Embryo Neural Tube Research Articles

Developmentally regulated expression and functional role of alpha 7 integrin in the chick embryo.

Integrin alpha 7 beta 1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the alpha 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. alpha 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The alpha 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of alpha 7 in the neural tube was transient suggesting that alpha 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. alpha 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense alpha 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, alpha 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of alpha 7 by blocking antibodies showed that alpha 7 integrin-laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with alpha 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that alpha 7 integrin-laminin signaling may have played a role in segment re-specification in the mesoderm.

The NeuronalMicrotubule-Associated Protein 1BIs under Homeoprotein Transcriptional Control

To identify genes regulated by homeoprotein transcription factors in postnatal neurons, the DNA-binding domain (homeodomain) of Engrailed homeoprotein was internalized into rat cerebellum neurons. The internalized homeodomain (EnHD) acts as a competitive inhibitor of Engrailed and of several homeoproteins (Mainguy et al., 2000). Analysis by differential display revealed that microtubule-associated protein 1B (MAP1B) mRNA is upregulated by EnHD. This upregulation does not require protein synthesis, suggesting a direct effect of the homeodomain on MAP1B transcription. The promoter region of MAP1B was cut into several subdomains, and each subdomain was tested for its ability to bind Engrailed and EnHD and to associate with Engrailed-containing cerebellum nuclear extracts. In addition, the activity, and regulation by Engrailed, of each subdomain and of the entire promoter were evaluated in vivo by electroporation in the chick embryo neural tube. These experiments demonstrate that MAP1B promoter is regulated by Engrailed in vivo. Moreover, they show that one promoter domain that contains all ATTA homeoprotein cognate binding sites common to the rat and human genes is an essential element of this regulation. It is thus proposed that MAP1B, a cytoskeleton protein involved in neuronal growth and regeneration, is under homeoprotein transcriptional regulation.

Antisense attenuation of nestin accumulation causes neural tube deformation in rat embryo cultures

ABSTRACT The roles of nestin in neural tube development were studied using immunostaining and antisense experiments in rat embryos. Nestin was detected in the neural tube of embryos of day 10.5 of gestation (E10.5), while no nestin staining was observed in E9.5 embryos in which the neural plate comprised 3 to 4 layers of neuroepithelial cells. As embryos developed, the neural tube became comprised of multiple cell layers and staining was observed in filamentous structures spanning from the ventricular surface to the pial surface of the neural tube. Nestin accumulation was suppressed in the neural tube of embryos treated with nestin antisense oligodeoxynucleotide (ODN). The treated embryos showed two types of neural tube deformation. One type was a thin neural tube which had 3 to 4 layers of neuroepithelial cells and the other was a local distribution of neuroepithelial cells near the basement membrane. While neuroepithelial cells in the neural tube were fewer in embryos treated with nestin antisense ODN than in controls, the percentage of Islet‐1‐positive neurons relative to the neuroepithelial cells was not different between the treated and control embryos. These results suggested that nestin plays roles in the proliferation of neural tube cells and in the formation and the maintenance of multiple cell layers in the neural tube but not in suppression of development of Islet‐1‐positive neurons.

Induction of apoptosis by beta radiation from tritium compounds in mouse embryonic brain cells.

Induction of apoptosis by tritium exposure was investigated in both cultured embryonic mid brain cells and brain sections of embryos and of newborns in mice. In the cultures of mid brain cells, addition of methyl-3H-thymidine (3H-TdR) (21 kBq mL(-1)) and tritiated water (5.616 MBq mL(-1)) induced late appearances and low percentages of apoptosis when compared to x-irradiation at the ID50 dose, the inhibitory dose that reduced cellular differentiation by 50% of the control. A significant increase in p53 protein was detected about 2 h before the marked appearance of apoptosis. The pregnant mice were given an intraperitoneal injection of tritiated water at the concentration of 481.8 kBq g(-1) of body weight on gestation day 12.5, by which treatment behavioral changes in the offspring occurred. Increased apoptotic cells were observed in the neural tube of embryos from 1 d after the injection to 1 wk postnatal age. Apoptosis induced by x-rays appeared 2 h after irradiation, with a peak at 4 h. Increase of apoptotic cells was also found in the brain cortexes of newborns. The percentage of apoptosis in the brain was higher in the prenatal tritiated water exposed mice than in the prenatal x-irradiated mice. Possible mechanisms on apoptosis and its relation to the higher relative biological effectiveness value of tritium beta-rays are discussed.

Chromatin structure and functional analysis of the mouse HNF3alpha gene.

