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PubMed2003

Developmental changes in contractility and sarcomeric proteins from the early embryonic to the adult stage in the mouse heart.

Developmental changes in force-generating capacity and Ca2+ sensitivity of contraction in murine hearts were correlated with changes in myosin heavy chain (MHC) and troponin (Tn) isoform expression, using Triton-skinned fibres. The maximum Ca2+-activated isometric force normalized to the cross-sectional area (FCSA) increased mainly during embryogenesis and continued to increase at a slower rate until adulthood. During prenatal development, FCSA increased about 5-fold from embryonic day (E)10.5 to E19.5, while the amount of MHC normalized to the amount of total protein remained constant (from E13.5 to E19.5). This suggests that the development of structural organization of the myofilaments during the embryonic and the fetal period may play an important role for the improvement of force generation. There was an overall decrease of 0.5 pCa units in the Ca2+ sensitivity of force generation from E13.5 to the adult, of which the main decrease (0.3 pCa units) occurred within a short time interval, between E19.5 and 7 days after birth (7 days pn). Densitometric analysis of SDS-PAGE and Western blots revealed that the major switches between troponin T (TnT) isoforms occur before E16.5, whereas the transition points of slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) and of beta-MHC to alpha-MHC both occur around birth, in temporal correlation with the main decrease in Ca2+ sensitivity. To test whether the changes in Ca2+ sensitivity are solely based on Tn, the native Tn complex was replaced in fibres from E19.5 and adult hearts with fast skeletal Tn complex (fsTn) purified from rabbit skeletal muscle. The difference in pre-replacement values of pCa50 (-log([Ca2+] M-1)) required for half-maximum force development) between E19.5 (6.05 +/- 0.01) and adult fibres (5.64 +/- 0.04) was fully abolished after replacement with the exogenous skeletal Tn complex (pCa50 = 6.12 +/- 0.05 for both stages). This suggests that the major developmental changes in Ca2+ sensitivity of skinned murine myocardium originate primarily from the switch of ssTnI to cTnI.

Animals↗
PubMed2002

[Retroperitoneal cystic lymphangioma. A silent disease in adults].

Primary retroperitoneal tumours may arise from different structures as neural, mesodermal, urogenital ridge, or embryonic remnant tissues. Lymphangioma is a rare benign tumour of the lymphatic tissue. They result from a developmental failure of the lymphatic system. Although benign, they can compress and infiltrative vital structures. The size of the lesion is more important than its location to the symptomatology development. Intraabdominal and retroperitoneal lymphangioma are the rarest tumour, specially when occurring in adults. The tumour can occur at any age and most are asymptomatic. Preoperative diagnosis is facilitated by ultrasonography and computed tomography. In order to correctly diagnose of these neoplasms it is essential to carry on ultrasound and CT examination. Treatment of choice is always surgical and a complete extirpation should be performed, unless vital structures were are involved.

Female↗
PubMed1989

Glycoprotein oligosaccharides as recognition structures.

A series of observations--the pronounced changes in the expression and distribution of oligosaccharide antigens during embryonic development, cell differentiation and oncogenesis, the prominence of these changing structures (oncodevelopmental antigens) on the receptor for epidermal growth factor, and the stimulation of receptor autophosphorylation following their perturbation with antibodies--has suggested that the oligosaccharides of growth factor receptors and complementary lectins may be intimately involved in molecular recognition events in growth and differentiation processes. For elucidating oligosaccharide recognition by diverse cellular and secreted proteins and microbial adhesins, a new technique has been developed which involves the overlay of immobilized oligosaccharide probes (neoglycolipids) derived from glycoproteins and other sources. New insights have been gained into carbohydrate recognition by several mammalian lectins, and a novel receptor system has been discovered in Escherichia coli isolated from patients with urinary tract infections. This new technique seems ideal for elucidating oligosaccharide recognition in diverse biological settings, and for 'quality control' of the sugar chains of recombinant glycoproteins engineered for the purpose of administration to man.

