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PubMed1985

Distinctive structural and cytoskeletal properties of the long-surviving neurons in cell cultures of embryonic spinal cord.

A distinctive population of neurons survives for longer than 3 months in cell cultures of chick or rat spinal cord. These neurons form a minor proportion (1%) of the neurons initially developing in the cultures, but are the only ones to survive longer than 30 days in vitro. In addition to their longevity, they share important morphological and cytoskeletal characteristics, which render them distinctive as a group even in early cultures which contain numerous other neurons of short-term viability. Each long-surviving neuron projects one neurite of great length relative to its other neurites, or to those of the shorter-lived neurons, and the length of this neurite is maintained constant for many weeks in vitro. This well-defined morphological feature may indicate the lineage(s) of these neurons. Structurally these cells are very different to the shorter-lived neurons. They are rich in neurofilaments and contain very few microtubules, whereas the shorter-lived neurons contain few neurofilaments but many microtubules. These differences in cytoskeleton coincide exactly with the distinction between limited and prolonged survival in vitro, and the possibility is considered that cytoskeletal stability in the presence of numerous small inflows of calcium might underlie "hardiness" in vitro. The state of development of the long-lived neurons is considered in the context of their shared features, and it is suggested that they may provide a model in which the regulation of development of neuronal function can be analysed.

Animals↗
PubMed2000

Programmed cell death and patterning in Drosophila.

Selective cell death provides developing tissues with the means to precisely sculpt emerging structures. By imposing patterned cell death across a tissue, boundaries can be created and tightened. As such, programmed cell death is becoming recognized as a major mechanism for patterning of a variety of complex structures. Typically, cell types are initially organized into a fairly loose pattern; selective death then removes cells between pattern elements to create correct structures. In this review, we examine the role of selective cell death across the course of Drosophila development, including the tightening of embryonic segmental boundaries, head maturation, refining adult structures such as the eye and the wing, and the ability of cell death to correct for pattern defects introduced by gene mutation. We also review what is currently known of the relationship between signals at the cell surface that are responsible for tissue patterning and the basal cell death machinery, an issue that remains poorly understood.

Animals↗
PubMed1998

Developmental remodeling and shortening of the cardiac outflow tract involves myocyte programmed cell death.

The embryonic outflow tract is a simple tubular structure that connects the single primitive ventricle with the aortic sac and aortic arch arteries. This structure undergoes a complex sequence of morphogenetic processes to become the portion of the heart that aligns the right and left ventricles with the pulmonary artery and aorta. Abnormalities of the outflow tract are involved in many clinically significant congenital cardiac defects; however, the cellular and molecular processes governing the development of this important structure are incompletely understood. Histologic and tissue-tagging studies indicate that the outflow tract tissues compact and are incorporated predominantly into a region of the right ventricle. The hypothesis tested in the current study was that cell death or apoptosis in the muscular portion of the outflow tract is an important cellular mechanism for outflow tract shortening. The tubular outflow tract myocardium was specifically marked by infecting myocytes of the chicken embryo heart with a recombinant replication-defective adenovirus expressing beta-galactosidase (beta-gal) under the control of the cytomegalovirus promoter. Histochemical detection of the beta -gal-labeled outflow tract myocytes revealed that the tubular structure shortened to become a compact ring at the level of the pulmonic infundibulum over several days of development (stages 25-32, embryonic days 4-8). The appearance of apoptotic cardiomyocytes was correlated with OFT shortening by two histologic assays, TUNEL labeling of DNA fragments and AnnexinV binding. The rise and fall in the number of apoptotic myocytes detected by histologic analyses paralleled the change in activity levels of Caspase-3, a protease in the apoptotic cascade, measured in outflow tract homogenates. These results suggest that the elimination of myocytes by programmed cell death is one mechanism by which the outflow tract myocardium remodels to form the proper connection between the ventricular chambers and the appropriate arterial trunks.

