Embryonic Hemispheres Research Articles

Novel genes expressed in the developing medial cortex.

Two cDNAs, M1 and M2, recently isolated by the differential display method from embryonic rat cerebral hemisphere were characterized and their patterns of spatiotemporal expression analysed in developing rat forebrain by in situ hybridization histochemistry and correlative immunocytochemistry. Neither gene bears any sequence homology to other known genes. Both genes are particularly expressed in medial regions of the cerebral hemisphere and M2 in the roof of the adjacent diencephalon. M1 expression is highly localized and confined to the neuroepithelium of the hippocampal rudiment from embryonic day (E) 12 onward. Its location corresponds to the fimbrial anlage, and immunocytochemical localization of M1 protein indicates its expression in radial glial cells. M2 expression at E12 is more extensive in the medial cerebral wall, extending into the preoptic region and beyond the hippocampus into dorsal hemisphere and into the dorsal diencephalon, with caudal extension along the dorsal midline and in the zona limitans intrathalamica. Later, M2 expression is found in association with the corpus callosum, hippocampal commissure, fimbria, optic nerve, stria medullaris, mamillothalamic tract and habenulopeduncular tract. M1 and M2 expression domains corresponding to the locations of fiber tracts are present prior to the arrival of the earliest axons, as identified by neuron specific markers. These findings suggest M1 and/or M2 genes are involved in early regional specification of the hippocampus and related structures in paramedian regions of the forebrain, and that cell populations expressing these genes in advance of developing axonal pathways may be involved in the early specification of tract location.

Gap-junctional coupling between neurons and astrocytes in primary central nervous system cultures.

Gap-junctional communication between neurons and astrocytes dissociated from rat brain was identified in culture by using dye-transfer assays and electrophysiological measurements. Cell types were identified by using antibodies against beta-tubulin III, glial fibrillary acidic protein, and 2',3'-cyclic-nucleotide phosphohydrolase, which are antigenic determinants of neurons, astroglia, and oligodendrocytes, respectively. Dye coupling was examined as a function of time after dissociated embryonic brain cells were plated onto confluent monolayers of postnatal astrocytes by intracellularly injecting the fluorochrome Lucifer yellow. Coupling of neurons to the astrocytic monolayer was most frequent between 48 h and 72 h in culture and declined over the next 4 days. This gradual uncoupling was accompanied by progressive neuronal maturation, as indicated by morphological measurements in camera lucida drawings. Dye spread was abolished reversibly by octanol, an agent that blocks gap junction channels in other systems. Double whole-cell voltage-clamp measurements confirmed the presence of heterocellular electrical coupling in these cocultures. Coupling was also seen between neurons and astrocytes in cocultures of cells dissociated from embryonic cerebral hemispheres but was rarely detectable in cocultures of postnatal brain cells. These data strongly suggest that junctional communication may provide metabolic and electrotonic interconnections between neuronal and astrocytic networks at early stages of neural development and that such interactions are weakened as differentiation progresses.

Neurons induce GFAP gene promoter of cultured astrocytes from transgenic mice

In order to investigate the influence of neuron-glia interaction on astrocyte differentiation, we used a transgenic mouse bearing part of the gene promoter of the astrocytic maturation marker GFAP linked to the beta-galactosidase (beta-gal) reporter gene. Addition of embryonic cerebral hemisphere (CH) neurons to transgenic CH astrocyte monolayers increased by 50-60% beta-gal positive cell number. Such event was dependent on the brain regional origin of the neurons and was followed by an arrest of astrocytes from the cell cycle and induction of glial differentiation. Time-course assays demonstrated that maximum effect was observed after 24 h of coculture. Addition of conditioned medium (CM) derived from CH neurons also increased beta-gal positive CH astrocytic cell number. However, such CM had no effect on midbrain and cerebellum astroglia. Together, these data suggest that neurons secrete brain region-specific soluble factors which induce GFAP gene promoter, as measured by beta-gal expression, thus suggesting that neuron-glia interaction might induce the astrocytic differentiation program.

An ultrastructural study of the role of an extracellular matrix during normal cleavage in a marsupial, the brushtail possum.

In marsupials, the mechanisms of lineage allocation into pluriblast and trophoblast are related to conceptus polarity and polarized discharge of extracellular matrix (ECM). The brushtail possum, Trichosurus vulpecula, a major pest species in New Zealand, is being intensively studied to develop an immunocontraceptive control method. Of 23 specimens examined, 11 were examined by electron microscopy to study the presence and role of the ECM in lineage allocation in the possum. A number of polarized features in the zygote identified the future embryonic and abembryonic poles. Pronuclei, in a broad band of mitochondrion-rich cortical cytoplasm, lay in the embryonic hemisphere, and numerous electron-lucent vesicles characterized the abembryonic cytoplasm. These vesicles seemed to contribute to the ECM. During cleavage, cells lay near the zona in the embryonic hemisphere, and ECM accumulated chiefly in the abembryonic hemisphere. Cell-zona adhesion facilitated by microvillous and club processes occurred at the early 4-cell stage, and cell-cell adhesion commenced at the late 4-cell stage. The first two cleavages were meridional, equal, and accompanied by elimination into the cleavage cavity of much of the electron-lucent vesicular material in the form of several membrane-bound yolk masses. The third cleavage was unequal, with both meridional and latitudinal planes. The first differences between trophoblast and pluriblast lineages appeared at the 8-cell stage. Later cleavage planes were latitudinal or oblique. Conceptus polarity, polarized discharge of ECM, and localized cell-zona adhesion were related to the first lineage allocation in the possum.

Embryonic brain tract formation in Drosophila melanogaster.

During embryogenesis in insects, the axonscaffold of the brain is built around the embryonic foregut which separates the anlagen of the brain hemispheres. Here, we investigate this process in Drosophila and show that the major longitudinal and horizontal tracts of the embryonic brain form superficially near the interface between the foregut and embryonic brain hemispheres. We identify three types of cellular structures which might be involved in tract formation. These are rows of glial cells at the medial brain margin, cellular bridges composed of neuronal somata and the epithelial surface of the foregut itself. The close proximity to the outgrowing axons suggests that the structures at the brain-foregut interface may play a role in the morphogenesis of embryonic brain tracts in Drosophila.

Differential expression in glial cells derived from chick embryo cerebral hemispheres at an advanced stage of development

Recently, we have characterized glial cultures derived from very early neurogenesis (E3) and found them to consist largely of early glioblastic or astroblastic cells with the capacity to differentiate into astrocytes given sufficient time in culture or with advancing age, i.e., cell passage. This study examines and compares the characteristics of astrocyte-enriched cultures derived from advanced embryonic ages (E15) in the chick embryonic cerebral hemispheres. We report several remarkable findings. 1) Mature astrocytes (GFAP+, vimentin-) appear as early as 5 days in vitro (DIV) in primary culture (P0). 2) Also apparent in primary cultures were extensive populations of neurons (neurofilament+; NF+) growing atop or in close proximity to mature astrocytes. 3) NF+ neurons disappeared after the first cell passage, and GFAP+ astrocytes were greatly diminished within two cell passages thereafter. 3) High concentrations of NGF were expressed, presumably by glial cells, in primary cultures through 14 DIV, declining to a low plateau through 27 DIV and remaining low, but measurable in subsequent cell passages. 4) At later cell passages (> 5) immature phenotypes of these same cell types continued to be expressed in E15CH cultures, i.e., positive staining for GFAP and vimentin and GFAP, GS, and NGF can all be detected on Western blots. We conclude from these findings that 1) mitotic multipotential neural cells are present within cerebral hemispheres even at late stages of development (E15); 2) neuroblasts and astroblasts have a reciprocal relationship requiring the presence of both cell types in order for mature expression of their phenotypes; 3) the NGF profile parallels the appearance and disappearance of neurons in E15 chick embryonic cerebral hemisphere primary cultures, strongly suggesting that this trophic factor may be involved in the mutually beneficial relationship between astrocytes and neurons.

Cultured rat neuronal and glial cells entrapped within hydrogel polymer matrices: a potential tool for neural tissue replacement

Cultured Schwann cells, neonatal astrocytes or cells dissociated from embryonic cerebral hemispheres were dispersed within poly-[ N-(2-hydroxypropyl)-methacrylamide]-based hydrogel matrices by gel entrapment and maintained in vitro for 1–6 days. Glial cells were pre-labelled with Hoechst 33342. Cell differentiation and viability were studied by immunocytochemistry. Up to 15% of Schwann cells initially entrapped within the polymer matrices were immunopositive for the low affinity nerve growth factor receptor, S100, glial fibrillary acidic protein (GFAP) and laminin; up to 10% of pre-labelled astrocytes were immunopositive for GFAP and laminin. Embryonic neurons displayed immunostaining for neurofilaments. Hydrogels containing entrapped Schwann cells were implanted into the rat neocortex. These polymers supported cellular and axonal ingrowth within parts of the polymer matrix.

Neuronal ganglioside increases dependent on the neuron--glia interaction in primary culture.

Dissociated neuronal cells from rat embryonic hemispheres were cultivated on astroglial layers. The increase in ganglioside content of the cocultures was more rapid than that of neuronal cultures seeded on polylysine surfaces for the first 24 h, and the extent of the increase was greater 7 days after inoculation, probably because of interaction between the preformed astroglial layers and the neuronal cells in vitro. The promoted expression of the a-pathway gangliosides, GM1 and GD1a, was recognized by TLC and the increase in GM1 was immunologically ascertained. The incorporation of 3H-labeled N-acetyl-D-mannosamine into GD3 and b-series gangliosides was elevated for the first 24 h. However, cocultures in which there was no contact between neuronal cells and the astroglial sheet showed no appreciable increase in incorporation. Thus, cell surface changes were induced at the membrane glycolipid level in the neuronal cells by contact with astroglial layers. The synthesis and expression of neuronal gangliosides are discussed in relation to the onset of neuron--glia interaction.

Catecholamine and indoleamine levels in chick cultured neurons and embryonic brain

Catecholamine and indoleamine levels were determined in cultured neurons from chick embryos and in the "homologous" embryonic cerebral hemispheres in order to study their neurotransmission systems. The seeding of a large number of cells resulted in a pure neuronal culture made of clusters interconnected by processes. Norepinephrine, which was absent from the starting material of the culture, appeared on the 2nd day and then decreased. A small amount of epinephrine was present on the 2nd day and decreased thereafter. Dopamine was not detected. In the cerebral hemispheres of chick embryos, dopamine appeared on the 10th day in ovo and increased steadily up to the 18th day. Epinephrine was also present in the cerebral hemispheres. Its level increased up to the 14th day and then decreased. Indoleamines were measured in the same material. The level of serotonin was markedly higher than that of catecholamines and it increased during cultivation. Tryptophan was already present in the starting material and its amount increased during cultivation. The level of 5-hydroxyindoleacetic acid changed like that of serotonin. In the embryonic cerebral hemispheres, the concentration of serotonin was highest on the 12th day after incubation and then decreased. Tryptophan level decreased steadily all during the embryogenesis. These results were discussed on the ground of differences in the synthesized neurotransmitters.