Animals↗
PubMed1986

Expression of the antigens recognized by monoclonal antibodies to human amnion (GB4, GB9, GB11) on the rabbit eye during embryonic development.

Three monoclonal antibodies to human amnion (GB4, GB9 and GB11) which recognized well-defined structures of the adult rabbit eye were used to study the embryonic eye development. The reactivity of GB4 could be identified on the migrating neural crest cells under the corneal epithelium at 2 weeks of gestation. At 3 weeks of gestation, the conjunctival epithelium, corneal epithelium and corneal endothelium reacted with GB4. In the newborn rabbit, the corneal epithelium was reactive with GB9 instead of GB4; corneal endothelium was positive with GB4 at this stage, but gradually became negative in the adult rabbit; the subcapsular epithelium of the lens was recognized by GB4 when it changed from pseudostratified to simple cuboidal epithelium. During early embryonic life, GB11 was positive on the external retinal layer of the optic cup and lens vesicle. In the adult, GB11 reacted only with the pigmented epithelium of the ciliary processes. The results of this study demonstrated that the expression of antigens recognized by GB4, GB9 and GB11 could be mapped according to the epithelial differentiation of the rabbit eye during embryogenesis.

Amnion↗
PubMed1996

Otoconial alterations after embryonic development in hypergravity.

The relation between prolonged hypergravity and structural adaptation of otoconia was studied in hamsters (n = 56). Three groups of hamsters (n = 27), were conceived and born in a centrifuge: group 1 (n = 10) 1 month under 2.5 G, group 2 (n = 9) 5 months under 2.5 G and 4 months under 1 G, group 3 (n = 8) 1 month under 2.5 G and 8 months under 1 G. Control hamsters (n = 29) were conceived and born under 1 G (1 month old, n = 7; 9 months old, n = 22). Histological study of the otoconial layers (energy dispersive x-ray element analysis and scanning electron microscopy) showed similar calcium content, size, and shape in utricular and saccular otoconia in all groups. Different were the utricular otoconial size classes, large, medium-sized, and small. The area with small otoconia increased in group 1 (p = 0.002). In group 2, the large otoconial area decreased (p = 0.001) and the medium-sized one increased (p < 0.001). In group 3, the large otoconial area decreased (p = 0.003) and the medium-sized one increased (p = 0.007). For age-related effects we found group 1 with an increased area of large otoconia (p = 0.001) and a decreased medium-sized one compared to groups 2 (p < 0.001) and 3 (p = 0.02). Hypergravity during formation of otoconia does not affect calcium content, size, or shape, but changes relative size of the areas with large, medium-sized, or small otoconia and the development of these areas. This resulted in a structural adaptation to hypergravity.

Animals↗
PubMed1993

Expression and structural studies of fasciclin I, an insect cell adhesion molecule.

Fasciclin I is a lipid-linked cell-surface glycoprotein that can act as a homophilic adhesion molecule in tissue culture cells. It is thought to be involved in growth cone guidance in the embryonic insect nervous system. To facilitate structure-function studies, we have generated Chinese hamster ovary (CHO) cell lines expressing high levels of cell surface grasshopper and Drosophila fasciclin I. Grasshopper fasciclin I released by phospholipase C cleavage was purified on an immunoaffinity column and single crystals were obtained that diffracted to approximately 5-A resolution. We also generated CHO and Drosophila S2 cell lines that produce a secreted form of fasciclin I. Fasciclin I expressed in S2 cells contains significantly less carbohydrate than the protein expressed in CHO cells, and may therefore be more suitable for crystallization. Biochemical characterization of purified fasciclin I indicates that the extracellular portion exists as a monomer in solution. Circular dichroism studies suggest that fasciclin I is primarily alpha-helical. Its structure is therefore different from other known cell adhesion molecules, which are predicted to be elongated beta-sheet structures. This suggests that fasciclin I may define a new structural motif used to mediate adhesive interactions between cell surfaces.