Adenoviridae↗
PubMed1993

Extinction of Oct-3/4 gene expression in embryonal carcinoma x fibroblast somatic cell hybrids is accompanied by changes in the methylation status, chromatin structure, and transcriptional activity of the Oct-3/4 upstream region.

In this study we evaluate, for the first time, the molecular mechanism that underlies the extinction of a tissue-specific transcription factor, Oct-3/4, in somatic cell hybrids and compared it with its down-regulation in retinoic acid (RA)-treated embryonal carcinoma (EC) cells. The Oct-3/4 gene, which belongs to the POU family of transcription factors and is abundantly expressed in EC (OTF9-63) cells, provides an excellent model system with which to study the extinction phenomenon. Unlike other genes whose expression has been repressed in hybrid cells but not during in vivo differentiation, Oct-3/4 expression is dramatically repressed in OTF9-63 x fibroblast hybrids and also during embryogenesis. The ectopic expression of Oct-3/4 in hybrid cells under a constitutive promoter is sufficient for transcriptional activation of an octamer-dependent promoter. These results argue against the possibility that fibroblasts contain a direct repressor which binds directly to the octamer sequence and prevents Oct-3/4 protein from binding. The extinction of Oct-3/4 binding activity in the hybrid cells occurs at the level of mRNA transcription, similarly to the repression of Oct-3/4 transcription during in vivo differentiation. This shutdown of Oct-3/4 transcription in hybrid cells and in RA-treated EC cells is accompanied by de novo methylation of its 1.3-kb upstream region. In contrast to EC cells, in which this region is sensitive to MspI digestion, in hybrid cells and in RA-treated EC cells, the Oct-3/4 upstream region is resistant to MspI digestion, which suggests a change in its chromatin structure. Furthermore, extinction is not restricted to the endogenous Oct-3/4 gene but is also exerted upon a transiently transfected reporter gene driven by the Oct-3/4 upstream region. Thus, changes in the cellular activity of trans-acting factors acting on the upstream region also contribute to the inability of the hybrid and RA-treated EC cells to generate Oct-3/4 transcripts. In conclusion, this study draws a connection between the shutdown of Oct-3/4 expression in RA-differentiated EC cells and its extinction in hybrid cells. In both systems, repression of Oct-3/4 expression is achieved through changes in the methylation status, chromatin structure, and transcriptional activity of the Oct-3/4 upstream regulatory region.

Animals↗
PubMed2004

Organization and function of microfilaments during late epiboly in zebrafish embryos.

We report that, during epiboly in zebrafish, three F-actin--based structures appear only after the blastoderm migrates past the embryonic equator. They are composed of two ring-like F-actin structures that form at the deep cell and enveloping layer margins of the blastoderm and a punctate actin band that develops in the external yolk syncytial layer. Treatment with cytochalasin B or the calcium chelator dibromo-BAPTA results in the disruption of all three of these actin-based structures, leading to the slowing or immediate arrest of epiboly, respectively, followed by a failure of yolk cell occlusion and the eventual lysis of the embryo through the vegetal pole region. We suggest, therefore, that these structures function in the occlusion of the vegetal portion of the yolk cell during the latter stages of epiboly. Possible roles for these new structures, their modulation by Ca2+, as well as the functions of other previously described F-actin--based structures observed throughout epiboly, are discussed.

Actin Cytoskeleton↗
PubMed1975

[Pineal organ and parietal eye of Lacerta vivipara: a qualitative and quantitative microscopic study of the uptake of 3H-5-hydroxytryptophan during ontogenesis].

After comparison of results obtained by different modalities of 3h-5HTP injections (involving intraallantodic, intraventricular and intraperitoneal injections), the intraperitoneal one was retaineed to study experimental cytochemcal biosynthesis of indoleamines, in the epiphysis of Lacerta vivpara J., during ontogenesis. From stage 36 of embryogenesis, the selective radioautographic labelling, significant of atritiated indoleamines storage, was located in the distal part of the epiphysis. From stage 36 to 40 (just before hatching) and 3 days after hatching, microscopic and microphotometric studies point out, in the epiphysis epithelium, a highly significant increase in tritiated infoleamines rate. In contrast, the weak nonspecific reactions, in the neighbouring brain structures (dorsal telencephalon, tectum opticum and paraphysis), stay constant. The metabolism and storage sites of indoleamines belong to sensory line cells. During ontogenesis, these sites appear successively in distal, middle and proximal epiphysis and from the basal to the upper part of the sensory line cells. The embryonic parietal eye, in spite of structural analogies, does not take up the radioactive compound.

5-Hydroxytryptophan↗
PubMed2001

Effect of the tetrapeptide RGDS on somatic embryogenesis in Daucus carota.

The present work examines the requirement for RGD-binding sites, such as those in the plasma membrane protein integrin during somatic embryogenesis in carrot (Daucus carota L. cv. Scarlet nantes). It is possible to assay for this requirement by competitively inhibiting binding of the site to the natural RGD-containing ligand by adding an excess of synthetic RGDS. We found that treatment of carrot callus cultures with RGDS (2.7 mM) inhibited the development of a normal shoot pole in carrot somatic embryos. The structures that formed contained separate zones of highly cytoplasmic and vacuolate cells and no evidence of embryonic organization, although occasionally a root-like structure was observed. If the aspartic acid residue in the peptide was replaced by a chemically similar amino acid (glutamic acid), the resultant somatic embryos were indistinguishable from those developing in untreated cultures. These effects are similar to those reported in animal systems where the protein receptor involved has been identified as integrin. Our results are thus consistent with the conclusion that a binding site for a motif similar to the integrin-binding site in a variety of animal extracellular matrix proteins exists in plants and appears to be important in somatic embryo development in carrot.

Journal Article↗
PubMed1994

Histotypic in vitro reorganization of dissociated cells from mouse fetal gonads.

Cell suspensions obtained from the testes of 13.5-14.5 day post coitum (dpc) mouse embryos reaggregate in cord-like structures following in vitro culture for 24 h on a reconstituted basement membrane (Matrigel). Ovarian cells of the same fetal age or cell suspensions from sex indifferent gonadal ridges (11.5 dpc embryos) form an organized cell network but not cord-like structures. Antibodies directed against laminin or against the alpha 6 subunit of its integrin receptor prevent such morphogenetic processes. The addition of 5 micrograms/ml cycloheximide, cytochalasin B or tunicamycin also inhibits the phenomenon. Interestingly, compounds that increase intracellular cyclic AMP (cAMP) (dbcAMP, forskolin and isobutyl-1-methylxanthine) induce embryonic testicular cells to organize into structures similar to those assembled by ovarian or sex indifferent cell suspensions. These findings offer a simple in vitro model for studying some aspects of early gonad development and provide novel experimental evidence that cell motility and cell-cell adhesion, possibly regulated by cAMP dependent mechanisms, are likely to play an important role in gonad morphogenesis.

1-Methyl-3-isobutylxanthine↗
PubMed1985

The trophotaenial placenta of a viviparous goodeid fish. I. Ultrastructure of the internal ovarian epithelium, the maternal component.

Embryos of goodeid fishes develop to term within the ovarian lumen, where they undergo considerable increase in weight due to transfer of maternal nutrients across a trophotaenial placenta. The placenta consists of an embryonic component, the trophotaeniae, and a maternal component, the ovarian lining. The latter was examined by transmission electron microscopy, scanning electron microscopy, and light microscopy in both gravid and nongravid ovaries of the viviparous goodeid fish, Ameca splendens. The single median ovary of A. splendens is a hollow structure whose lumen is divided into lateral chambers by a highly folded longitudinal ovarian septum. Germinal tissue occurs within folds of the ovarian lining that extend into each of the two lateral chambers. Matrotrophic embryonic development takes place within ovarian chambers. During gestation, the lining of the ovarian lumen is in direct apposition to body surfaces and trophotaenial epithelia of developing embryos. The ovarian lining consists of a simple cuboidal epithelium, termed the internal ovarian epithelium (IOE), overlying a well-vascularized bed of connective tissue. Cells of the IOE are apically convex. Well-developed granular and agranular endoplasmic reticula and numerous large membrane-bound vesicles with electron-dense content occupy the apical cytoplasm of IOE cells. Two functional states of the same cell type are distinguished within the IOE. Phase I cells contain few, if any, large apically situated vesicles; Phase II cells contain many. Secretory products of the IOE are presumed to be an important source of nutrients for embryonic development. Structural and functional relationships of the IOE to the trophotaenial epithelium of developing embryos are discussed in relation to maternal-embryonic nutrient transfer processes.

Animals↗
PubMed1997

Development of hepatic sinusoidal structure with special reference to the Ito cells.

To elucidate sinusoidal cell structure and function under normal conditions and their behavior in diseased settings, an understanding of their developmental aspects is needed. At day 10 of gestation in mice and rats or at 5 weeks of gestation in humans, the hepatic cords grow into the mesenchymal tissue of the septum transversum, and the primitive sinusoidlike structure is simultaneously observed between the liver cell cords. In the margin of the growing liver primordium, mesenchymal cells in the septum transversum are trapped in the subendothelial space. These subendothelial cells are at the early stages of organogenesis and become progenitors of the Ito cells. By days 12-14 of gestation in mice and rats or 8 weeks of gestation in humans, the basic structure of the sinusoids has developed. Embryonic hepatic sinusoids are usually lined by a continuous endothelium without basement membranes, and an incompletely fenestrated sinusoid appears at the middle gestational stage. In the late gestational stages, the Ito cells exhibit myofibroblastlike features in humans, mice, and rats. In association with this event, perisinusoidal reticular networks are gradually intensified. After birth until days 4-5 in mice and rats, the sinusoidal and perisinusoidal structures are almost completely formed, although slight morphological differences from those in adult livers still exist. What happens to sinusoidal endothelial cells and Ito cells in hepatic fibrosis-cirrhosis of the adult may be a deviated or uncontrolled occurrence of what goes on during the fetal period, i.e., a continuous nonfenestrated sinusoidal lining in the early embryonic stage and a myofibroblastlike transformation of Ito cells in late fetal life.

Animals↗
PubMed1991

A novel modular mosaic of cell adhesion motifs in the extracellular domains of the neurogenic trk and trkB tyrosine kinase receptors.

The extracellular domains of the human trk protooncogene and the closely homologous murine trkB share two highly conserved regions as well as several invariant cysteines which are supposed to be important for ligand recognition. Detailed comparative sequence analyses have now revealed the presence of a novel combination of distinct cell adhesion motifs corresponding precisely with these conserved regions. The N-terminal part consists of an array of three tandem leucine-rich motifs of 24 amino acids which is flanked by two distinct cysteine clusters. Significantly homologous structural features are found in the toll gene product of Drosophila, a transmembrane protein mediating specific cell adhesion events involved in the dorsoventral embryonic pattern formation. Directly adjacent to these structures we additionally identified two repeats of the immunoglobulin-like C2 type, which are significantly similar to repeats found in the neural cell adhesion molecules (N-CAMs) and in the platelet-derived growth factor receptor (PDGFR)-like tyrosine kinase receptor family. Our findings indicate that the trk/trkB protein tyrosine kinase receptors are involved in the transmembrane signalling of growth factor binding as well as of specific cell adhesion events during neuronal development. Additionally, we propose that the specifically expressed truncated forms of the trkB receptor, lacking the tyrosine kinase domain, are functioning as neural cell adhesion molecules. The knowledge of this domain structure will facilitate the elucidation of the molecular reasons why alterations in the extracellular region lead to oncogenic activation of the human trk proto-oncogene, which is a major cause of papillary thyroid carcinoma.

Amino Acid Sequence↗
PubMed1983

Major changes in the 5' and 3' chromatin structure of sea urchin histone genes accompany their activation and inactivation in development.

The major histone gene repeat (h22) of the sea urchin Psammechinus miliaris is transiently expressed for several hours during early embryonic development. Several major alterations in chromatin structure coincide with changes in the pattern of early histone gene expression and are reversed later. During the early (128-cell) blastula stages when h22 DNA is maximally expressed, promoter regions of all five histone genes are sensitive to both micrococcal nuclease and DNAase I. Hypersensitivity to micrococcal nuclease remains the same throughout the early hours of development, but disappears abruptly at hatching blastula stage when transcription has completely ceased. Some sequences near the 3' end of active histone genes are very resistant to micrococcal nuclease cutting and map just downstream of the inverted DNA repeats essential for generating faithful 3' ends of histone mRNAs. These same histone DNA sequences are readily cut in free DNA or when histone genes are transcriptionally inactive.

Animals↗
PubMed2006

Enzymatically active N-deacetylase/N-sulfotransferase-2 is present in liver but does not contribute to heparan sulfate N-sulfation.

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

Amidohydrolases↗
PubMed2001

Recombinant expression of human mannan-binding lectin.

Mannan-binding lectin (MBL) constitutes an important part of the innate immune defence by effecting the deposition of complement on microbial surfaces. MBL deficiency is among the most common primary immunodeficiencies and is associated with recurrent infections and symptoms of poor immune complex clearance. Plasma-derived MBL has been used in reconstitution therapy but concerns over viral contamination and production capacity point to recombinant MBL (rMBL) as a future source of this protein for clinical use. Natural human MBL is an oligomer of up to 18 identical polypeptide chains. The synthesis of rMBL has been accomplished in several mammalian cell lines, however, the recombinant protein differed structurally from natural MBL. In this, study we compare rMBL produced in myeloma cells, Chinese hamster ovary (CHO) cells, human hepatocytes, and human embryonic kidney (HEK) cells. We report that rMBL structurally and functionally similar to natural MBL can be obtained through synthesis in the human embryonic kidney cells followed by selective carbohydrate affinity chromatography.

Carrier Proteins↗
PubMed2004

Developmental changes in integrated ultrasound backscatter from embryonic blood in vivo in mice at high US frequency.

Mouse blood imaged using high-frequency ultrasound (US) is more echogenic in embryos than in adults. Studying changes in blood echogenicity in embryos may be of fundamental interest in studies on the genetic regulation of normal and abnormal blood development in mutant mice. Embryonic red blood cells (RBCs) are large and nucleated in midgestation but decrease in size and become enucleated as they mature. We therefore hypothesised that these structural alterations are responsible for variations in echogenicity of embryonic blood with gestational age and development. The objective of the current study was to quantify these structural changes in echogenicity (echo brightness) and apparent integrated backscatter (AIB) from embryonic blood at high US frequencies in vivo in mice. Results from anaesthetised pregnant mice studied using transcutaneous US showed that echogenicity of embryonic blood in the heart, aorta and umbilical cord and AIB within the heart chambers peaked at embryonic day (ED) 13.5 and then decreased progressively toward term. Between EDs 13.5 and 17.5 (near term), RBC mean cell volume decreased from 133 to 109 fL, haematocrit increased from 12 to 34%, and the percentage of nucleated RBCs decreased from 59 to 2%. Relative to younger ages, RBC nuclei at ED 13.5 were small and dense (pyknotic) which may have contributed to the peak in echogenicity and AIB at this age. To calculate the AIB, radiofrequency (RF) signals with centre frequencies of 28 MHz and 35 MHz were integrated over the 16- to 35-MHz and 21- to 42-MHz frequency range, respectively. At 28 MHz, mean apparent integrated backscatter of blood in the embryonic heart increased significantly from 0.0023 +/- 0.0004 Sr.cm(-1) (mean +/- SEM) at ED 12.5 to peak at 0.0037 +/- 0.0005 Sr.cm(-1) at ED 13.5. The mean AIB then decreased progressively with advancing gestation to 0.0002 +/- 0.0001 Sr.cm(-1) at ED 17.5. At 35 MHz, the mean AIB changed similarly with gestational age, except that values were lower than at 28 MHz at all ages. Higher attenuation of US at 35 MHz than at 28 MHz in tissue likely accounted for the lower AIB of blood insonified at 35 MHz. We speculate that developmental changes in red cell morphology are responsible for the observed changes in echogenicity and AIB of embryonic blood with gestational age in mice.