The transcription factor HNF3alpha is a member of the winged-helix family of regulatory proteins. It is expressed in the definitive endoderm, notochord, and neural tube in embryos, but in the adult is expressed primarily in endoderm-derived tissues such as liver, lung, and pancreas. We present here the cloning of the mouse HNF3alpha gene and a characterization of its chromatin structure and regulatory sequences. The HNF3alpha gene is encoded by two exons and its transcription initiates at multiple start sites at a TATA-less promoter that is highly conserved between mouse and rat. We found different patterns of DNaseI hypersensitive sites in HNF3alpha gene chromatin in different adult tissues in which HNF3alpha is expressed, suggesting distinct regulatory mechanisms occurring within different tissue derivatives of the endoderm germ layer. Cell transfection data indicate that sequences spanning certain upstream hypersensitive sites can enhance transcription from the HNF3alpha promoter, but only when stably integrated into chromatin and not when transiently transfected. The results suggest a complex regulatory interplay between distinct genetic regulatory sequences that function specifically in chromatin.

Requirement for LIM Homeobox Gene Isl1 in Motor Neuron Generation Reveals a Motor Neuron– Dependent Step in Interneuron Differentiation

Motor neuron differentiation is accompanied by the expression of a LIM homeodomain transcription factor, Islet1 (ISL1). To assess the involvement of ISL1 in the generation of motor neurons, we analyzed cell differentiation in the neural tube of embryos in which ISL1 expression has been eliminated by gene targeting. Motor neurons are not generated without ISL1, although many other aspects of cell differentiation in the neural tube occur normally. A population of interneurons that express Engrailed1 (EN1), however, also fails to differentiate in Isl1 mutant embryos. The differentiation of EN1 + interneurons can be induced in both wild-type and mutant neural tissue by regions of the neural tube that contain motor neurons. These results show that ISL1 is required for the generation of motor neurons and suggest that motor neuron generation is required for the subsequent differentiation of certain interneurons.

Programmed cell death during the earliest stages of spinal cord development in the chick embryo: A possible means of early phenotypic selection

The spatiotemporal distribution of cell death in the chick embryo neural tube and spinal cord (brachial region) was examined between stage (St.) 12 and 22, in plastic semithin sections. Between St. 12 and 16, the total number of pycnotic cells per segment was low, whereas after St. 16 the number of pycnotic cells was substantially increased. Between St. 17 and 19 three cell death foci or regions could be recognized. One region, the dorsal pycnotic zone, was located in the most dorsal part of the spinal cord, including the neural crest, with the highest number of pycnotic cells observed at St. 18. The second region, or ventral pycnotic zone, was located between motoneurons and the floor plate and had the highest number of dying cells at St. 17. The third region, the floor plate pycnotic zone, was located in the midportion of the floor plate and had the greatest amount of cell death at St. 19. Although low numbers of pycnotic cells were also observed in other regions between St. 17 and 19, no pycnotic cells were found in the ventrolateral region that gives rise to motoneurons. Ultrastructural observations as well as data from in situ nick end labeling indicate that the pycnotic cells observed in the neural tube die by apoptosis and that the debris from the dead cells is phagocytized primarily by adjacent healthy neuroepithelial cells. Although the spatiotemporal distribution of pycnotic cells suggests that cell death at these early stages could play a role in establishing the pioneer axonal pathway for spinal commissural neurons, preliminary observations following perturbations of cell death do not support this notion. Alternatively, early cell death may be involved in the regulation of cellular patterning along the dorsoventral axis of the neural tube by a kind of negative selection of specific progenitor cells.

Methionine and neural tube closure in cultured rat embryos: Morphological and biochemical analyses

When headfold-stage rat embryos were cultured on cow serum, their neural tubes failed to close unless the serum was supplemented with methionine. Methionine deficiency did not appear to affect the ability of the neural epithelium to fuse as a type of fusion was observed between anterior and posterior regions of the open neural tube in methionine-deficient embryos. Although methionine deficiency reduced the cell density and mitotic indices of cranial mesenchyme and neural epithelial cells, this did not appear to be a factor in failure of the neural tube to close. For example, embryos cultured on diluted cow serum also had fewer mesenchymal cells yet could complete neural tube closure if provided with methionine. Examination of the tips of the neural folds suggested that microfilament contraction could be involved; in the absence of methionine the neural folds failed to turn in. This possibility was supported by the reductions in neurite extension of isolated neural tubes cultured without methionine and by the reductions in microfilament associated methylated amino acids contained in embryo neural tube proteins.

Comparative responsiveness of early chick neural tube neurons to muscle-conditioned medium, laminin, NGF and fibronectin

Dissociates from the metencephalic basal plate of early chick embryo neural tubes containing the trigeminal (V) motor nucleus were cultured on substrates conditioned with appropriate target-derived muscle conditioned medium (MCM), laminin (LAM), MCM with nerve growth factor (NGF) in the medium, and fibronectin (FN). Comparisons were made of neuronal survival, the number of neurons with processes, and the length of processes elaborated. It was found that both MCM and LAM significantly enhanced survival and neuritic production from this population when compared to controls grown on a collagen-polyornithine substrate, but MCM surpassed LAM in these measures. When the neurons were grown on an MCM-conditioned substrate with an NGF-supplemented medium, no improvements were produced over the MCM or the NGF conditions alone. Therefore, the two do not appear to act in synergy, as NGF and LAM have been shown to do. FN produced no enhancement of any of the measures taken from this population. An ELISA analysis revealed no detectable LAM in the early target MCM. These results indicate that the specific responsiveness of this early neural tube population to its target MCM is not mediated by LAM, but the growth-enhancing component acts in a similar manner, although its influence is more potent.

Influence of laminin on the responsiveness of early chick embryo neural tube neurons to nerve growth factor.

Dissociated neurons from the neural tube containing the trigeminal motor nucleus from early chick embryos were cultured on laminin or collagen-polyornithine substrates, with and without nerve growth factor (NGF). Control cultures were grown in similar conditions with cytochrome-C. It was found that neuronal survival was not affected by NGF or cytochrome-C, but it was enhanced by laminin. The expression of neuritic processes, however, was significantly enhanced in the presence of NGF on both laminin and collagen-polyornithine surfaces, with the greatest number of neurons producing processes seen in the laminin-plus-NGF group. The length of processes was similarly enhanced by laminin and by NGF. Cytochrome-C did not influence any of these measures. The results indicate that while laminin potentiates the NGF effect on these early neuronal populations, NGF in conjunction with other substrata can have similar, though less dramatic, effects. These results, together with prior evidence of NGF receptors and specific NGF retrograde transport, suggest that this growth factor may play a significant role in the normal ontogeny of early motoneuron populations.

Species specificity in the responsiveness of chick embryo neural tube explants to target-conditioned medium

Explants from the metencephalic region of 40-h chick embryo neural tubes containing the trigeminal (V) motor nucleus were cultured in appropriate target muscle-conditioned media (MCM) derived from chick, quail and rat embryos. Enhanced neuritic outgrowth was found only in the presence of chick MCM, indicating that this early, initial responsiveness to target-released materials within this system is species-specific.

The effect of 6-aminonicotinamide on energy metabolism in rat embryo neural tube.

6-Aminonicotinamide (6-AN) is a potent metabolic inhibitor which produces central nervous system malformations when administered to pregnant rats. Since the antimetabolite interferes with energy yielding pathways, the present study was undertaken to determine the effect of maternally administered 6-AN on energy metabolism of the neural tube in developing embryos. On day 9 of gestation, pregnant rats were injected with 2 mg/kg 6-AN, and embryos removed 12 or 24 hours later. Glucose, glycogen, lactate, ATP, PCr, and GABA were measured in neural tube sections using fluorometric techniques. Only ATP and PCr showed changes as compared to appropriate controls. These two metabolites were increased 29% and 37%, respectively, by 6-AN administration. The mechanism of these alterations in metabolites is unclear, but may be related to decreased metabolic demand.

The effect of 5-fluorouracil on [ 3H]nucleoside incorporation into the DNA of mouse embryos and maternal tissues

Pregnant albino mice (ICR) were administered, ip, 40 mg/kg 5-fluorouracil (5-FU) on Day 10 of gestation. This dosage produced 96.3% embryolethality and 100% of the surviving fetuses were malformed. This dosage also produced a pronounced inhibition of 6-[ 3H]dUrd incorporation into the DNA of the embryo, chorioallantoic placenta, maternal intestine, and maternal spleen. There was no recovery in the embryo or placenta over the 48 hr time period analyzed. In the maternal tissues, however, incorporation levels returned to control values by 48 hr after 5-FU administration. 5-FU had no effect on 6-[ 3H]dThd incorporation into the DNA of any of the mitotically active tissues with the exception of the embryo at the 48 hr time point. Concomitant histological studies of the embryo neural tube showed that 40 mg/kg 5-FU produced mitotic inhibition 2 hr after treatment. Cell death began at 4 hr with heteropycnosis in the DNA synthetic zone of the primitive ependymal layer. As with the incorporation experiment, there was no histological recovery in the neural epithelium and the pycnosis became progressively more severe with time after 5-FU treatment.