Amino Acid Sequence↗
PubMed2003

Caenorhabditis elegans nucleoporins Nup93 and Nup205 determine the limit of nuclear pore complex size exclusion in vivo.

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate communication between the nucleus and the cytoplasm. To obtain insight into the structure and function of NPCs of multicellular organisms, we have initiated an extensive analysis of Caenorhabditis elegans nucleoporins. Of 20 assigned C. elegans nucleoporin genes, 17 were found to be essential for embryonic development either alone or in combination. In several cases, depletion of nucleoporins by RNAi caused severe defects in nuclear appearance. More specifically, the C. elegans homologs of vertebrate Nup93 and Nup205 were each found to be required for normal NPC distribution in the nuclear envelope in vivo. Depletion of Nup93 or Nup205 caused a failure in nuclear exclusion of nonnuclear macromolecules of approximately 70 kDa without preventing active nuclear protein import or the assembly of the nuclear envelope. The defects in NPC exclusion were accompanied by abnormal chromatin condensation and early embryonic arrest. Thus, the contribution to NPC structure of Nup93 and Nup205 is essential for establishment of normal NPC function and for cell viability.

Animals↗
PubMed1999

Ventrally located commissural neurons express the GABAergic phenotype in developing rat spinal cord.

Early-forming commissural neurons are studied intensively as a model of axonal outgrowth and pathfinding, yet the neurotransmitter phenotype of the majority of these neurons is not known. The present study has determined that a substantial number of commissural neurons express the 65-kDa isoform of glutamic acid decarboxylase (GAD65) as early as embryonic day 12 (E 12). Patterns of GAD65 localization were compared with those of TAG-1, the Transiently expressed Axonal Glycoprotein that is the best known marker of commissural axons. On E13, both GAD65- and TAG-1-labeled commissural axons emanate from similar lateral and ventromedial regions. However, dorsally located TAG-1-positive commissural axons were GAD65-negative. These results suggest that commissural neurons have both gamma-aminobutyric acid (GABA)ergic and non-GABAergic phenotypes. The intensity of GAD65 staining within commissural somata and axons decreased between E14-15 and continued to decline during embryonic development, whereas terminal-like structures in surrounding neuropil increased dramatically. This sudden loss of somatic and axonal GAD65 staining was unexpected and could be interpreted as commissural neurons only transiently expressing the GABAergic phenotype. Further experiments were undertaken to identify commissural neurons with other established GABAergic markers, GAD67 and GABA. When antibody labeling of the two GAD isoforms was compared, GAD67 was detected 1 day later than GAD65, and in a different subcellular distribution. In contrast to GAD65, GAD67 intensely stained somata but labeled few commissural axons. GABA immunoreactivity also was detected in commissural axons 1 day after GAD65, and the labeling pattern between E13 and E16 resembled that of GAD67 rather than GAD65. When GAD and GABA results were compared, it was clear that a number of ventrally located commissural neurons expressed and maintained the GABAergic phenotype during embryonic development. However, the early expression and subcellular redistribution of GAD65 suggests that the GAD isoforms are differentially regulated. The function of the transient GAD65 expression in commissural somata and axons is unknown, but its temporal expression pattern parallels the transient expression of TAG-1, as both are expressed during the early stages of commissural axon outgrowth and pathfinding.

Animals↗
PubMed1998

Effects of mesenchyme on epithelial tissue architecture revealed by tissue recombination experiments between the submandibular gland and lung of embryonic mice.

Lung epithelium during morphogenesis maintains a sheet structure of polarized cells lining a lumen, in which E-cadherin, beta-catenin and tight junctional proteins are localized at the cell-cell contact sites. On the other hand, the submandibular gland epithelium at early stages of development forms a non-cavitated mass of cells where E-cadherin/beta-catenin are present on the entire cell surfaces and tight junctional proteins are almost absent or weakly scattered. In the present study, tissue recombination experiments were performed between the two organs to explore roles of mesenchyme in the architectural development of the epithelium. Homotypic recombinants of both submandibular gland and lung showed the tissue architecture as observed in the intact organs. In contrast, 11-day lung epithelium cultured with 13-day submandibular mesenchyme formed multilayers of cells with the lumen being less visible. It was accompanied by redistribution of E-cadherin/beta-catenin along the entire cell surfaces and by an irregular distribution of tight junctional proteins. A similar redistribution of these molecules was observed in 15-day lung epithelium cultured with the submandibular mesenchyme, although the epithelial sheet structure lining the lumen was formed. On the other hand, the tissue architecture of submandibular gland epithelium was little affected by lung mesenchyme, although the epithelium was flattened and showed branching morphogenesis.

