摘要:

AbstractWe have investigted the developmental capacity of mouse embryos in which one blastomere was destroyed by lysis at the 2‐cell stage. The allocation of cells to the trophectoderm and inner cell mass (ICM) was documented by differential cell counts on single embryos after 2 days under different culture conditions. Viability and further developmental potential were tested by embryo transfer to foster mothers. The conditions used were: (1) in vitro culture in modified BMOC‐2 medium, (2) in vivo oviduct transfer to immature (prepuberal) females, and (3) in vivo oviduct transfer to pseudopregnant females. Half embryos almost always fared less well for all parameters of development than control embryos developing under the same conditions. Lower total cell numbers in half embryos were accounted for by decreases in both ICM and trophectoderm with a disproportionate decrease in ICM in smaller embryos. In both half and control embryos, the growth conditions affected the rate of morphological development, the total cell number, and embryo viability. Unlike the effect of halving embryos, the growth condition effects on total cell number can be accounted for primarily by differences in ICM cell number, with trophectoderm cell number remaining constant. These results provide new information on the ability of the mouse embryo to differentially regulate ICM and trophectoderm cell number under different conditions, and confirm our previous work showing the advantage of short‐term development in vivo over short‐term in vitro culture (Papaioannou and Ebert [1986] J. Reprod. Fertil. 76:603–608). They also clearly document that lower total cell number and a lower proportion of ICM is correlated with compromised de

ISSN:1058-8388    

DOI:10.1002/aja.1002030402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractVascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate endothelial cell proliferation, migration, and vascular tube formation. We tested the hypotheses that these growth factors stimulate (1) cell migration and (2) assembly into cord‐like structures in embryonic rat heart explants cultured on collagen gels. Atrial and ventricular explants from rat embryos at 12 (E12, avascular) and 14 (E14, early vascularization stage) days of gestation were cultured on a collagen substrate. Western blot analysis of the explants indicated that endogenous VEGF was present in both atria and ventricles during incubation. Addition of bFGF to E12 explants markedly increased cell migration, whereas VEGF had no significant effect. In E14 explants neither growth factor influenced cell migration. Cotreatment with VEGF and bFGF did not have a synergistic effect on the migration distance of cells from either E12 or E14 embryonic hearts. However, VEGF stimulated the appearance of cord‐like structures in E14, but not E12, explants. Transmission electron microscopy analysis showed that these cord‐like structures consist of elongated cells, some of which aggregate into clusters, or form tube‐like structures, similar to capillaries. Serial sections of monolayers revealed that tube formation occurs beneath the surface of collagen gel. We conclude that in this model system VEGF and bFGF play distinct roles, at specific time points, in coronary vascular tube formation in the developing heart. ©1995 Wiley

ISSN:1058-8388    

DOI:10.1002/aja.1002030403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractLaminin‐1 is an extracellular matrix protein composed of three polypeptide chains that are designated α1, βT1, and γ1. We investigated the expression of laminin α1, β1, and γ1 subunit chains during several stages of rabbit fetal lung development. Utilizing polyclonal antibodies directed against human placental laminin and immunoblot analysis, we found that the highest levels of laminin α1, β1, and γ1 subunit chains in the fetal lung were present on day 26 of gestation (term = 31 days), coincident with the initiation of alveolar epithelial cell differentiation. Levels of the laminin chains were approximately five times higher in fetal lung at day 26 of gestation than in adult lung tissue. Different temporal patterns of laminin α1, β1, and γ1 subunit chain expression were observed, data suggestive that the chains are independently regulated during lung development. Laminin was localized to the basement membranes of bronchi, bronchioles, preal‐veolar ducts, and blood vessels in fetal lung tissue, as shown by immunostaining with polyclonal laminin antibodies. A similar staining pattern was observed in adult lung tissue, but the alveolar wall was also stained. Laminin was also observed surrounding a few mesenchymal cells in fetal lung on day 19 of gestation; the number of positive mesenchymal cells increased with lung development. Laminin α1 subunit chains, detected using a monoclonal antibody, were present in the basement membranes of bronchi, bronchioles, preal‐veolar ducts, and blood vessels in fetal lung tissue. No laminin α1 chain staining was observed in the mesenchyme of early fetal lung tissue. Using a monoclonal antibody, laminin β1 subunit chains were immunolocalized in the basement membranes of bronchi, bronchioles, in prealveolar ducts, and surrounding some mesenchymal cells in fetal lung tissue. Laminin α1 and β1 subunit chains in adult lung tissue were present in basement membranes of airways, blood vessels, and alveoli. Thus, changes in the localization and accumulation of laminin near the time of alveolar type I and type II epithelial cell differentiation suggest that laminin may play a role in mediating the differentiation of these cell types during rabbit fetal lung development.

