural tube favors proliferation at the expense of neuronal differentiation. These results contribute to our understanding of how the cell cycle control can be linked to the patterning programs to influence the balance between proliferation and neuronal differentiation in discrete progenitors domains.     

Prion protein function and the disturbance of early embryonic development in zebrafish

Transmissible Spongiform Encephal-opathies (TSE) or prion diseases are a threat to food safety and to human and animal health. The molecular mechanisms responsible for prion diseases share similarities with a wider group of neurodegenerative disorders including Alzheimer disease and Parkinson disease and the central pathological event is a disturbance of protein folding of a normal cellular protein that is eventually accompanied by neuronal cell death and the death of the host. Prion protein (PrP) is a constituent of most normal mammalian cells and its presence is essential in the pathogenesis of TSE. However, the function of this normal cellular protein remains unclear. The prevention of PRNP gene expression in mammalian species has been undramatic, implying a functional redundancy. Yet PrP is conserved from mammals to fish. Recent studies of PrP in zebrafish have yielded novel findings showing that PrP has essential roles in early embryonic development. The amenability of zebrafish to global technologies has generated data indicating the existence of “anchorless” splice variants of PrP in the early embryo. This paper will discuss the possibility that the experimentalist''s view of PrP functions might be clearer at a greater phylogenetic distance.Key words: prion protein, zebrafish, gene expression, embryo development, neurogenesis     

Prion protein function and the disturbance of early embryonic development in zebrafish

Transmissible Spongiform Encephalopathies (TSE) or prion diseases are a threat to food safety and to human and animal health. The molecular mechanisms responsible for prion diseases share similarities with a wider group of neurodegenerative disorders including Alzheimer disease and Parkinson disease and the central pathological event is a disturbance of protein folding of a normal cellular protein that is eventually accompanied by neuronal cell death and the death of the host. Prion protein (PrP) is a constituent of most normal mammalian cells and its presence is essential in the pathogenesis of TSE. However, the function of this normal cellular protein remains unclear. The prevention of PRNP gene expression in mammalian species has been undramatic, implying a functional redundancy. Yet PrP is conserved from mammals to fish. Recent studies of PrP in zebrafish have yielded novel findings showing that PrP has essential roles in early embryonic development. The amenability of zebrafish to global technologies has generated data indicating the existence of “anchorless” splice variants of PrP in the early embryo. This paper will discuss the possibility that the experimentalist’s view of PrP functions might be clearer at a greater phylogenetic distance.     

Effects of temperature during early life history on embryonic and larval development and growth in haddock

The effect of incubation temperature (2, 4, 6, 8 and 10° C) on haddock Melanogrammus aeglefinus development and growth during the embryonic period and in subsequent ontogeny in a common post‐hatch thermal environment (6° C) was investigated. Hatching times were inversely proportional to incubation temperature and ranged from 20·3 days at 2° C to 9·1 days at 10° C. Growth rates were directly proportional to incubation temperature during both the embryonic and larval periods. There was a significant decline in growth rates following hatch in all temperature groups. Compared to the endogenously feeding embryos, growth rates in the exogenous period declined by 4·4‐fold at 4° C to 3·9‐fold at 8° C, indicative of the demarcation between the endogenous and exogenous feeding periods. Yolk utilization varied from 17 days at 2° C to 6 days at 10° C and followed a three‐stage sigmoidal pattern with the initial lag period inversely proportional to incubation temperature. Time to 50% yolk depletion varied inversely with temperature but occurred 1–1·5 days post‐hatch at all temperatures. Additionally, the period between 10 and 90% yolk depletion also decreased with increased temperature. Overall developmental rate was sequential with and directly proportional (2·3‐fold increase) to incubation temperature while the time spent in each developmental stage was inversely proportional to temperature. Larger embryos tended to be produced at lower temperatures but this pattern reversed following hatch, as larvae from higher temperature groups grew more rapidly than those from other temperature groups. Larvae from all temperatures achieved a similar length (c.total length 4·5 mm) upon complete yolk absorption. The study demonstrated the significant impact that temperature has upon developmental and growth rates in both endogenous and exogenous feeding periods. It also illustrated that temperature changes during embryogenesis had significant and persistent effects on growth in subsequent ontogeny.     

