Cortex Of Embryos Research Articles

Combinatorial expression patterns of individual TLE proteins during cell determination and differentiation suggest non-redundant functions for mammalian homologs of Drosophila Groucho.

The Drosophila protein Groucho is involved in the regulation of cell-determination events during insect neurogenesis and segmentation. A group of mammalian proteins, referred to as transducin-like Enhancer of split (TLE) 1 through 4, share with Groucho identical structures and molecular properties. The aim was to determine whether individual TLE proteins participate in the regulation of cell determination in mammals like their Drosophila counterpart. It is here reported that TLE family members are expressed in combinatorial ways during the in vitro differentiation of mouse P19 embryonic carcinoma cells (a model for neural determination) and rat CFK2 cells (a model for chondrocytic determination). TLE1 is up-regulated and TLE2 and TLE4 are down-regulated to different extents during early stages of differentiation. In contrast, later stages correlate with up-regulation of TLE2 and TLE4, and decreased expression of TLE1. Individual TLE proteins are also expressed in combinatorial as well as complementary patterns during the development of the cerebral cortex and spinal cord of mouse embryos. In particular, TLE1 is robustly expressed in both neural progenitor cells and postmitotic neurons of the outer layers of the cortical plate, whereas TLE4 expression marks preferentially postmitotic neurons of the inner layers. Taken together, these results strongly suggest non-redundant roles for individual TLE proteins during both cell-determination and cell-differentiation events.

Simultaneous expression of brain-derived neurotrophic factor and neurotrophin-3 in Cajal–Retzius, subplate and ventricular progenitor cells during early development stages of the rat cerebral cortex

To identify production sites and action targets of neurotrophins during neurogenesis, we investigated immunoreactivities of neurotrophins and their tyrosine kinase receptors in the cerebral cortex of rat embryos. Two sets of ligand–receptor systems, brain-derived neurotrophic factor/TrkB and neurotrophin-3/TrkC, were expressed simultaneously in Cajal–Retzius, subplate neurons and ventricular multipotent stem cells at embryonic days 13 and 15. Intraventricular administration of brain-derived neurotrophic factor or neurotrophin-3 at embryonic day 16 markedly modulated microtubule-associated protein II and/or Hu protein expression in different ways in the cortical plate cells by embryonic day 20. These observations indicate the involvement of autocrine and/or local paracrine action of brain-derived neurotrophic factor and/or neurotrophin-3 during formation of the cerebral cortex.

Expression of a novel aristaless related homeobox gene ‘ Arx’ in the vertebrate telencephalon, diencephalon and floor plate

We have isolated a novel homeobox gene that is expressed in the vertebrate central nervous system and which shows striking similarity to the Drosophila al gene in the homeodomain (85% identity) and in a 17 amino acid-sequence near the carboxyl-terminus. This gene was designated Arx ( a ristaless r elated homeobo x gene) in consideration of its structural similarity to the al gene. Arx was highly conserved between mouse and zebrafish. Neuromeric expression in the forebrain and longitudinal expression in the floor plate were observed in mouse and zebrafish. The expression of Arx in the ganglionic eminence and ventral thalamus overlapped regionally with that of Dlx1, but the cell layer where Arx is expressed differed from that of the Dlx1. This gene was also found to be expressed in the dorsal telencephalon (presumptive cerebral cortex) of mouse embryos. The structure and expression pattern of Arx with respect to any possible relationship to al and Dlx1, as well as the function of Arx in the floor plate are discussed.

Isolation of actin-associated proteins from Caenorhabditis elegans oocytes and their localization in the early embryo.

The actin cytoskeleton plays an important, but poorly understood, role in the development of multicellular organisms. To help illuminate this role, we used actin filament affinity chromatography to isolate actin binding proteins from large quantities of Caenorhabditis elegans oocytes. To examine how these proteins might be involved in early development, we prepared antibodies against some of them and determined their distribution in fixed embryos. Three of these proteins co-localize with different subsets of the embryonic actin cytoskeleton. One co-localizes with actin to all cell cortices. The second oscillates between the nucleus and cortex in a cell-cycle-dependent manner. The third is asymmetrically enriched at the anterior cortex of one-cell embryos, showing a temporal and spatial localization suggestive of a function in generating developmental asymmetry. We conclude that biochemistry is a feasible and useful approach in the study of early C. elegans development, and that the embryonic actin cytoskeleton is regulated in a complex fashion in order to carry out multiple, simultaneous functions.