Neuronal and glial differentiation in reaggregation cultures.

Dissociation and reaggregation cultures from different portions of the chick embryo neural tube were made, and the resulting aggregates were fixed for electron microscopy after 1, 5, 8, 14, 16 and 22 days in vitro. All cultures (pure aggregates of telencephalon, optic lobe or neural retina, and combined aggregates made from mixtures of optic lobe plus neural retina or optic lobe plus telencephalon) show a common timing of neuronal and glial morphological differentiation. During the first week in vitro, some cells developed neuronal characteristics in the absence of morphological evidence of glial differentiation. Numerous axonic processes usually formed fascicles with all the fibers running parallel to each other. Axonic growth cones were abundant and a few immature synapses were also persent. The second week in culture was characterized by the disappearance of growth cones and the increase in number and morphological maturation of synapses. Morphologically detectable glial differentiation began by the end of this week, and during the third week almost every neuronal element, including the axonic fascicles, became associated with glial cells showing astrocytic features.

A stereological study of the effect of 2,4-dinitrophenol on chick embryo neural tube Mitochondria

A stereological study of the effect of 2,4-dinitrophenol on chick embryo neural tube Mitochondria

Cell shape and morphology of the neural tube: Implications for microtubule function

Cell shape-determining factors were investigated in the asymmetric cells of the chick embryo neural tube. In order to determine their relative importance in maintenance of cell shape, cytoplasmic microtubules were degraded experimentally by colchicine and cold treatments. The results indicate that concurrent with degradation of microtubules there was reduction of normal cell asymmetry, though the pseudostratified epithelial structure of the neural tube was preserved. It was concluded that microtubules contribute to the maintenance of cell shape, and that other factors, particularly cell contacts and the structural specializations of the apical region of the cells, are also important in maintaining cell shape, the relations of cells to one another, and the total morphology of the neural tube. This work also revealed a differential stability of cytoplasmic microtubules in reaction to experimentally induced degradation.

Cell proliferation in embryonic mouse neural tube following urethane exposure

A comparative autoradiographic study of the cell cycle in normal 10-day mouse embryos and in embryos exposed to urethane showed an increase of 4.5 hours in the cycle time of the experimental group. Initially urethane interfered with the migration of some unlabeled premitotic nuclei to the germinal zone, resulting in extraventricular mitoses. A transient increase in the mitotic index and proportion of metaphases suggested a lengthened mitotic time but this was of brief duration and would not contribute significantly to the overall increased cycle time. The G 2 period of cells which incorporated thymidine- 3H in the presence of urethane was significantly lengthened as were the S and G 1 periods and some injured cells died in interphase or mitosis. The new lengthened generation time of embryo neural tubes exposed to urethane resembles that of older, 11-day embryos. This suggests the possibility that cells with a short cycle may be preferentially killed by urethane, leaving the determination of mean cycle time to more differentiated cells.

Submicroscopical study of chick embryo neural tube experimentally reopened "in vitro".

Chick embryo neural tubes experimentally reopened by culturing in a medium containing submaxillary gland extracts (SGE) were studied under the electron microscope. While in control explants the neural tube cells are in close contact whith each other, in SGE treated explants they appear widely separated. However, in the apical zones, cells remain in contact by means of tight junctions and desmosomes, thus forming a continuous surface. Neural tube cells in control and SGE treated explants have abundant free ribosomes, scanty endoplasmic reticulum and mitochondria; SGE treated explants have in addition well developed Golgi complexes and abundant microtubules.

Experimental production of chick embryo neural tube opening ‘in vitro’

La reapertura del tubo neural de embrion de pollo, obtenida «in vitro» mediante la accion de extractos de glandula submaxilar de rata, o de tripsina, es analizada.

An autoradiographic study of the generation cycle in the ten-day mouse embryo neural tube

An autoradiographic study of the generation cycle in the 10-day-old mouse embryo neural tube shows a total generation time of 8.5 hr, and a DNA synthetic period of approximately 4.0 hr. Though growth appears to be almost exponential over the early part of the time studied, increasing migration and differentiation of labeled cells becomes evident towards the end of the observation period. Between day 10 and day 11, the mean Ts increases by approximately 1 hr. This increase in the S period appears to correlate with the transition between the phase of predominate cell proliferation in the 10 day neural tube and accelerated migration and differentiation of the 11 day neural tube.