ISSN:1058-8388    

DOI:10.1002/aja.1002030404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractMutation of theAPC(adenomatous polyposis coli) gene is an early event in colon tumor development in humans. Mice carryingMin(multiple intestinal neoplasia), a mutant allele ofApc, develop intestinal and mammary tumors as adults. To study the role of theApcgene in development, we have investigated the phenotype of embryos homozygous forApcMin(Min). Development of the primitive ectoderm fails prior to gastrulation in homozygousMinembryos. By midgestation, the presumed homozygotes consist of a mass of trophoblast giant cells with an additional cluster of much smaller embryonic cells. These results indicate that functional Apc is required for normal growth of inner cell mass derivatives. ©1995 Wiley‐Liss, I

ISSN:1058-8388    

DOI:10.1002/aja.1002030405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractOP‐1, also known as BMP‐7, is a member of the TGF‐β superfamily of proteins and was originally identified on the basis of its ability to induce new bone formation in vivo. OP‐1 mRNA is found in the developing kidney and adrenal gland as well as in some brain regions (Özkaynak et al. [1991] Biochem. Biophys. Res. Commun. 179:116–123). We have tested the effect of recombinant human OP‐1 on quail trunk neural crest cultures. The number of catechol‐amine‐positive cells which developed after 7 days in vitro in the presence of OP‐1 was increased in a dose‐dependent manner, with a greater than 100‐fold maximal stimulation observed. The increase in the number of catecholamine‐positive cells in the presence of OP‐1 was paralleled by an increase in the number of tyrosine hydroxylase (TH)‐positive cells. In contrast, total and melanocyte cell number were unaffected by the presence of OP‐1. The number of Islet‐1‐immunoreactive cells was also increased by OP‐1, but to only about half the value seen for TH. Double label experiments revealed these Islet‐1‐positive cells were a subset of the TH‐positive cells. Inhibitors of DNA synthesis prevented the OP‐1‐mediated increase in adrenergic cell number, indicating that OP‐1 does not act on a postmitotic cell population. However, labeling studies with bromodeoxyuridine indicated that OP‐1 did not increase the proportion of the cell population engaged in DNA synthesis. Thus, the OP‐1‐mediated increase in adrenergic cell number most likely occurs as a result of the enhanced survival of a subpopulation of adrenergic precursors or an increase in their probability of adrenergic differentiation, but not by increasing the mitotic rate of adrenergic precursors or adrenergic cells themselves. In contrast to OP‐1, TGF‐β1 decreased adrenergic cell number. When OP‐1 and TGF‐β1 were added simultaneously, TGF‐β1 antagonized the OP‐1‐mediated increase in

ISSN:1058-8388    

DOI:10.1002/aja.1002030406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractWe have determined the localization and developmental expression of glutamate decarboxylase (GAD67) in the rat lens. Immunofluorescence experiments showed that GAD67 was transiently expressed in the nuclear fiber cells of the lens between embryonic days (E) 15 and 20, with maximal immunostaining occurring on E17 and E18. γ‐amino butyric acid (GABA) co‐localized with GAD67 in the embryonic nuclear fiber cells. Reverse transcription‐polymerase chain reaction (RT‐PCR) tests showed that at least three alternatively spliced forms of GAD67 mRNA, including mRNAs with and without the I80 and the I86 insert, were transiently co‐expressed with GAD67 in the embryonic lens. The major GAD67 protein in the lens was 67 kDa. We conclude that enzymatically active GAD67 is transiently expressed in the lens nuclear fiber cells of the embryonic rat. The transient expression is regulated by transcriptional and/or posttranscriptional processes. We speculate on the basis of possible common gene regulatory elements for glutamate and ornithine decarboxylases and the involvement of these enzymes with polyamine synthesis, that the transient expression of GAD67 may be connected to nuclear and/or DNA breakdown during lens fiber cell differentiation. ©1995 Wile

ISSN:1058-8388    

DOI:10.1002/aja.1002030407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractWe have shown that unidirectional pumping of Na+out of the neural tube's luminal fluids in amphibian embryos produces a large potential difference (40–90 mV, lumen negative to the abluminal surface). This transneural tube potential (TNTP) is analogous to the Na+dependent transepithelial potential (TEP) that exists across surface ectoderm. This TEP is retained in ectoderm after it is internalized when the neural folds fuse to form the neural tube. The TNTP can be markedly reduced for several hours by injection of the Na+channel blockers amiloride or benzamil into the lumen by iontophoresis through microelectrodes. Here we describe the effect of TNTP modification on developmental anatomy. Axolotl embryos possessing a fused and closed neural tube (stage 21–23) were injected with either amiloride or benzamil and allowed to continue development for 36–52 hr. These were compared to control embryos injected with vehicle alone, or to embryos in which amiloride or benzamil was iontophoresed just beneath surface ectoderm. All embryos in which the TNTP was reduced were grossly defective. These were characterized by a disaggregation of the cells comprising the structures that had already begun to form (otic primordia, brain, spinal cord, notochord) as well as a failure in the development of new structures. Remarkably, some of these embryos displayed continuing development of external form in the complete absence of concomitant internal histogenesis. We discuss the ways in which a large endogenous voltage gradient associated with an epithelial potential difference (the TNTP) may be required both for the structural integrity of the early neuroepithelium, and a prerequisite for normal morphogenesis. ©1995 Wiley‐L