Animals↗
PubMed2003

Atlas of the embryonic brain in the pygmy squid, Idiosepius paradoxus.

Gross structural changes and neuropil formation in the brain during development were described in Idiosepius paradoxus, a sepioid that we chose as a model cephalopod. The brain originates in 4 pairs of ectodermal placodes, which occur separately in the embryonic surface undergoing epiboly. In the final period of epiboly, neuroblasts internalize from the placodes and gather into 4 pairs of ganglionic masses. The ganglionic masses assemble into a ring-like cluster encircling the inner yolk and the foregut anlage, gradually integrated into the 4 domains of a massive brain, a subesophageal mass (SBM), a supraesophageal mass (SPM), and a pair of optic lobes. In the early brain, neuropil forms a framework composed of a longitudinal ladder lying in the SBM, and a transverse arch standing on the lateral sides of the SBM and crossing the SPM. Differentiation of brain lobes proceeds from ventral to dorsal along this framework; first the magnocellular lobes and the posterior pedal lobe appear first in the SBM, the other lobes in the SBM and the basal lobes follow in the proximal region of the SPM, and the accessory lobes develop last in the most dorsal zone of the SPM. In the hatchlings, the brain lobes show almost the same arrangement as in the adults, but the accessory lobes, particularly the vertical lobe, are much smaller than those in the adults. Comparison of the present results with those in the teuthoid and the octopod indicates that developmental sequences of the brain are highly conserved in the coleoid cephalopods.

Animals↗
PubMed2004

Heterozygous PAX6 mutation, adult brain structure and fronto-striato-thalamic function in a human family.

Recent progress in developmental neurobiology and neuroimaging can be drawn together to provide new insight into the links between genetically specified processes of embryonic brain development and adult human brain structure and function. We used magnetic resonance imaging (MRI) to show that individuals with aniridia and deficits in executive and social cognition, due to heterozygous mutation of the neurodevelopmental control gene PAX6, have structural abnormalities of grey matter in anterior cingulate cortex, cerebellum and medial temporal lobe, as well as white matter deficits in corpus callosum. Functional MRI demonstrated reduced activation of fronto-striato-thalamic systems during performance of overt verbal fluency and nonsense sentence completion; the most consistent abnormality of verbal executive activation was located in the thalamus. These results provide the first evidence for brain functional differences in humans with PAX6 mutation that are compatible both with anatomical abnormalities in the same subjects and, more circumstantially, with the known roles of murine Pax6 in regional differentiation, axonal guidance and other aspects of early forebrain development. Highly conserved homeobox genes may be critical for normal ontogenesis of large-scale neurocognitive networks supporting phylogenetically advanced mental functions.

Adolescent↗
PubMed2006

Antiangiogenic activity of brown algae fucoxanthin and its deacetylated product, fucoxanthinol.

The antiangiogenic effects of fucoxanthin and a deacetylated product, fucoxanthinol, were examined. Fucoxanthin significantly suppressed HUVEC proliferation and tube formation at more than 10 microM, but it had no significant effect on HUVEC chemotaxis. The formation of blood vessel-like structures from CD31-positive cells was evaluated using embryonic stem cell-derived embryoid bodies. Fucoxanthin effectively suppressed the development of these structures at 10-20 microM, suggesting that it could suppress differentiation of endothelial progenitor cells into endothelial cells involving new blood vessel formation. Fucoxanthin and fucoxanthinol suppressed microvessel outgrowth in an ex vivo angiogenesis assay using a rat aortic ring, in a dose-dependent manner. These results imply that fucoxanthin having antiangiogenic activity might be useful in preventing angiogenesis-related diseases.

Angiogenesis Inhibitors↗