Animals↗
PubMed1986

A monoclonal antibody against meningococcus group B polysaccharides distinguishes embryonic from adult N-CAM.

The neural cell adhesion molecules (N-CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular polysaccharides of meningococcus group B (Men B) which specifically distinguishes embryonic N-CAM from adult N-CAM. This antibody recognizes alpha 2-8-linked N-acetylneuraminic acid units (NeuAc alpha 2-8). Immunoblot together with immunoprecipitation experiments with cell lines or tissue extracts showed that N-CAM are the major glycoproteins bearing such polysialosyl units. Moreover we could not detect any sialoglycolipid reactive with this antibody in mouse brain or in the neural cell lines examined. By indirect immunofluorescence staining this anti-Men B antibody decorated cells such as AtT20 (D16/16), which expressed the embryonic forms of N-CAM, but not cells that expressed the adult forms. In primary cultures this antibody allowed us to follow the embryonic-to-adult conversion in individual cells. In addition, the existence of cross-reactive polysialosyl structures on Men B and N-CAM in embryonic brain cells for caution in efforts to develop immunotherapy against neonatal meningitis.

Animals↗
PubMedDate not supplied

[Embryonic development of the hip joint].

Articular elements, joint cavity and intra-articular structures of the human hip joint are completely developed at the end of the embryonic period (8. week). The fetal period is characterized by growth of articular elements, by vascularization of the primordial skeleton and by the ossification of the acetabulum.

Cartilage, Articular↗
PubMed2006

Engineering three-dimensional tissue structures using stem cells.

The worldwide status of rapidly increasing demand for organ and tissue transplantation has promoted tissue engineering as a promising alternative, in particular the use of stem cells. Human embryonic stem cells (hESCs) have the advantage of differentiating to all cell types in the body and high proliferation capabilities. Considering their ability to organize into complex multi-cell-type structures during embryonic-like differentiation, hESCs can potentially provide a source of cells for tissue engineering applications and meet the growing demand for viable human tissue structures in therapeutic clinical application. This chapter describes first steps toward realizing this goal gathered from our experience in growing and differentiating hESCs in three dimensions.

Cell Differentiation↗
PubMed1997

The C. elegans MEX-1 protein is present in germline blastomeres and is a P granule component.

In the nematode Caenorhabditis elegans, germ cells arise from early embryonic cells called germline blastomeres. Cytoplasmic structures called P granules are present in the fertilized egg and are segregated into each of the germline blastomeres during the first few cleavages of the embryo. Mutations in the maternally expressed gene mex-1 disrupt the segregation of P granules, prevent the formation of germ cells, and cause inappropriate patterns of somatic cell differentiation. We have cloned the mex-1 gene and determined the distribution pattern of the mex-1 gene products. The MEX-1 protein contains two copies of an unusual 'finger' domain also found in the PIE-1 protein of C. elegans. PIE-1 has been shown to be expressed in germline blastomeres, and is a component of P granules. We show here that MEX-1 also is present in germline blastomeres and is a P granule component, although MEX-1 is a cytoplasmic protein while PIE-1 is present in both the nucleus and cytoplasm. We further show that MEX-1 is required to restrict PIE-1 expression and activity to the germline blastomeres during the early embryonic cleavages.

Amino Acid Sequence↗
PubMed2006

Mannose 6-phosphate receptors in an ancient vertebrate, zebrafish.

The endosome/lysosome system plays key roles in embryonic development, but difficulties posed by inaccessible mammalian embryos have hampered detailed studies. The accessible, transparent embryos of Danio rerio, together with the genetic and experimental approaches possible with this organism, provide many advantages over rodents. In mammals, mannose 6-phosphate receptors (MPRs) target acid hydrolases to endosomes and lysosomes, but nothing is known of acid hydrolase targeting in zebrafish. Here, we describe the sequence of the zebrafish cation-dependent MPR (CD-MPR) and cation-independent MPR (CI-MPR), and compare them with their mammalian orthologs. We show that all residues critical for mannose 6-phosphate (M6P) recognition are present in the extracellular domains of the zebrafish receptors, and that trafficking signals in the cytoplasmic tails are also conserved. This suggests that the teleost receptors possess M6P binding sites with properties similar to those of mammalian MPRs, and that targeting of lysosomal enzymes by MPRs represents an ancient pathway in vertebrate cell biology. We also determined the expression patterns of the CD-MPR and CI-MPR during embryonic development in zebrafish. Both genes are expressed from the one-cell stage through to the hatching period. In early embryos, expression is ubiquitous, but in later stages, expression of both receptors is restricted to the anterior region of the embryo, covering the forebrain, midbrain and hindbrain. The expression patterns suggest time- and tissue-specific functions for the receptors, with particular evidence for roles in neural development. Our study establishes zebrafish as a novel, genetically tractable model for in vivo studies of MPR function and lysosome biogenesis.

Amino Acid Sequence↗
PubMed1990

Extracellular matrix production by embryonic epithelium cultured on type IV collagen. Deposition of a primary corneal stroma-like structure containing large irregular type I fibrils without type II collagen.

The corneal stroma of the chick embryo is deposited in two steps. The primary stroma is laid down by the corneal epithelium and it contains type I, type II and type IX collagens. Its formation is subsequent to the presumptive epithelial cells' migration onto the lens capsule (which is rich in type IV collagen). The secondary, ultimate stroma is synthesized by fibroblasts which, on day 5 of development, invade the swollen primary stroma. It is composed of a matrix of thin (25 nm), regular fibrils containing type I and type V collagens. We found that a chick corneal epithelium isolated from either a 6-day or a 14-day embryo was able to produce, in vitro, stroma-containing type I collagen fibrils. However, the amount of collagen deposited and its organization were highly dependent on the substratum used. Plastic or purified bovine type I collagen substrata led to the release of very few fibrils. Purified human type IV collagen induced the production of an abundant matrix made of large irregular collagen fibrils. When compared to native corneal stroma, there were two aspects in which this matrix differed: (1) it contained only type I collagen, as shown by indirect immunofluorescence, and (2) there were numerous large, irregular fibrils of about 100 to 130 nm in diameter. In conclusion, it is suggested that purified type IV collagen substitutes, in part, for the basement membrane and allows the production of a corneal stroma-like matrix by an embryonic corneal epithelium in culture. This production is possible even with a 14-day epithelium which, in vivo, is no more involved in the synthesis of the stroma collagens. Moreover, the regulatory effect of type II collagen, previously suggested by in vivo observations, may be confirmed in this in vitro system by the appearance of large fibrils in the newly deposited stroma that are made only by type I collagen.

Animals↗
PubMed1997

Cardiotrophin-1 and the role of gp130-dependent signaling pathways in cardiac growth and development.

