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PubMed2004

Immunocytochemical detection of synaptophysin in enteric neurones during prenatal development in the rat stomach.

Summary In this study, the localization and appearance of synaptophysin-immunoreactive (IR) nerve cells and their relationships with the developing gastric layers were studied by immunocytochemistry and light microscopy in the embryonic rat stomach. The stomachs of Wistar rat embryos aged 13-21 days were used. The first neuronal bodies and their processes containing synaptophysin-immunoreactivity were observed on embryonic day 13. In contrast, synaptophysin-IR nerve terminals were first observed between mesenchymal cells on embryonic day 14. These results indicate that synaptophysin is expressed in growing neurits and neuronal cell bodies before these neurones have established synaptic connections. The occurrences of mesenchymal cell condensation near synaptophysin-IR neuroblasts on embryonic day 15 reflect an active nerve element-specific mesenchymal cell induction resulting in the morphogenesis of muscle cells. Similarly, the appearance of glandular structures after synaptophysin-IR neuroblasts, on embryonic day 18, suggests that the epithelial differentiation may be closely related to the neuronal maturation as well as other factors. Finally, synaptophysin is functionally important in neuronal development and maturation, together with the establishment of neuroneuronal and neuromuscular contacts and in epithelial differentiation.

Animals↗
PubMed2002

Immunocytochemical detection of neuronal nitric oxide synthase (nNOS)-IR in embryonic rat stomach between days 13 and 21 of gestation.

In this study, the localization and appearance of neuronal nitric oxide synthase-immunoreactive (nNOS-IR) nerve cells and their relationships with the developing gastric layers were studied by immunocytochemistry techniques and light microscopy in embryonic rat stomach. The stomachs of Wistar rat embryos aged 13-21 days were used. The first nerve cells containing nNOS-IR were seen on embryonic Day 14. The occurrence of mesenchymal cell condensation near nNOS-IR neuroblasts on embryonic Day 15 may reflect an active nerve element-specific mesenchymal cell induction causing the morphogenesis of muscle cells. Similarly, the appearance of glandular structures after nNOS-IR neuroblasts, on embryonic Day 18, suggests that the epithelial differentiation may depend on inputs coming from nNOS-IR neuroblasts, as well as other factors. Observation of nNOS-IR nerve fibers on embryonic Day 21 demonstrates that at this stage they contribute to nonadrenergic noncholinergic relaxation. In conclusion, depending on this study's results, it can be said that cells and tissues might be affected by NO secreted by nNOS-IR nerve cells during the development and differentiation of embryonic rat stomach.

Animals↗
PubMed1994

Inhibition of prolyl 4-hydroxylase by oxalyl amino acid derivatives in vitro, in isolated microsomes and in embryonic chicken tissues.

The potency of oxalyl amino acid derivatives as inhibitors of prolyl 4-hydroxylase was studied in vitro, in isolated microsomes and in chicken embryonic-tissue culture. These compounds represent structural analogues of 2-oxoglutarate in which the -CH2- moiety at C-3 is replaced by -NH-, with or without further structural modifications. The most efficient inhibitor of purified prolyl 4-hydroxylase was oxalylglycine. Its mode of inhibition was competitive with respect to 2-oxoglutarate. The Ki value varied between 1.9 and 7.8 microM, depending on the variable substrate used. Oxalylalanine inhibited purified enzyme with a Ki of 40 microM. Other oxalyl amino acid derivatives showed little inhibitory activity. In microsomes isolated from embryonic chicken bone, oxalylglycine and oxalylalanine inhibited prolyl hydroxylation with IC50 values of 23 and 120 microM respectively. Dimethyloxalylglycine was not an inhibitor of purified prolyl 4-hydroxylase and only weakly active in the microsomal system, but efficiently suppressed hydroxyproline synthesis in embryonic chicken calvaria and lung. The data suggest that dimethyloxalyl amino acids are converted into active inhibitors in intact cells, most likely in the cytoplasmic compartment.

Amino Acid Sequence↗
PubMed2002

A missense mutation (R565W) in cirhin (FLJ14728) in North American Indian childhood cirrhosis.