Membrane-anchorage of Cripto protein by glycosylphosphatidylinositol and its distribution during early mouse development

cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell–cell interactions that play a critical role in the early patterning of the embryo.     

Macrophages of hemangioblastic lineage invade the lens vesicle-ectoderm interspace during closure and detachment of the avian embryonic lens

Summary An area of cell death is apparent in the lens vesicle margin and the lens stalk during closure and detachment of the lens anlage from the cephalic ectoderm. Free phagocytic cells closely associated with this area of cell death have been interpreted as cells migrating from the lens epithelium. Scanning and transmission electron microscopy, light-microscopic histochemical staining for acid phosphatase and immunostaining using MB1 (a monoclonal antibody specific for quail endothelial and hemopoietic cells) of chimeras of chick embryo and quail yolk sac were used to analyze these lens vesicle-associated free phagocytic cells. The cells have morphological features identical to those of macrophages in other embryonic tissues. In contrast to epithelial cells phagocytosing cell debris, they exhibit strong acid phosphatase activity, a feature typical of macrophages. In addition, free phagocytic cells are MB1 positive in chick embryo-quail yolk sac chimeras, hence they proceed from cells of hemangioblastic lineage originating in the yolk sac. These results indicate that the lens vesicle-associated free phagocytic cells are macrophages. Observations of MB1 positive amoeboid cells in the juxta-retinal mesenchyme and on the borders of the optic cup suggest that these macrophages migrate through the mesenchyme surrounding the eye primordium. Macrophages are seen in both the interspace between lens vesicle and ectoderm and in the lumen of the lens as well as within both the ectoderm and the lens epithelium. In these locations they remove cell debris, and thereby contribute to the complete disappearance of the area of cell death. Macrophages remain in the lens vesicle-ectoderm interspace until developmental stages at which it is invaded by corneal endothelial cells.     

Impact of embryonic expression of enhanced green fluorescent protein on early mouse development

The impact of embryonic enhanced green fluorescent protein (EGFP)-expression on development is not clear. In this study, we comprehensively assessed EGFP-expression pattern and its effect on early mouse development, following pronuclear-microinjection of the EGFP-transgene, containing chicken-beta-actin promoter and cytomegalovirus enhancer. Preimplantation embryos exhibited differential EGFP-expression patterns. While blastocyst development of non-expressing embryos was 77.3+/-1.8%, that of expressing embryos was only 43.9+/-1.6% (P<0.0001). Developmental competence of embryos negatively correlated (r=-0.99) with the levels of EGFP-expression. Faint-, moderate-, and intense-expressing embryos developed to 83.1+/-5.3%, 50+/-5%, and 9.5+/-3.9% blastocysts, respectively (P<0.002). Interestingly, blastocysts expressing faint-moderate levels of EGFP were developmentally competent through the post-implantation period and delivered viable transgenic 'green' mice, following embryo transfer. These results indicate that hyper-expression of EGFP affects preimplantation development and faint-moderate level of its expression is compatible with normal embryogenesis in the mouse.     

The expression of c-kit protein during oogenesis and early embryonic development.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and was shown to be allelic with the white-spotting locus (W) of the mouse. Mutations at the W locus have pleiotropic effects on the development of hematopoietic stem cells, melanoblasts, and primordial germ cells. In order to elucidate the role of c-kit protein in gametogenesis and oocyte maturation, we have examined immunohistochemically the expression of c-kit in the ovaries of mice at late fetal and postnatal stages, and in early embryos. By the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody, the c-kit protein was detected in ovaries after the time of birth, but not before. The expression of c-kit was observed mainly on the surface of oocytes, but not in granulosa cells nor in interstitial regions. Oocytes of primordial to fully grown Graafian follicles showed the c-kit protein. When ovulation was induced by hCG, the expression of c-kit in ovulated unfertilized oocytes was weaker than in oocytes of Graafian follicles. In 1-cell embryos the c-kit protein was still observed, but with cell division its expression further decreased, and it was not detected in embryos of 4-cell, 8-cell, and morula stages. In summary, the highest expression of c-kit was observed on the surface of oocytes arrested in the diplotene stage of meiotic prophase. With ovulation and the resumption of meiotic maturation, its expression declined. These results suggest that the c-kit protein may play some role in meiotic arrest, oocyte growth, and oocyte maturation.