ISSN:1058-8388    

DOI:10.1002/aja.1002030408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractIn the process of endochondral ossification, chondrocytes progress through a series of maturational changes, including division and hypertrophy, that culminate in chondrocyte loss and cartilage resorption. From an investigation of morphology, DNA fragmentation, and collagen synthesis in the developing chick sterna we have characterized chondrocytes death in this process. Light microscopy of resorbing sterna demonstrated chondrocyte condensation at the interface with the invading vasculature and electron microscopy demonstrated a range of chondrocyte morphologies, including retraction from the pericellular matrix, cytoplasmic and nuclear condensation, and vesiculation suggestive of sequential changes characteristic of apoptosis. Isolation and end‐labeling of DNA from chick primary ossification centers demonstrated fragmentation to nucleosome sized units, only in primary ossification centers exhibiting active resorption, and in situ detection of DNA fragmentation showed a restriction to chondrocytes at the interface with invading blood. We conclude that terminal differentiation of chondrocytes results in death by an apoptotic process prior to resorption of the tissue and invasion by blood vessels. The extent of DNA fragmentation correlated closely with the proportion of cells displaying a condensed phenotype in contralateral primary ossification centers and peaked at an early stage of resorption, suggesting that chondrocyte apoptosis may be an initiating event in tissue resorption and vascular invasion. Comparison of DNA fragmentation with expression of the hypertrophic chondrocyte phenotype, as indicated by type X collagen synthesis, suggested that DNA fragmentation was a late event in the process of chondrocyte hypertrophy and probably corresponded with chondrocyte condensation. ©1995 Wiley‐Liss,

ISSN:1058-8388    

DOI:10.1002/aja.1002030409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe thrombospondins are a family of related glycoproteins found in the embryonic extracellular matrix. To date, five members of this family have been identified. Thrombospondin‐1 and thrombospondin‐2 have similar primary structure, but are expressed in different tissues at different times during development. Thrombospondins‐3, ‐4, and cartilage oligomeric protein belong to a second thrombospondin subgroup in which the carboxyl‐half of each molecule is most similar to thrombospondin‐1 and ‐2. Here, we report the cloning and sequencing of a novel probe to avian thrombospondin‐4. We have used this probe to determine the origins of thrombospondin‐4 in the chick embryo by in situ hybridization. Thrombospondin‐4 transcripts first appear in the mesenchyme surrounding bone anlage coinciding with the initial stages of osteogenesis. The expression in osteogenic tissues is transient: thrombospondin‐4 mRNAs are not seen in the osteoblasts of bone collars in developing long bones. This pattern is distinct from avian thrombospondin‐2, which is expressed in perichondrium and embryonic fibrous connective tissues. Our observations indicate that connective tissues are the principal site of thrombospondin‐4 expression in the chick. The diverse origins of different thrombospondin gene family members imply distinctive roles for these proteins related to the growth and differentiation of cartilage, tendons, and bo

ISSN:1058-8388    

DOI:10.1002/aja.1002030410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractTenascin‐X (TN‐X) is the newest member of the tenascin family of extracellular matrix proteins and it is highly expressed in muscular tissues during development. To gain insight into the possible functions of TN‐X during development, we evaluated its expression in the rat embryo. Using an 800 bp cDNA encoding the fibrinogen‐like domain of TN‐X, we show that TN‐X expression begins in migrating cells of the epicardium in the E12 heart. The epicardium provides progenitors of fibrous and vascular tissue to the developing heart. After the epicardium is complete, TN‐X is expressed in the sub‐epicardial space in association with developing blood vessels, and later by non‐myocytes dispersed through the myocardial wall. A similar pattern of TN‐X expression, first in connective tissue surrounding muscle, and then by a subset of cells within muscle, was seen in para‐axial, body wall, craniofacial, and appendicular muscle. This pattern suggests a role in connective tissue cell migration and late muscle morphogenesis. TN‐X is also highly expressed in the interdigital space at E15 and surrounding developing tendons, suggesting an additional role in cell fate determination. Although the pattern of TN‐X expression is distinct from that of tenascin C, they are frequently expressed in close proximity. Indirect genetic evidence in humans suggests an essential function for TN‐X, and the pattern of TN‐X expression in heart, skeletal muscle, and limb is consistent with this hypo

ISSN:1058-8388    

DOI:10.1002/aja.1002030411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY