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PubMed1999

Induction of matrix metalloproteinases and collagenolysis in chick embryonic membranes before hatching.

The membranes surrounding the chick embryo undergo striking morphological changes before hatching, which include structural degradation of the allantoic membrane. The fibrillar collagen content of the membranes declined by embryonic day (ED) 20 (the day of hatching). By ED 19, a 55-kDa matrix metalloproteinase (MMP) activity appeared in the extraembryonic fluid, and by ED 20 there was substantial 55-kDa MMP activity in embryonic membrane extracts. Reverse transcription-polymerase chain reaction was employed to clone a partial cDNA representing the chicken homologue of MMP-13, a 55- to 57-kDa enzyme. MMP-13 mRNA dramatically increased in abundance in embryonic membranes by ED 19, reaching a peak on ED 20. Introduction of the MMP inhibitor batimastat into the extraembryonic fluid prevented the structural changes in the embryonic membranes before hatching. We conclude that, like mammalian fetal membranes, chick embryonic membranes undergo terminal remodeling before hatching, in part as a result of increased MMP activity. The chicken egg system represents a novel in vivo model for exploring biochemical events leading to embryonic membrane remodeling prior to birth and to test inhibitors of MMPs for their ability to prevent collagenolysis and fetal membrane rupture.

Allantois↗
PubMed1999

[The concept of branchial apparatus and cervico-facial embryology].

The so-called "branchial" embryonic organization corresponds to a segmental disposition, for which a determining role of the aortic arch, vascular component of the constitutive unit of this organization, has never been disputed. This attitude is only justified by virtue of the conception of this organization as preparatory to a branchial primordium, although this initial conception, which raises major objections, has now been discarded. Moreover, direct objections against this theory of a vascular preeminence are presented. Therefore, a revision of cervico-facial morphogenesis (formations cranial to the mandibular arch) is mandatory, based on somitomeres, neural systematization, evidence obtained from heterograft experiments and genetics, allowing to recognize in the "branchial" organization its proper somatic participation. The repartition of elements deriving from the metameric segmentation in the "branchial" segmentation suggests that both types of segmentation represent two coordinate expressions of a single process. The primitive characters of the "branchial" segmentation (tendency to be total, paleontological data, cartilaginous skeleton) are suggestive for a peripheral and limited expression of the segmentation, which appears as an intermediary type between that of the invertebrates and that metameric segmentation which predominates in the para-axial region. In this perspective, the "embryonic branchial apparatus" represents a structural link between invertebrates and vertebrates. This type of segmentation applied to the "pharynx" of chordates has allowed the outgrowth of vertebrates, acting as a major key in their organization.

Animals↗
PubMed1984

Expression of a carbohydrate differentiation antigen, stage-specific embryonic antigen 1, in human colonic adenocarcinoma.

The expression of the carbohydrate structure defined by monoclonal antibody to murine stage-specific embryonic antigen 1 (SSEA-1) was examined, using immunofluorescence, in formalin-fixed, paraffin-embedded sections of normal fetal and adult human colon and human colonic adenocarcinoma. SSEA-1 was expressed in all human colonic adenocarcinoma tissues examined, although in some cases the staining was heterogeneous. In normal human colonic mucosa, under the conditions used, faint staining was seen in the lower crypts and in only 26% of the crypts examined. When human fetal colon was tested, SSEA-1 was expressed in much larger amounts and in over 50% of all crypts. Transitional mucosa, immediately adjacent to human colonic adenocarcinomas, was also tested, and in this case, increased SSEA-1 expression was seen not only in the lower crypts but also in the upper crypts and surface epithelium. These results show that the increased expression of SSEA-1 in human colonic adenocarcinoma is an oncodevelopmental marker for this cancer. In addition, the results suggest that increased expression of SSEA-1 may be a preneoplastic change in human colon.

Adenocarcinoma↗
PubMed1995

An unusual arrangement of 13 zinc fingers in the vertebrate gene Z13.

The zinc finger is a protein domain that imparts specific nucleic acid-binding activity on a wide range of functionally important proteins. In this paper we report the molecular cloning and characterization of a novel murine zinc-finger gene, mZ13. Analysis of mZ13 cDNAs revealed that the gene expresses a 794-amino-acid protein encoded by a 2.7 kb transcript. The protein has an unusual arrangement of 13 zinc fingers into a 'hand' of 12 tandem fingers and a single isolated finger near the C-terminus. This structural organization is conserved with the probable chicken homologue, cZ13. mZ13 also contained an additional domain at the N-terminus which has previously been implicated in the regulation of zinc-finger transcription factor DNA-binding, via protein-protein interactions. mZ13 expression was detected in a wide range of murine embryonic and adult tissues. The structural organization of mZ13 and its expression profile suggest that it may function as a housekeeping DNA-binding protein that regulates the expression of specific genes.

Amino Acid Sequence↗
PubMed2003

Extracellular matrix in vascular morphogenesis and disease: structure versus signal.

The vascular system matures during embryonic development to form a stable, well-organized tubular network. In vivo data have established that the extracellular matrix (ECM) is crucial in providing structural support to the vascular system. In vitro studies are defining the involvement of ECM-smooth-muscle cell signaling in establishing and maintaining the mature tubular structure. However, correlating cell signaling with established structural functions for the ECM and determining the relative importance of these two roles in vivo is often difficult. Here, we examine human genetics, murine gene targeting and cell biology to better understand the relationship between structural and signaling roles for the ECM in vascular morphogenesis and disease.

Animals↗
PubMed1987

Diphenylhydantoin and mitotic spindle abnormalities in cultured mouse and human cells.

The present study demonstrates that the antiepileptic drug diphenylhydantoin (DPH) is capable of inducing aneuploidy but not structural aberrations in cultured mouse embryonic fibroblasts. A high concentration of 200 micrograms/ml was found to increase the percentage of hyperdiploidy from 4.8 (control) to 16.0. The treatment was found to increase mitotic indices as a consequence of a mitotic-arresting action of the drug. These effects are probably due to the effect of the drug on the structure of the mitotic apparatus. Abnormal cell divisions and mitotic disturbance were found to increase in a dose-dependent manner after DPH treatment. In a parallel study, human amnion cells were found to show similar response to DPH treatment.

Aneuploidy↗
PubMed1980

Changes in lectin-mediated agglutinability during primary embryonic induction in the amphibian embryo.

The present study was undertaken to investigate structural alterations at the surface of presumptive neural cells after primary embryonic induction. For this purpose, plant lectin-mediated agglutinability of dissociated cells from the epiblast of Bufo arenarum gastrulae was tested. Two fragments of epiblast were excised from the same mid-gastrula: one from the dorsal side of the egg, making contact with the invaginating chordamesoblast and assumed to be composed of determined cells and the other from the ventral region of the egg, facing the blastocoele cavity and assumed to be composed of undetermined cells. Cells of the pooled fragments were dissociated in calcium-free Holtfreter's solution with potassium oxalate and incubated in the presence of different concentrations of phytohemagglutinin and concanavalin A. Epiblast cells overlying the archenteron roof are less agglutinated with both lectins than undetermined cells. On the other hand, when egg fragments were removed from the dorsal and ventral regions of early gastrulae before the archenteron was formed, no significant difference in lectin-mediated agglutinability was observed, even after having been cultured in vitro in absence of inducing tissue. These results suggest that the target of the inducing signal generated in the mesoblast is likely to be located on the surface of epiblast cells. Additional experiments showed that cells pretreated with colchicine, cytochalasin B or colchicine and cytochalasin B simultaneously exhibit no significant variation in agglutinability, suggesting that the cytoskeleton was not be involved in the cell surface alteration here described. Treatment of whole embryos or sandwich explants with concanavalin A or phytohemagglutinin has no effect on neural tube formation, suggesting that the carbohydrate-containing binding sites for these lectins are not involved in primary embryonic induction. Changes in cell agglutinability described in this paper are to be interpreted thus as a secondary expression of structural alterations in the cell surface concomitant with neural determination.

Animals↗
PubMed2004

Protein related to DAN and cerberus is a bone morphogenetic protein antagonist that participates in ovarian paracrine regulation.

Bone morphogenetic proteins (BMPs) are important for body patterning and morphogenesis, whereas several BMP antagonists regulate the functions of BMPs during embryonic development and tissue differentiation. Protein related to DAN and cerberus (PRDC) is a secreted protein with a cystine knot structure identified by gene trapping in embryonic stem cells. Although PRDC shows sequence homology with proteins of the BMP antagonist family, its biological activity and physiological functions are unclear. We generated recombinant PRDC and its paralog, gremlin, and tested their ability to suppress actions initiated by diverse BMP proteins. Similar to the known BMP antagonist, gremlin, PRDC blocked ligand signaling induced by BMP2 and BMP4 but had minimal effects on reporter gene activation induced by GDF-9, activin, or transforming growth factor-beta. Co-precipitation assays further demonstrated the direct protein-protein interactions between PRDC and BMP2 or BMP4. Reverse transcriptase-PCR analyses indicated that PRDC transcripts are widely expressed showing higher levels in ovary, brain, and spleen. In mouse ovary, PRDC transcripts were increased following gonadotropin treatment. In situ hybridization analyses further indicated that ovarian PRDC transcripts are localized in granulosa cells of selective follicles. In addition, co-treatment with PRDC antagonized the inhibitory effects of BMP4 on the follicle-stimulating hormone stimulation of progesterone production by cultured rat granulosa cells. Thus, PRDC is a potent BMP antagonist with a wide tissue expression pattern, and ovarian PRDC expressed in granulosa cells could be involved in follicular development by antagonizing the actions of theca cell-derived BMPs.

Animals↗
PubMed1991

Spatial domains in the developing forebrain: developmental regulation of a restricted cell surface protein.

We have isolated a monoclonal antibody, mAb 52G9, that recognizes a 55-kDa cell surface protein restricted to the early embryonic rat forebrain and to placode-derived structures. In the central nervous system (CNS), 52G9 immunoreactivity appears at Embryonic Day 11 (E11) in the rostral-most area of the telencephalon. It then spreads to the neuroepithelium of the telencephalon and basal diencephalon. Most strikingly, it appears at E14 in a distinct zone at the caudal end of the ventral diencephalic neuroepithelium. This area is sharply defined by strong 52G9 immunoreactivity bounded by unlabeled neuroepithelium. The pattern revealed by 52G9 is the first biochemical demonstration of spatial domains in the forebrain at a time prior to neuronal differentiation. By E18, 52G9 immunoreactivity has progressively disappeared from the forebrain; the glomerular layer of the olfactory bulb is the only 52G9-positive area in the CNS. The olfactory, otic, and hypophyseal placodes, which can be identified as early as E10, are also 52G9 positive as are their derivatives, the sensory epithelial of the nasal passage and inner ear, and also Rathke's pouch. The distribution and regulation of the 52G9 protein suggests that this novel cell surface molecule may be involved in the formation of spatial domains in the developing forebrain.

Animals↗
PubMed1997

In vitro-generated neural precursors participate in mammalian brain development.

During embryogenesis, pluripotent stem cells segregate into daughter lineages of progressively restricted developmental potential. In vitro, this process has been mimicked by the controlled differentiation of embryonic stem cells into neural precursors. To explore the developmental potential of these cell-culture-derived precursors in vivo, we have implanted them into the ventricles of embryonic rats. The transplanted cells formed intraventricular neuroepithelial structures and migrated in large numbers into the brain tissue. Embryonic-stem-cell-derived neurons, astrocytes, and oligodendrocytes incorporated into telencephalic, diencephalic, and mesencephalic regions and assumed phenotypes indistinguishable from neighboring host cells. These observations indicate that entirely in vitro-generated neural precursors are able to respond to environmental signals guiding cell migration and differentiation and have the potential to reconstitute neuronal and glial lineages in the central nervous system.

Animals↗
PubMed2006

Anisocotyly and meristem initiation in an unorthodox plant, Streptocarpus rexii (Gesneriaceae).

In common with most Old World Gesneriaceae; Streptocarpus Lindl. shows anisocotylous growth, i.e., the continuous growth of one cotyledon after germination. Linked to this phenomenon is an unorthodox behaviour of the shoot apical meristem (SAM) that determines the growth pattern of acaulescent species (subgenus Streptocarpus). In contrast caulescent species develop a conventional central post-embryonic SAM (mainly subgenus Streptocarpella). We used S. rexii Lindl. as a model to investigate anisocotyly and meristem initiation in Streptocarpus by using histological techniques and analyses of the expression pattern of the meristematic marker SrSTM1 during ontogeny. In contrast to Arabidopsis thaliana (L.) Heynh., S. rexii does not establish a SAM during embryogenesis, and the first evidence of a SAM-like structure occurs during post-embryonic development on the axis (the petiolode) between the two cotyledons. The expression pattern of SrSTM1 suggests a function in maintaining cell division activity in the cotyledons before becoming localized in the basal meristem, initially at the proximal ends of both cotyledons, later at the base of the continuously growing macrocotyledon, and the groove meristem on the petiolode. The latter is equivalent to a displaced SAM seemingly originating de novo under the influence of endogenous factors. Applied cytokinin retains SrSTM1expression in the small cotyledon, thus promoting isocotyly and re-establishment of a central post-embryonic SAM. Hormone-dependent delocalization of the process of meristem development could underlie anisocotyly and the unorthodox SAM formation in Streptocarpus.

Cotyledon↗
PubMed1992

Nephrogenic adenofibroma. A novel kidney tumor of young people.

Nephrogenic adenofibroma is a novel kidney tumor of young people (mean age of presentation, 13 years), who present with polycythemia, hypertension, or hematuria, which resolve following nephrectomy. The typical nephrectomy specimen contains a solitary, nonencapsulated, vaguely circumscribed, irregularly shaped or spherical, firm mass with either tan, gray-white, or pale yellow coloration. Cysts are sometimes present within the tumor. The histologic appearance is distinctive and characterized by a marked proliferation of spindled mesenchymal cells resembling the classical type of congenital mesoblastic nephroma, encasing discrete nodules of embryonal epithelium similar to the hyperplastic nephrogenic rests (nephroblastomatosis) usually associated with Wilms' tumor. The mesenchymal component consists of a fascicular proliferation of tightly interlaced, uniform, benign-appearing spindled cells that immunostatin for vimentin and fibronectin, but not desmin or actin. The epithelial component consists of discrete islands of blastemal cells that are partially or fully differentiated toward tubular, tubulopapillary, or papillary structures. Psammoma bodies are plentiful. Embryonal epithelium immunostains for cytokeratin but not epithelial membrane antigen. The overall histologic appearance of the mesenchymal and epithelial components is benign, and preliminary clinical data suggest that the tumor has a benevolent course. Two cases, however, contained small, well-circumscribed papillary lesions near the renal pelvis that resembled low-grade collecting duct carcinoma. The clinical implications of the latter finding are unclear.

Adenofibroma↗
PubMed2005

Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca).

In white spruce, an improvement of somatic embryo number and quality can be achieved through experimental manipulations of the endogenous levels of reduced (GSH) and oxidized (GSSG) glutathione. An optimal protocol for embryo production included an initial application of GSH in the maturation medium, followed by replacement with GSSG during the remaining maturation period. Under these conditions, the overall embryo population more than doubled, and the percentage of fully developed embryos increased from 22% to almost 70%. These embryos showed improved post-embryonic growth and conversion frequency. Structural studies revealed remarkable differences between embryo types, especially in storage product deposition pattern and organization of the shoot apical meristem (SAM). Compared with their control counterparts, glutathione-treated embryos accumulated a larger amount of starch during the early stages of development, and more protein and lipid bodies during the second half of development. Differences were also noted in the organization of SAMs. Shoot meristems of control embryos were poorly organized and were characterized by the presence of intercellular spaces, which caused separation of the subapical cells. Glutathione-treated embryos had well-organized meristems composed of tightly packed cells which lack large vacuoles. The improved organization of the shoot apical meristems in treated embryos was ascribed to a lower production of ethylene. Differences in meristem structure between control and treated embryos were also related to the localization pattern of HBK1, a shoot apical meristem 'molecular marker' gene with preferential expression to the meristematic cells of the shoot pole. Expression of this gene, which was localized to the apical cells in control embryos, was extended to the subapical cells of treated embryos. Overall, it appears that meristem integrity and embryo quality are under the direct control of the glutathione redox state.

Germination↗
PubMed2006

De novo methylation, long-term promoter silencing, methylation patterns in the human genome, and consequences of foreign DNA insertion.