The reactivation of an embryonic pattern of gene expression is a central feature common to virtually all forms of cardiac hypertrophy. Unraveling the regulatory mechanisms, growth factors and cytokines controlling gene expression and cell fate during cardiac development may therefore have implications for our understanding of cardiac hypertrophy in the adult. Along this line, a cDNA expression library was established from an embryonic stem cell-based in vitro model of cardiogenesis, and screened for clones that would induce an increase in cell size in cultured cardiomyocytes. This experimental strategy resulted in the isolation of a novel cytokine, cardiotrophin-1 (CT-1), that activates several features of cardiomyocyte hypertrophy in vitro, including sarcomeric organization and embryonic gene expression. CT-1 displays structural similarities to the interleukin (IL)-6 related cytokines. Furthermore, receptor binding studies and functional studies reveal that CT-1 shares the signal transducing receptor components gp130 and LIFR with the previously identified members of the IL-6 cytokine family. CT-1 rapidly activates gp130 and LIFR tyrosine phosphorylation in cultured cardiac myocytes. The growth promoting effects of CT-1 therefore indicate that signaling pathways emanating from gp130 and LIFR are coupled to cardiomyocyte hypertrophy. In support of this notion, the simultaneous overexpression of IL-6 and the IL-6 receptor in transgenic mice has been shown to result in a constitutive tyrosine phosphorylation of gp130 in the myocardium and cardiac hypertrophy. The striking phenotype of gp130 null-mutant mice, generated by homologous recombination, implies gp130 in cardiac development as well: mutant mice exhibit severe ventricular hypoplasia, suggesting a role for gp130-dependent signaling pathways in the expansion of the compact layer of the ventricular myocardium. CT-1 is expressed at high levels in the myocardium during the course of cardiogenesis, and promotes the proliferation and survival of embryonic cardiomyocytes. CT-1 may therefore represent a candidate cytokine to activate gp130 during cardiac development. In summary, cytokines signaling through gp130 are emerging as potent regulators of embryonic heart development and adult cardiac hypertrophy.

Animals↗
PubMed1989

Fine structure and organ culture of chick embryo dorsal aorta.

Embryonic blood vessels have not been grown in organ culture, in a way which might easily submit them to studies of vascular organogenesis. The chick embryo dorsal aorta is easily accessible and relatively simple to explant to culture. Its organ culture may provide a model for wounding and repair of the intima and/or media and provide a model for studies of growth (or maintenance) and differentiation. Fresh dorsal aorta and its organ culture from the chick embryo was characterized morphologically by light microscopy scanning (SEM) and transmission electron microscopy (TEM) from days 4 to 18. Explants were incubated up to 4 days at 37.5 degrees C in normal atmosphere in media including whole chick embryo extract (EE) diluted to 20%. The normal fine structure by SEM shows that as the embryo ages the endothelial cells of the aorta tend to elongate, their microvilli lengthen and increase in size and number up to about 11 days, after which they decrease. TEM also demonstrates an increased differentiation of smooth muscle cells as embryonic age advances. The best culture medium was EE20-Tyrode's. Most of the explants from 14 and 17 days cultured in EE20-Tyrode's contracted into small balls of tissue, endothelial side out. None of these explants showed any significant cellular degeneration. None of the explants survived in EE20-DMEM. Addition of insulin to each medium had no effect on the explants. Intimal cells resembling myofibroblasts were observed in the contracted explants. Using a simple culture condition the chick embryo dorsal aorta can be sustained in organ culture for at least 4 days. The most suitable age for organ culture studies is 9 days (or perhaps somewhat younger); however, it is the most difficult to obtain.

Animals↗
PubMed1998

Murine branched chain alpha-ketoacid dehydrogenase kinase; cDNA cloning, tissue distribution, and temporal expression during embryonic development.

These studies were designed to demonstrate the structural and functional similarity of murine branched chain alpha-ketoacid dehydrogenase and its regulation by the complex-specific kinase. Nucleotide sequence and deduced amino acid sequence for the kinase cDNA demonstrate a highly conserved coding sequence between mouse and human. Tissue-specific expression in adult mice parallels that reported in other mammals. Kinase expression in female liver is influenced by circadian rhythm. Of special interest is the fluctuating expression of this kinase during embryonic development against the continuing increase in the catalytic subunits of this mitochondrial complex during development. The need for regulation of the branched chain alpha-ketoacid dehydrogenase complex by kinase expression during embryogenesis is not understood. However, the similarity of murine branched chain alpha-ketoacid dehydrogenase and its kinase to the human enzyme supports the use of this animal as a model for the human system.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