Subcultured astrocytes suppress the proliferation of neuroblasts in vitro

The effects of subcultured astrocytes on the proliferation of neuronal precursor cells (neuroblasts) from rat embryonic cerebral hemispheres were examined. The survivability of neurons and the neurite outgrowth were significantly enhanced by the subcultured astrocytes compared to those of neurons plated on poly- l-lysine-coated coverslips. The incorporation of [ 3H]thymidine into neuroblasts was remarkably suppressed by the subcultured astrocytes indicating that the astrocytes inhibit the proliferation of neuroblasts. These results suggest that astrocytes enhance the maturation of neuroblasts possibly via either cell-cell contact or trophic substances.

Septal deafferentation enhances the neurotrophic effects of rat hippocampus on cultured neural cells from the central nervous system

Neurotrophic effects (NTEs) of various brain regions of 4-week-old rats were examined in primary culture of rat embryonic cerebral hemispheres. Extracts of the hippocampus, brainstem and septal nucleus highly enhanced the survivability of neuronal cells and the division of non-neuronal cells by 9 days. The septohippocampal tract (fimbria fornix) was cut and the effect on the neurotrophic activity in the hippocampus was examined. The NTEs of hippocampal extracts remained unchanged 3 days after septal deafferentation, was significantly increased by 7 days, peaked at 14 days and returned to the basal level by 21 days.

Localization of ganglioside GM1 on myelin, in dissociated cells from rat embryonic cerebral hemispheres, using biotinylated choleragen and avidin peroxidase

The distribution of ganglioside GM1 in primary cultured cells from rat cerebral hemispheres was studied. These dissociated cell cultures are capable of forming myelin. 2′,3′-Cyclic nucleotide 3′-phosphodiesterase (CNPase) activity, which is considered to be a good marker for myelin, increased with the onset of myelin formation. Ganglioside GM1 was localized on the myelin-related structures using a biotinylated choleragen and avidin peroxidase technique. The distribution of ganglioside GM1 resembled that of immunostained CNPase.

Myelin formation in dissociated cell cultures of rat embryonic cerebral hemispheres.

Developing cultures of dissociated cerebral hemispheres obtained from 18-day-old embryonic rats synthesized, or activated, a myelin-related enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). This increase in activity coincided with the onset of myelination. The presence of CNPase in oligodendrocytes and myelin was demonstrated using immunocytochemical staining techniques. Myelinated axons and myelin sheaths were clearly made visible by electron microscopy of 28-day-old cultures.

Morphological maturation and survival of chicken and rat embryonic neurons on different culture substrata

Neuronal cells from chicken and rat embryonic cerebral hemispheres were plated at a low cell concentration and cultured either on collagen or on a supporting continuous glial layer for periods of up to 21 days. The glial layer was either homologous or heterologous with regard to the animal species; the survival and maturation of the neuronal cells in these different conditions were investigated by light and electron microscopy.Neuronal cells cultured on collagen formed aggregates similar to those formed by neuronal cells plated at high cell density as described in a previous paper; a few aggregated neurons formed processes after 24 h and, only after 48 h of culture, more fibres had developed; the majority of the cells progressively degenerated between days 7 and 21 of culture. In contrast to this, neuronal cells cultured on a supporting glial layer, whether homologous or heterologous, progressively differentiated: neuronal perikarya remained well separated from each other and many processes were already formed after 24 h; later on, networks of fibres developed. At the electron microscopic level, microtubules and neurofilaments were present at a high density in the cells and fibres; immature synapses could be found, but infrequently. Differentiated cells were represented mostly by neurons; oligodendroglial cells were absent, and myelinated fibres could not be detected.The highest positive effect on the maturation and survival of neuronal cells was observed in the presence of a layer of glial cells from the same species. These results emphasize the essential role of glial cells for the neuronal maturation in the absence of contact between neuroblasts.

Histological study of implantation in the Natal clinging bat (Miniopterus schreibersii natalensis).