North American Indian childhood cirrhosis (CIRH1A, or NAIC), a severe autosomal recessive intrahepatic cholestasis described in Ojibway-Cree children from northwestern Quebec, is one of several familial cholestases with unknown molecular etiology. It typically presents with transient neonatal jaundice, in a child who is otherwise healthy, and progresses to biliary cirrhosis and portal hypertension. Clinical and physiological investigations have not revealed the underlying cause of the disease. Currently, liver transplantation is the only effective therapy for patients with advanced disease. We previously identified the NAIC locus by homozygosity mapping to chromosome 16q22. Here we report that an exon 15 mutation in gene FLJ14728 (alias Cirhin) causes NAIC: c.1741C-->T in GenBank cDNA sequence NM_032830, found in all NAIC chromosomes, changes the conserved arginine 565 codon to a tryptophan, altering the predicted secondary structure of the protein. Cirhin is preferentially expressed in embryonic liver, is predicted to localize to mitochondria, and contains WD repeats, which are structural motifs frequently associated with molecular scaffolds.

Amino Acid Motifs↗
PubMed1997

[Recovery of motor function of spinal cord with embryonic spinal cord graft in adult rat].

The capacity of embryonic spinal cord tissue in the repair of injured structure of spinal cord has been noted for years. In order to investigate the embryonic spinal cord graft in the repair of motor function of injured spinal cord, the embryonic spinal cord tissue was transplanted to the hemisection cavity in spinal cord in adult rat. One hundred adult Wistar Rats were used to simulate the hemisectional injury of spinal cord by drilling 2-3 mm cavity in lumbar enlargement. Sixty rats were treated with rat embryonic spinal cord tissue grafting while the other forty were chosen as control. The outcome was evaluated according the combined behavioural score (CBS) and motor evoked potential (MEP) in the 1, 2, 4 and 12 weeks. The grafting group was superior to the control as assessed by CBS (P < 0.05), especially within 4 weeks. (P < 0.01). The restoration of the latent peak of early wave(P1, N1) was better in the grafting group, too. This suggested that embryonic spinal cord graft could improve the recovery of motor function of injured spinal cord in adult rat. The effect of the embryonic spinal cord tissue graft might be concerned with its secretion of several kinds of neurotrophic factors, nerve growth factor, nerve transmitted factor, or adjustment of hormone.

Animals↗
PubMed1975

Tracing of cells of the avian thymus through embryonic life in interspecific chimeras.

Differences in the structure of the interphase nucleus between two species of birds, the Japanese quail (Coturnix coturnix japonica) and the chick (Gallus gallus) has been used to distinguish cells from different origins in interspecies combinations. This biological cell marking technique was applied to thymus histogenesis. Using various combinations between components of quail and chick thymic rudiments, the respective contribution of endodermal epithelium, mesenchyme, and blood-borne extrinsic elements to the histogenesis of thymus was analyzed. It was demonstrated that the whole lymphoid population of the thymus is derived from immigrant blood-borne stem cells which are chemically attracted by the endoderm of the 3rd and 4th pharyngeal pouch. The latter is determined to differentiate into thymic epithelial reticulum as soon as the 15-somite stage, and is able to attract blood stem cells even when transplanted in an heterotopic position such as the ventral body wall of the embryo. It was shown that the thymic mesenchyme originates from the neural crest mesectoderm which colonizes early the 3rd and 4th branchial arches. It participates in the formation of perivascular mesenchyme, but does not give rise to lymphocytes. From heterospecific transplantations of quail thymuses into chick embryo (and inversely) at various stages of development is appeared that the thymic rudiment becomes attractive for lymphoid stem cells at a precise stage of its evolution for each species. The attractivity period lasts about 24 h for the quail and 36 h for the chick. Then, the inflow of stem cells becomes very low until the end of the incubation period. At this time, a second wave of lymphocytoblasts invades the thymus and the primitive embryonic lymphoid population is completely renewed around the hatching time. Competent thymic stem cells are present in the blood before and after the period of physiological thymic attractivity. The identity of basophilic cells appearing in the thymus during its histogenesis and lymphoid stem cells has been demonstrated from the analysis of quail-chick chimeric thymuses.

Animals↗
PubMed2001

Three-dimensional imaging of embryonic mouse kidney by two-photon microscopy.

Developing mammalian embryonic kidney becomes progressively more elaborate as the ureteric bud branches into undifferentiated mesenchyme. Morphological perturbations of nephrogenesis, such as those seen in inherited renal diseases or induced in transgenic animals, require careful and often tedious documentation by multiple methodologies. We have applied a relatively quick and simple approach combining two-photon microscopy and advanced three-dimensional (3-D) imaging techniques to visualize and evaluate these complex events. As compared with laser confocal microscopy, two-photon microscopy offers superior optical sectioning deep into biological tissues, permitting analysis of large, heterogeneous, 3-D structures such as developing mouse kidney. Embryonic and newborn mouse kidneys were fluorescently labeled with lectins, phalloidin, or antibody. Three-dimensional image volumes were then collected. The resulting volume data sets were processed using a novel 3-D visualization technique. Reconstructed image volumes demonstrate the dichotomous branching of ureteric bud as it progresses from a simple, symmetrical structure into an elaborate, asymmetrical collecting system of multiple branches. Detailed morphology of in situ cysts was elucidated in a transgene-induced mouse model of polycystic kidney disease. We expect this integration of two-photon microscopy with advanced 3-D image analysis will provide a powerful tool for illuminating a variety of complex developmental processes in multiple dimensions.

Animals↗
PubMed1992

Liver development in rats during the embryonic period (Carnegie stages 11-14).

The hepatic structures appearing during Carnegie stages 11-14 were analyzed in a series of 61 OFA rat embryos. The group of embryos (crown-rump length 2-7 mm, 10th-12th days after coitus) was composed of 52 specimens of the somite period (stages 11 and 12) and of 9 specimens of the postsomite period (stages 13 and 14). The embryos were submitted to serial histological sectioning with graphic reconstruction. Stage 11 was characterized by the development of the hepatic diverticulum induced by differential growth of the endodermal plate and fixed contact between the entoderm and endothelium lining the heart. Stage 12 presented obvious signs of cellular differentiation, the septum transversum giving the liver stroma and the hepatic diverticulum the epithelial trabecula. At stage 13 the epithelial cords enmeshed the stromal capillaries, while hepatocardiac veins drained the hepatic flow into the sinus venosus. Stage 14 was recognized by the presence and development of the hepatic lobes and the enlargement of vascular channels. The cystic bud was never observed. The identification of these features permits to precise the hepatic developmental staging in rats and to obtain accurate criteria for the characterization of the end of the somite period and the beginning of the postsomite period.

Animals↗
PubMed2006

Physiopathology of the embryonic heart (with special emphasis on hypoxia and reoxygenation).

The adaptative response of the developing heart to adverse intrauterine environment such as reduced O2 delivery can result in alteration of gene expression with short- and long-term consequences including adult cardiovascular diseases. The tolerance of the developing heart of acute or chronic oxygen deprivation, its capacity to recover during reperfusion and the mechanisms involved in reoxygenation injury are still under debate. Indeed, the pattern of response of the immature myocardium to hypoxia-reoxygenation differs from that of the adult. This review deals with the structural and metabolic characteristics of the embryonic heart and the functional consequences of hypoxia and reoxygenation. The relative contribution of calcium and sodium overload, pH disturbances and oxidant stress to the hypoxia-induced cardiac dysfunction is examined, as well as various cellular signaling pathways (e.g. MAP kinases) involved in cell survival or death. In the context of the recent advances in developmental cardiology and fetal cardiac surgery, a better understanding of the physiopathology of the stressed developing heart is required.

Animals↗
PubMed1976

The embryology of the common marmoset (Callithrix jacchus).

Earlier studies have noted the unusual placental anatomy of marmosets and the high frequency of biovular, synchorial twinning. The maintenance of a captive colony of Callithrix jacchus has enabled further studies to be made of embryonic development in this species. Thirty-six embryos from seventeen pregnancies were examined in detail and assigned to horizons on the basis of Streeter's classification of external form and internal structure. The specimens represented tha major part of the embryonic period and, with few exceptions, the structural development of the marmoset closely resembled that of the other primates that have been described. Marmoset embryos of a given horizon were consistently shorter than those of man. The timing of morphogenesis in C. jacchus was different from that observed in man and other primates. There was a considerable delay in early embryogenesis in the marmoset, although subsequent to stage XI, development proceeded at approximately the same rate as other primates. The available evidence suggested that this anomaly was not due to delayed implantation or embryonic diapause but to a generally slower rate of early development. The evolution of synchorial twinning is considered as a possible reason for this mode of development.