Histological changes during development of the cerebellum in the chick embryo exposed to a static magnetic field

Few studies have been performed to evaluate the ultrastructural changes that exposure to static magnetic fields (SMF) can cause to the processes of cell migration and differentiation in the cerebellum during development. Thus, we have studied the development of the cerebellum in the chick embryo (n = 144) under a uniform SMF (20 mT). All of our observations were done on folium VIc of Larsell's classification. The cerebella of chick embryos, which were exposed solely on day 6 of incubation and sacrificed at day 13 of incubation [short exposure (S)1; n = 24], showed an external granular layer (EGL) that was less dense than the EGL in the control group (n = 24). The molecular layer (ML) exhibited a low number of migratory neuroblastic elements. Moreover, the internal granular layer (IGL) was immature, with the cellular elements less abundant and more dispersed than in controls. In chick embryos exposed on day 6 of incubation and sacrificed at day 17 (S2; n = 24), the outstanding feature was the regeneration of the different layers of the cerebellar cortex. The cerebellar cortex of chick embryos exposed continuously to an identical field from the beginning of the incubation up to day 13 [long exposure (L)1; n = 24] or day 17 (L2; n = 24) of incubation showed a higher number of alterations than that of group S1. Electron microscopy confirmed the findings from light microscopy and, at the same time, showed clear signs of cell degeneration and delay in the process of neuronal differentiation. This was more apparent in groups L1 (100%) and L2 (100%) than in groups S1 (95.4%) and S2 (65.2%). In conclusion, the present study showed that SMF can induce irreversible developmental effects on the processes of cell migration and differentiation of the chick cerebellar cortex.

Cortical actin movements during the first cell cycle of the Caenorhabditis elegans embryo.

The first division of the Caenorhabditis elegans embryo is unequal, generating daughter cells with distinct fates. The differences between the cells are believed to result from the partitioning of cytoplasmic determinants during the first cell cycle. Actin microfilaments play a critical, but poorly defined, role in this event. In this paper, the actin cortex in live embryos is studied during cytoplasmic localisation by fluorescently labelling microfilaments in oocytes and then using in vivo fluorescence microscopy to observe their behaviour. This reveals that there is a concerted movement of cortical actin to the anterior of the embryo at the time cytoplasmic localisation takes place. Furthermore, it is demonstrated that endogenous foci of F-actin are asymmetrically distributed following this event; these structures have previously been seen in fixed cortices. A model for the participation of the actin cytoskeleton in cytoplasmic localisation is presented based on these results.

13 Cortical Cytoskeleton of the Xenopus Oocyte, Egg, and Early Embryo

Publisher Summary The cortex of eggs and embryos is a highly specialized region of the cell that undergoes rapid changes in structure and function, in response to biochemical signals, throughout oogenesis and development. Progesterone triggers resumption of meiosis, which results in alignment of the cortical granules beneath the plasma membrane, formation of the endoplasmic reticulum, and conversion of the unfertilizable oocyte into the fertilizable egg. Biochemical signals at fertilization trigger further modifications of the cortex. The cortical cytoskeleton is an extremely dynamic structure that undergoes repeated reorganizations as the function of the egg changes. The importance of these cytoskeletal changes during meiotic maturation, fertilization, and early development is clear since disrupting these rearrangements results in abnormal growth and development. A cell as large as the Xenopus egg requires specialized regions with distinct functions. Delivery of unique components to these regions via the cytoskeleton can resolve the developmental challenges faced by such a large egg. As the tools of molecular biology and confocal microscopy provide increasing amounts of new information about the repositioning of these unique components, the role of the cytoskeleton in early development will become better understood.

Downregulation of in vitro neurotoxicity of brain macrophages by prostaglandin E2 and a beta-adrenergic agonist.

Brain macrophages (BM), a subpopulation of microglia, have the ability to kill neurons by producing reactive oxygen intermediates. Cocultures of neurons and macrophages derived from the cerebral cortex of rat embryos were used to look for regulation of BM neurotoxicity. Isoproterenol (10(-7) M), a beta-adrenergic agonist, induced a significant inhibition of BM neurotoxicity and this effect was abolished in the presence of propranolol, a beta-adrenergic antagonist. BM neurotoxicity was also reduced in the presence of prostaglandin E2 (10(-8), 10(-6) M), a metabolite derived from arachidonic acid. These results suggest endogenous mechanisms of neuroprotection operating either during development or following lesions.