This chapter presents a personal account of the work on DNA methylation in viral and mammalian systems performed in the author's laboratory in the course of the past 30 years. The text does not attempt to give a complete and meticulous account of the work accomplished in many other laboratories; in that sense it is not a review of the field in a conventional sense. Since the author is also one of the editors of this series of Current Topics in Immunology and Microbiology on DNA methylation, to which contributions by many of our colleagues in this field have been invited, the author's conscience is alleviated that he has not cited many of the relevant and excellent reports by others. The choice of viral model systems in molecular biology is well founded. Over many decades, viruses have proved their invaluable and pioneering role as tools in molecular genetics. When our interest turned to the demonstration of genome-wide patterns of DNA methylation, we focused mainly on the human genome. The following topics in DNA methylation will be treated in detail: (1) The de novo methylation of integrated foreign genomes; (2) the long-term gene silencing effect of sequence-specific promoter methylation and its reversal; (3) the properties and specificity of patterns of DNA methylation in the human genome and their possible relations to pathogenesis; (4) the long-range global effects on cellular DNA methylation and transcriptional profiles as a consequence of foreign DNA insertion into an established genome; (5) the patterns of DNA methylation can be considered part of a cellular defense mechanism against foreign or repetitive DNA; which role has food-ingested DNA played in the elaboration of this mechanism? The interest in problems related to DNA methylation has spread-like the mechanism itself-into many neighboring fields. The nature of the transcriptional programs orchestrating embryonal and fetal development, chromatin structure, genetic imprinting, genetic disease, X chromosome inactivation, and tumor biology are but a few of the areas of research that have incorporated studies on the importance of the hitherto somewhat neglected fifth nucleotide in many genomes. Even the fly researchers now have to cope with the presence of this nucleotide, in however small quantities it exists in the genome of their model organism, at least during embryonal development. The bulk of the experimental work accomplished in the author's laboratory has been shouldered by many very motivated undergraduate and graduate students and by a number of talented postdoctoral researchers. Their contributions are reflected in the list of references in this chapter. We have also had the good luck to receive funding through a number or organizations as acknowledged.

Adenoviridae↗
PubMedDate not supplied

5,8,11,14-eicosatetraynoic acid-induced differentiation of U937 cells.

U937 cells, transformed human monocyte/macrophage precursors, incubated with 5,8,11,14 eicosatetraynoic acid (ETYA), a competitive in vitro and in vivo inhibitor of arachidonic acid, develop properties consistent with a form of cellular differentiation. In order to define the extent of this differentiation and decide whether a monocyte or macrophage-like cell developed, control cells and cells cultured for three days with 40 microns ETYA were examined by transmission electron microscopy. Control cells were characterized by oval nuclei, a high nuclear to cytoplasmic ratio, a cytoplasm with a relative paucity of mitochondria, vesicles and vacuoles, and a Golgi apparatus that was not extensively developed. Ribosomes and putative ribosome-like granules were frequently "grouped" in the cytoplasm, rather than as single granules or in association with the endoplasmic reticulum. The plasma membrane included a discrete region of fimbriated structures that were distinct from pseudopodia. Such structures frequently are expressed in embryonic and transformed cells and may be associated with motility, metastases, with immunologic reactions or with endocytosis. After culture with 40 microns ETYA for three or more days, a morphology consistent with that of immature monocytes developed. These characteristics included lobulated nuclei, a reversal of the nuclear to cytoplasmic ratio, an increased complexity and development of the cytoplasmic components and the disappearance of the fimbriated plasma membrane structures. In addition, "grouped" ribosomes were less evident.(ABSTRACT TRUNCATED AT 250 WORDS)

5,8,11,14-Eicosatetraynoic Acid↗
PubMed2005

Role of Folbp1 in the regional regulation of apoptosis and cell proliferation in the developing neural tube and craniofacies.

Folic acid is essential for many cellular reactions, including synthesis of nucleotides and regulation of cell cycle. Folic acid-binding protein one (Folbp1), a membrane-bounded protein, is the primary mediator of folic acid transport. Mice deficient in Folbp1 gene die in utero with multiple malformations, including severe exencephaly and craniofacial defects. Fusion of the neural tube and craniofacies require precisely regulated interactions of apoptosis, cell proliferation, and differentiation. To understand the role of Folbp1 in regulating the fusions of these primordia, levels of dead and proliferating precursor cells from Folbp1 embryos were quantified before the fusion processes. Massive apoptosis was detected in the Folbp1-/- defective tissues, with Bax and activated caspase-3 distributed evenly across the apico-basal axis of the lateral neural plate. 5-Bromodeoxyuridine (BrdU) and PCNA labeling assays revealed a reduced cell proliferation as well. However, telomerase activity was unaltered, arguing against telomere shortening and consequently, chromosomal instability, as the cause of the apoptosis. Notably, Islet-1 and 2H3 immunohistochemistry demonstrated the presence of differentiating neuronal cells, albeit in decreased numbers. Interestingly, Folbp1-/- embryos also elaborated novel neural structures that sprouted orthogonally from the embryonic neuraxis. Assays on the defective craniofacies exhibited similar phenomena, suggesting the neural crest precursor population that gives rise to both these structures is selectively vulnerable to Folbp1 inactivation. The results demonstrate a prominent role of Folbp1 in the regional regulation of apoptosis and cell proliferation that underlies the aberrant neural tube and craniofacial defects.

Animals↗
PubMed2000

Evolution of placentation among squamate reptiles: recent research and future directions.

Squamate reptiles are uniquely suited to study of evolution of reproductive mode and pattern of embryonic nutrition. Viviparous species have evolved from oviparous ancestors on numerous occasions, patterns of nutritional provision to embryos range widely from lecithotrophy, at one end of a continuum, to placentotrophy at the other, and structure and function of the maternal-embryonic relationship is highly constrained resulting in parallel evolutionary trajectories among taxa. Embryos of oviparous species primarily receive nourishment from yolk, but also mobilize a significant quantity of calcium from the eggshell. Most viviparous species also are predominantly lecithotrophic, yet all viviparous species are placentotrophic to some degree. Similarities in embryonic development and nutritional pattern between oviparous species and most viviparous species suggest that the pattern of nutrition of oviparous squamates is an exaptation for the evolution of viviparity and that placentotrophy and viviparity evolve concomitantly. The few species of squamates that rely substantially on placentotrophy have structural modifications of the interface between the embryo and mother that are interpreted as adaptations to enhance nutritional exchange. Recent studies have extended understanding of the diversity of embryonic nutrition and placental structure and have resulted in hypotheses for transitions in the evolution of placentotrophy, yet data are available for few species. Indirect tests of these hypotheses, by comparison of structural-functional relationships among clades in which viviparity has evolved, awaits further study of the reproductive biology of squamates.

Animals↗
PubMed2000

Behavior of M-phase synchronized blastomeres after nuclear transfer in cattle.

M-phase synchronized bovine blastomeres were used to study the effect of nuclear-cytoplasmic synchronization on the developmental potential after nuclear transfer (NT). The capacity of nocodazole and benomyl to reversibly synchronize blastomeres from embryos in M-phase was evaluated. Nocodazole reversibly arrested bovine embryos at the studied stages and induced high rates of M-phases in morulae and compact morulae. In contrast, benomyl was less efficient than nocodazole to synchronize in M-phase. After transfer of an M-phase blastomere, premature chromatin condensation was the prevalent finding 1 hr post-fusion (hpf). Condensed chromosomes non-arranged in the equatorial plate (1-3 hpf) that acquired an organized structure over time (3-7 hpf) were subsequently observed. Anaphase-telophase structures were predominantly recorded at 4-9 hpf. About 50% of the embryos activated at both 3-4 and 6-7 hpf extruded a polar body-like structure 5 hr after activation, but this was not observed in embryos activated immediately after fusion. A significantly lower activation rate was observed for oocytes activated 3-4 hpf compared to those activated 6-7 hpf. However, the ability to undergo first cleavage was significantly lower in the latter group. Reconstructed embryos activated immediately after fusion showed no difference in the rate of activation compared to those activated 6-7 hpf, although the cleavage rate was higher. DNA synthesis was observed at a significantly higher rate in embryos activated both immediately and 3-4 hpf that did not extrude a PB-like structure than in those activated 3-4 hpf that extruded a polar body-like structure. Under the conditions tested M-phase donor cells cannot be properly remodeled after NT in cattle to trigger normal embryonic development. Our observations of chromatin structures together with DNA synthesis suggest that the failure in the development may be due to improper chromatin remodeling of mitotic nuclei after NT, which may result in chromosomal abnormalities incompatible with normal embryo development.

Animals↗