Abortion, Induced↗
PubMed1991

Analysis of the role of hormones and growth factors in growth control and tissue differentiation using transplanted mammalian embryos and fetal structures.

Results obtained from our analyses of the role of hormones and growth factors in rat embryonic and fetal development are reviewed. Whole 10-day embryos or structures from 14-16-day fetuses (paws, intestines, reproductive tracts) were transplanted under the kidney capsule of syngeneic host rats of different age, sex, physiological state or level of nutrition. In intact hosts, fetal transplants grew almost as much as they would have if left in situ in the fetuses, and tissue differentiation in them was essentially normal. By contrast, growth of embryo transplants was severely depressed relative to their growth in utero, but tissue differentiation was only slightly retarded. Fetal paws grew equally well in female hosts widely diverging in age and growth rate. Thus, in such hosts growth of the fetal paw was highly independent of the growth rate of the hosts. In hosts in which growth was arrested or reduced by diabetes, hypophysectomy or food restriction, growth of paw transplants was impaired, but not as severely as that of the hosts themselves. Fetal skeletal structures were relatively independent of thyroid hormones (TH) for growth; TH dependence developed progressively postnatally. In pregnant hosts, fetal paw transplants grew as well during the first half of gestation as they did in virgin females. By contrast, during the second half of pregnancy and during both halves of the lactational period, growth of the transplants was significantly reduced. Host skeletal growth was also inhibited during late pregnancy and throughout lactation. The impaired growth during the second half of gestation was associated with a large reduction in serum IGF-I concentration and a resistance to the growth-promoting actions of GH. In the lactating hosts, serum IGF-I concentration returned almost to prepregnancy levels and the resistance to GH persisted, but at a reduced level. The direct effects of hormones, growth factors, and antibodies to them on growth and tissue differentiation in the transplants were assessed using infusion methods. Substances were infused into the renal artery of transplant-bearing kidneys via catheters attached to osmotic minipumps. The direct effects of insulin, GH, IGFs, basic FGF and EGF on transplant growth and tissue differentiation were evaluated. Insulin directly stimulated growth of transplants in diabetic hosts but GH did not have a direct effect in hypophysectomized rats. The growth-restorative effects of GH observed in hypophysectomized hosts were apparently mediated indirectly via IGF-I. Infused rat IGF-II (MSA) was more effective at stimulating growth of embryo transplants than was recombinant human IGF-I.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

[Influence of the activator of transcription GAL4 on growth and development of embryos and embryonic cells in primary cultures of sand dollar].

In order to solve many tasks of biotechnology, constant lines of the cells of marine invertebrates with a high growth potential are required, which are absent at present. We used the universal activator of transcription gal4 to change the degree of expression of genes of growth factors in embryonic sea urchin cells and, thereby, increase their proliferative activity. The fertilized sea urchin eggs and dissociated embryonic cells at the blastula stage were treated with plasmids containing both the functional gene gal4 and the gene devoid of the regions encoding the activator domain. The transfection of embryonic sea urchin eggs with the functional gene led to cell dedifferentiation and formation of tumor-like structures in the embryos or increased number of embryonic cells in culture. In the cells obtained from the transfected embryos, the pigments were found within two months of cultivation, whose absorption spectrum coincided with that of echinochrome.

Animals↗
PubMed1998

Identification of the developmental marker, JB3-antigen, as fibrillin-2 and its de novo organization into embryonic microfibrous arrays.

The monoclonal antibody JB3 was previously shown to react with a protein antigen present in the bilateral primitive heart-forming regions and septation-stage embryonic hearts; in addition, primary axial structures at primitive streak stages are JB3-immunopositive (Wunsch et al. [1994] Dev. Biol. 165:585-601). The JB3 antigen has an overlapping distribution pattern with fibrillin-1, and a similar molecular mass (Gallagher et al. [1993] Dev. Dyn. 196:70-78; Wunsch et al. [1994] Dev. Biol. 165:585-601). Here we present immunoblot and immunoprecipitation data showing that the JB3 antigen is secreted into tissue culture medium by day 10 chicken embryonic fibroblasts, from which it can be harvested using JB3-immunoaffinity chromatography. A single polypeptide (Mr = 350,000), which was not immunoreactive with an antibody to fibrillin-1, eluted from the affinity column. Mass spectroscopy peptide microsequencing determined the identity of the JB3 antigen to be an avian homologue of fibrillin-2. Live, whole-mounted, quail embryos were immunolabeled using a novel microinjection approach, and subsequently fixed. Laser scanning confocal microscopy indicated an elaborate scaffold of fibrillin-2 filaments encasing formed somites. At more caudal axial positions, discrete, punctate foci of immunofluorescent fibrillin-2 were observed; this pattern corresponded to the position of segmental plate mesoderm. Between segmental plate mesoderm and fully-formed somites, progressively longer filamentous assemblies of fibrillin-2 were observed, suggesting a developmental progression of fibrillin-2 fibril assembly across the somite-forming region of avian embryos. Extensive filaments of fibrillin-2 connect somites to the notochord. Similarly, fibrillin-2 connects the mesoderm associated with the anterior intestinal portal to the midline. Thus, fibrillin-2 fibrils are organized by a diverse group of cells of mesodermal or mesodermally derived mesenchymal origin. Fibrillin-2 microfilaments are assembled in a temporal and spatial pattern that is coincident with cranial-to-caudal segmentation, and regression of the anterior intestinal portal. Fibrillin-2 may function to impart physical stability to embryonic tissues during morphogenesis of the basic vertebrate body plan.

Actin Cytoskeleton↗
PubMed1998

An alternative splice variant of the tissue specific transcription factor HNF4alpha predominates in undifferentiated murine cell types.

The transcription factor hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue specific transcription factor mainly expressed in the liver, kidney, intestine and the endocrine pancreas, but is also an essential regulator for early embryonic events. Based on its protein structure HNF4alpha is classified as an orphan member of the nuclear receptor superfamily. Comparing HNF4alpha transcription factors in the differentiated and dedifferentiated murine hepatocyte cell line MHSV-12 we identified in dedifferentiated cells the novel splice variant HNF4alpha7. This variant is characterized by an alternative first exon and has a lower transactivation potential in transient transfection assays using HNF4 dependent reporter genes. HNF4alpha7 mRNA and the corresponding protein are expressed in the undifferentiated pluripotent embryonal carcinoma cell line F9, whereas HNF4alpha1 only appears after differentiation of F9 cells to visceral endoderm. HNF4alpha7 mRNA is also found in totipotent embryonic stem cells. However, the function of HNF4alpha7 seems not to be restricted to embryonic cells as the HNF4alpha7 mRNA is also present in adult tissues, most notably the stomach. All these features suggest that the presence of distinct splice variants of HNF4alpha modulates the activity of HNF4alphain a cell type specific way.

Alternative Splicing↗
PubMed2003

Effects of desiccation on the physiology and biochemistry of Quercus alba acorns.

Seeds that lose viability when dried to a water content of less than 12% are said to be recalcitrant. We subjected acorns of Quercus alba L., a species with recalcitrant seeds, to desiccation to determine the effects of drying on lipids, proteins and carbohydrates of the embryonic axis and cotyledon tissues. Samples of fresh seed and seed dried for selected intervals were analyzed for water content and germination, and for lipids, proteins and carbohydrates by Fourier transform-infrared (FT-IR) spectroscopy. Carbohydrates were further analyzed by gas chromatography (GC). The FT-IR analysis revealed that membrane lipid structure initially exhibited reversible shifts between gel and liquid crystalline phases in response to drying and rehydration; however, reversibility declined as viability was lost. Changes in carbohydrate concentration were observed based on peak height comparisons; sucrose concentration in the embryonic axis increased dramatically after 5 days of drying. The most sensitive indicator of desiccation damage was the irreversible change in protein secondary structure in embryonic axes and cotyledon tissue. These changes were illustrated by shifts in amide absorbance near 1650 cm(-1). Gas chromatography indicated an abundance of sucrose in both the embryonic axes and the cotyledon tissue. Although sucrose concentrations in these tissues were initially similar, sucrose concentration in the embryonic axes became significantly greater than in the cotyledons as the acorns dried. We hypothesize that, in drying acorns, increased concentration of sucrose does not prevent loss of viability, but acts as a glycoprotectant against cell collapse and cell wall membrane damage as water stress increases.

Chromatography, Gas↗
PubMed2000

Inhibiting proteasome activity causes overreplication of DNA and blocks entry into mitosis in sea urchin embryos.

The proteasome has been shown to be involved in exit from mitosis by bringing about destruction of mitotic cyclins. Here, we present evidence that the proteasome is also required for proper completion of S phase and for entry into mitosis in the sea urchin embryonic cleavage cycle. A series of structurally related peptide-aldehydes prevent nuclear envelope breakdown in their order of inhibitory efficacies against the proteasome. Their efficacies in blocking exit from S phase and exit from mitosis correlate well, indicating that the proteasome is involved at both these steps. Mitotic histone HI kinase activation and tyrosine dephosphorylation of p34(cdc2) kinase are blocked by inhibition of the proteasome, indicating that the proteasome plays an important role in the pathway that leads to embryonic p34(cdc2 )kinase activation. Arrested embryos continued to incorporate [(3)H]thymidine and characteristically developed large nuclei. Pre-mitotic arrest can be overcome by treatment with caffeine, a manoeuvre that is known to override the DNA replication checkpoint. These data demonstrate that the proteasome is involved in the control of termination of S phase and consequently in the initiation of M phase of the first embryonic cell cycle.

Animals↗
PubMed1993

Contribution of the pentose phosphate pathway to glucose utilization by preimplantation sheep embryos.

The activity of the pentose phosphate pathway of glucose metabolism in early sheep embryos and in the structures of the advanced conceptus from Day 13 to Day 19 of pregnancy was measured quantitatively during a 2.5-h incubation with glucose as sole energy source. For embryos during cleavage, activity of this pathway accounted for 6-9% of total glucose utilized. The proportion of glucose metabolized through the pentose pathway fell progressively with development and by Day 19 represented 1-2% of glucose turnover. However, total turnover of glucose increased eight fold between the 2-cell and blastocyst stage and the amount of glucose processed through the pentose pathway increased over this time despite the fall in the proportion utilized in this way. In contrast, glucose turnover by the advanced embryo and its extra embryonic membranes progressively decreased as the structures developed. As a result, estimates of the amount of glucose utilized through the pathway per microgram dried weight per hour declined to low values at Day 19 following the peak in activity at about the time of blastulation. Trophoblast and yolk sac processed less glucose through the pentose pathway per microgram dried weight than embryonic tissue but the allantois was similar to the embryo. Overall, the pentose pathway accounted for a relatively constant proportion of the CO2 produced from glucose under these experimental conditions with values generally between 15 and 20% of total CO2 produced. When activities in the components of the advanced conceptus were expressed as the total amount of glucose processed through the pathway per hour, turnover in the embryo, allantois and yolk sac increased progressively with time. By contrast, there was a substantial trough in the activity of the trophoblast on Day 17 of pregnancy.

Allantois↗
PubMed2001

When body segmentation goes wrong.

The segmented or metameric aspect is a basic characteristic of many animal species ranging from invertebrates to man. Body segmentation usually corresponds to a repetition, along the anteroposterior (AP) axis, of similar structures consisting of derivatives from the three embryonic germ layers. In humans, segmentation is most obvious at the level of the vertebral column and its associated muscles, and also in the peripheral nervous system (PNS). Functionally, segmentation is critical to ensure the movements of a rod-like structure, such as the vertebral column. The segmented distribution of the vertebrae derives from the earlier metameric pattern of the embryonic somites. Recent evidence from work performed in fish, chick and mouse embryos indicates that segmentation of the embryonic body relies on a molecular oscillator called the segmentation clock, which requires Notch signaling for its proper functioning. In humans, mutations in genes required for oscillation, such as Delta-like 3 (DLL3), result in abnormal segmentation of the vertebral column, as found in spondylocostal dysostosis syndrome, suggesting that the segmentation clock also acts during human embryonic development.

Animals↗