Ventral mesencephalic and cortical transplants into the rat striatum display enhanced activity for neutral endopeptidase 24.11 (‘enkephalinase’; CALLA)

A role for neutral endopeptidase 24.11 (NEP) in growth and development is supported by the demonstration that NEP hydrolyses and inactivates a number of peptide growth factors including atrial natriuretic peptide, endothelins, bombesin-like peptides, and opioid peptides, including the enkephalins. In the present study, suspensions of cells obtained from the ventral mesencephalon or cortex of rat embryos (ED14) were implanted into the striatum of the adult rat brain. Three to 15 weeks after transplantation the relative distribution of NEP-positive cellular elements was visualized histochemically. NEP staining in the transplants consistently appeared before NEP staining in the surrounding host striatum supporting a relative increase in NEP activity in the transplants. The NEP staining richly visualized cells of varying size and morphology which lacked the normal organization of the host striatum. The histochemical staining in the transplants and the surrounding host tissue was completely blocked by a 100 nM concentration of the selective NEP inhibitors phosphoramidon or JHF-26, supporting the exclusive localization of NEP by this method. NEP localization in the embryonic (ED14) cortex and ventral mesencephalon was also confirmed, suggesting one possible origin for the NEP-positive cells visualized in the transplants. Fluurescent double-labeling studies for NEP and glial fibrillary acidic protein (GFAP) or transforming growth factor alpha precursor (TGFαp) revealed the presence of rich glial labeling within the transplants for both GFAP and TGFap. NEP-labeled cells in the transplants were closely associated with glial elements, however, only occasional glial elements in the transplants stained for NEP; supporting a non-astrocytic localization for the NEP in the transplants. The marked enhancement of NEP staining in the transplants may have significance for controlling the rate or pattern of growth of the transplanted cells through inactivation of peptide growth factors produced by, or in response to, the transplants.

Downstream DNA sequences are required to activate a gene expressed in the root cortex of embryos and seedlings.

We showed previously that a gene, designated AX92, which is expressed at an early stage of cortex differentiation in the root apex of oilseed rape seedlings, is also expressed in embryos. To compare AX92 gene regulation during embryo-genesis and postembryonic growth, we constructed a chimeric gene consisting of AX92 5' and 3' untranslated and flanking regions fused with a beta-glucuronidase protein coding region. We showed that the chimeric gene is active in both developing cortex cells in the root apical meristems of transgenic oilseed rape seedlings and in cortex cells at the root end of embryonic axes. To determine whether the AX92 gene is regulated by a common mechanism in embryos and seedlings, we analyzed the expression of modified chimeric genes. We showed that the AX92 chimeric gene is regulated combinatorially and that DNA sequences located 3' of the protein coding region are necessary for its activation in the root cortex of both embryos and seedlings. Our results suggest that common regulatory sequences are required to activate the gene in the embryonic and postembryonic root cortex.

Downstream DNA Sequences Are Required to Activate a Gene Expressed in the Root Cortex of Embryos and Seedlings

Downstream DNA Sequences Are Required to Activate a Gene Expressed in the Root Cortex of Embryos and Seedlings

Developmental and Age‐Dependent Changes of 28‐kDa Calbindin‐D in the Central Nervous Tissue Determined with a Sensitive Immunoassay Method

For the quantitative analysis of vitamin D-dependent 28-kDa calcium-binding protein (calbindin-D) in the CNS, we have established a highly sensitive immunoassay method. The antisera were raised in rabbits with purified calbindin-D from rat kidneys, and the antibodies were purified with a calbindin-D-coupled Sepharose column. The purified antibodies were specific for calbindin-D, showing a single band on the immunoblot with the extract of rat kidney or cerebellum. The sandwich-type immunoassay system was prepared by the use of purified monospecific antibodies, and the minimum detection limit of the assay was 0.1 pg or 3.6 amol of calbindin-D, which was sufficiently sensitive for the measurement of calbindin-D content in isolated Purkinje cell bodies at the level of single cells. The average content of calbindin-D in a single Purkinje cell was 0.05 pg. Calbindin-D was detected in most of the rat tissues examined, but it was present predominantly in the kidney and CNS, especially in the cerebellum. Calbindin-D was detected at a similarly low level in the cerebral cortex, cerebellum, and brainstem of rat embryos of 15 gestational days, and it increased gradually but differently in these regions, reaching the respective adult levels by 4-5 weeks of postnatal age. In contrast, kidney calbindin-D increased sharply between 15 gestational days and 3 postnatal days, reaching the adult level by 6 days of age. Calbindin-D levels in the adult rat CNS were affected little by age, whereas the concentrations in human cerebral cortices were significantly low in the aged brain as compared with those in the young brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxic Effect of Brain Macrophages on Developing Neurons.

Brain macrophages are transiently present in different regions of the central nervous system during development or in the course of tissue remodelling following various types of injuries. To investigate the influence of these phagocytes on neuronal growth and survival, brain macrophages stemming from the cerebral cortex of rat embryos were added to neuronal primary cultures. A neurotoxic effect of brain macrophages was demonstrated by the reduction of the number of neurons bearing neurites within two days of contact between the two cell types. Neuronal death and phagocytosis were also directly observed in video recordings of living cultures. This toxicity involved the production by brain macrophages of reactive oxygen intermediates, as shown by the protective effect of catalase, a scavenger of H2O2. In addition, the respiratory bursts of brain macrophages were stimulated in the presence of neurons. These results suggest that brain macrophages could favour the appearance of neuroregressive events which occur either during neurogenesis or in neurodegenerative diseases, implying intracerebral recruitment of mononuclear phagocytes.

Retarded nuclear migration in Drosophila embryos with aberrant F-actin reorganization caused by maternal mutations and by cytochalasin treatment

Three X-linked mutations of Drosophila melanogaster, gs(1)N26, gs(1)N441 and paralog, had a common maternal-effect phenotype. Mutant embryos show reduced egg contraction that normally occurs at an early cleavage stage in wild-type embryos. In addition, the mutants exhibited retarded nuclear migration while synchronous nuclear divisions were unaffected. The retarded migration causes nuclei to remain in the anterior part of the embryo retaining their spherical distribution even in a late cleavage stage. This consequently results in an extreme delay in nuclear arrival in the posterior periplasm. A mutant phenocopy was induced in wild-type embryos that were treated with cytochalasin B or D at a very early cleavage stage. Remarkable differences were noticed in the organization of cortical F-actin between the mutants and the wild type throughout the cleavage stage: obvious F-actin aggregates were dispersed in the cortex of mutant embryos, in contrast to the wild type where the cortical F-actin layer was smooth and underlying F-actin aggregates were smaller than those in the mutants; the transition of the distribution pattern of F-actin in the yolk mass, from the centralized to the fragmented type, occurred later in the mutants than in wild type. The results suggest that these mutations affect the mechanism underlying establishment and transition of F-actin organization required for normal egg contraction and nuclear migration in the cleavage embryos.

Electron Microscopic Visualization of Actin Filaments in the Early Embryo of <italic>Drosophila melanogaster</italic>: The Use of Phalloidin and Tropomyosin

Various specimen preparations for thin-section electron microscopy were tested to better preserve and visualize actin filaments in the cortex of the early embryos of Drosophila melanogaster. When embryos were treated with phalloidin prior to fixation, many actin filaments were observed in their cortex comparable to the staining with fluorescently labeled phalloidin in light microscopy. Then we used various fixatives containing phalloidin. As far as we examined, actin filaments were best preserved in the specimen fixed with 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium phosphate buffer or in 0.1 M PIPES buffer (1 mM EGTA and 1 mM MgCl2) containing 10 microM phalloidin and 0.1% saponin. When embryos were glycerinated and then treated with tropomyosin before fixation, actin filaments were well visualized as thicker, uniform-sized filaments, though the number of filaments decreased probably owing to glycerination. This suggests that, like heavy meromyosin and its subfragment-1, this protein may protect actin filaments from being disrupted by chemical fixation. Using these improved fixation procedures, we successfully examined the distribution of actin filaments in the Drosophila embryo cortex during cellularization. These methods may be applicable to stabilize labile actin filaments in other types of cells.

Fetal cortical transplants reduce motor deficits resulting from neonatal damage to the rat's frontal cortex

Motor deficits in the execution of grasping movements of the right forelimb were compared in normal female Wistar rats, in animals which sustained a neonatal lesion of the left frontal cortex and in animals which received immediately after the lesion a transplant obtained from the frontal cortex of E16 embryos. Behavioral testing was carried out from postnatal day 48 (D48) to D108. The animals were placed at the center of a circular wire grid which was turned upside down so that they hung by their 4 paws at a distance of 40 cm above the floor. The precision of grasping movements of the right limb and the number of falls were recorded during a 1 min session of active moving across the grid. The lesioned subjects were most impaired on both motor indices whereas the grafted animals although performing slightly poorer than the controls were, however, less impaired than the lesioned animals. Fetal cortical transplants, therefore, seem to promote functional recovery from neonatal cortical damage.

Horizontal compartmentation in the germinal matrices and intermediate zone of the embryonic rat cerebral cortex

Cellular compartmentation was studied in the germinal matrices and the intermediate zone of the cerebral cortex of rat embryos that survived for 1 or more days after injection with [ 3H]thymidine. In contrast to the vertical compartmentation seen in the neuroepithelium with short-survival thymidine autoradiography, sequential-survival autoradiography revealed a horizontal compartmentation both in the germinal matrices and the intermediate zone. In the neuroepithelium of embryos that survived for 24 h, the differentially labeled cells form two distinct horizontal bands. The band overlapping with the mitotic zone is composed of heavily labeled cells, whereas the band overiapping with the synthetic zone is composed of lightly labeled cells. This indicates that there are two proliferative cell populations within the neuroepithelium, one turning over fast and the other more slowly. In the cortical intermediate zone of the same embryos several horizontal bands are present. Of these, the dispositions of two bands of heavily labeled cells—the superior band and the inferior band—were followed for several days. The superior band is apparently composed of glial cells that disperse in the direction of the internal capsule and the corpus callosum. In contrast, the inferior band (which overlaps with the subventricular zone where many cells are horizontally oriented) is apparently composed of sojourning young neurons. The cells of the inferior band resume their migration toward the cortical plate after a pause of 1–2 days. These observations call for a reappraisal of the view that young cortical neurons follow a direct radial path to the cortical plate.

Spontaneous DNA breaks in the rat brain during development and aging

The level of spontaneous DNA breaks in nuclei isolated from the cerebral cortex of rat embryos at 12, 15 and 19 days of gestation, and from cerebral cortex and cerebellum of 24-day-, 6-month- and 36-month-old rats was measured by alkaline elution. A constant low level of DNA breaks was found in brain DNA during development from an embryo at day 12 of gestation to a 24-day-old rat. During aging the level of DNA breaks remained at the same low level, as shown by comparing nuclei from the cerebral cortex and cerebellum of 6- and 36-month-old animals. By contrast, an almost 2-fold increase in the level of DNA breaks was observed in rat liver nuclei between 6 and 36 months of age, confirming our earlier findings on isolated liver cells. Although there were no changes in the level of DNA breaks in rat brain during development or during aging, breaks accumulated rapidly post mortem. The rate of this process was not age-dependent. Our data suggest that the level of spontaneous DNA breaks in the brain is not likely to be of fundamental importance in the complex cellular alterations associated with brain development and aging.

Centrosomes, and not nuclei, initiate pole cell formation in Drosophila embryos

An injection of aphidicolin into early Drosophila embryos inhibits DNA synthesis and nuclear division, while centrosome replication and many other aspects of the mitotic cycle continue. If aphidicolin is injected at nuclear cycle 7–8, the normal migration of nuclei to the embryo cortex is completely inhibited. In most of these embryos, however, centrosomes continue to migrate in a coordinated manner to the cortex, where they reorganize tubulin, actin, and the overlying plasma membrane. Remarkably, the centrosomes that migrate to the posterior pole of such embryos initiate pole cell formation in the absence of nuclei. These observations demonstrate that centrosomes alone are able to direct a major reorganization of the cortical cytoskeleton when they arrive at the surface of the embryo. They also suggest that the coordinated movement of nuclei to the embryo cortex is mediated by forces acting on the centrosome rather than on the nucleus itself.

Levels and sub-cellular distribution of physiologically important metal ions in neuronal cells cultured from chick embryo cerebral cortex.

Mg2+, Ca2+, Mn2+, Zn2+, and Cu content of neurons from chick embryo cortex cultivated in chemically defined serum free growth medium was determined by energy dispersive X-ray fluorescence and atomic absorption spectroscopy. The intracellular volume of cultured neurons was determined to be 2.73 microliters/mg. Intracellular Mn2+, Fe2+, Zn2+, and Cu2+ in the cultivated neurons were 100-200 times the concentrations in the growth medium: Mg2+ and Ca2+ were 0.9 and 1.7 mM respectively, around 20 fold higher than in growth medium. Mg2+, Fe2+, Cu2+ and Zn2+ concentrations in neurons were in the range of ca. 300-600 microM, approximately 2-3 times the values previously reported in glial cells; Ca2+ and Mn2+ content of the neurons were higher by 5 and 10 fold respectively compared to glial cells. In neurons, the subcellular distribution of Fe2+, Cu2+, and Mn2+ follows the rank order: cytosol greater than microsomes greater than mitochondria; for Zn2+ the distribution differs as following: cytosol greater than mitochondria greater than microsomes. Determination of the superoxide dismutase activities in the cultivated neurons indicated that the Mn2+ linked activity predominates whereas, the Cu-Zn dependent enzyme is dominant in glial cells. Enrichment of the culture medium with Mn2+ to 2.5 microM enhanced the Mn-SOD by approximately 33% but the Cu2+-Zn2+ enzyme activity was not modified. The high Mn2+ content, the capacity to accumulate Mn2+, and the predominancy of the Mn-SOD form observed in neurons is in accord with a fundamental functional role for this metal ion in this type of brain cells.