In the Natal clinging bat, Miniopterus schreibersii natalensis, implantation is centric and superficial in the right uterine horn. The blastocyst implants during the bilaminar stage with the inner cell mass orientated antimesometrially. During the preimplantation period the blastocyst becomes completely enclosed by uterine mucosae with the trophoblast in close contact with the surrounding uterine epithelium. Just before implantation the trophoblast becomes bilaminar over the greater part of the embryonic hemisphere of the blastocyst. The uterine epithelium is very persistent and parts were still present mesometrially although penetration of the uterine endometrium had already occurred on the antimesometrial side.

Developmental formation and cortisol regulation of gamma-glutamyl transpeptidase in brain and spinal cord of chick embryo.

The developmentally conditioned changes in gamma-glutamyl transpeptidase (GGT) activity in chick embryonic hemispheres, optic lobes, cerebellum, brain stem and in lumbar and brachial enlargements of the spinal cord were studied. A marked increase in GGT activity was found in all tissues between embryonic day 11 and hatching. The application of cortisol enhanced GGT activity particularly on embryonic days 15 and 17. This effect of cortisol was reversed by the concomitant administration of actinomycin D or cycloheximide. The results are discussed in relation to the potential metabolic and transport role of GGT and its regulation in the developing chick embryonic brain.

Early development and implantation of the blastocyst in the bat,Hipposideros speoris (Schneider)—Hipposideridae—Microchiroptera

The embryo ofHipposideros speoris enters the uterus at the morula stage of development. The blastocyst implants superficially and circumferentially and the embryonic mass is so oriented during implantation that it faces between the antimesometrial and the lateral sides of the uterus. The most conspicuous progestational change in the uterus concerns the uterine glands which undergo hypertrophy and become wide thereby reducing the endometrial stroma to thin strands. During implantation the glands undergo destruction, accompanied by the destruction of endometrial cells, and the formation of symplasma. The trophoblast on the embryonic hemisphere is more invasive than that in the other regions of the blastocyst.

Subcellular distribution of glutamyltransferase activities in embryonic cerebral hemispheres

Glutamyltransferase (GT) activities were examined in cell groups from embryonic cerebral hemispheres. During normal development, GT activities were highest in a mixed population of small neurons and non-neuronal cells (small cell group). In culture, in response to hydrocortisone, activities exceeded normal levels only in this small cell group. In view of these findings, the small cell population was used to study the subcellular distribution of the enzyme. At the subcellular level, GT activities in the small cell group were distributed differentially but increased generally during normal development. In vitro, hydrocortisone promoted a precocious increase in GT activity, under experimental conditions regarded as optimum, only in the subcellular fraction characterized by synaptic endings.

Trophoblastic factors involved in lysis of the blastocyst coverings and in implantation in the rabbit: observations on inversely orientated blastocysts

ABSTRACT Lysis of the blastocyst coverings in the rabbit normally starts at the abembryonic hemisphere of the blastocyst facing the antimesometrial endometrium. Theoretically, this restricted effect could be caused by factors locally produced by abembryonic trophoblast, anti-mesometrial endometrium, or a combination of them. Blastocysts abnormally implanting with reversed orientation exhibit lysis of blastocyst coverings in the abembryonic hemisphere despite the absence of antimesometrial endometrium, and they do not show lysis in the embryonic hemisphere despite its contact with antimesometrial endometrium. Accordingly, it appears that lysis of blastocyst coverings is attributable to a factor localized in the abembryonic hemisphere of the blastocyst. Blastocyst protease activity was found to be maximal there in all blastocysts, whether oriented normally or reversed. The origin and role of this enzyme and other factors are discussed.

Distribution and development of glutamine synthetase in the embryonic cerebral hemisphere.

AbstractGlutamine synthetase (GS) activity is preferentially localized in a distinct cell mass, the ectostriatum, in cerebral hemispheres of late chick embryos. GS activities rise in the ectostriatum coincident with the growth of this prominent nucleus. GS development in the ectostriatum may be controlled by normal increases of 11β‐hydroxyl corticosteroids in late embryonic serum. It is clear in any case that GS activities in this area of the hemisphere are susceptible to precocious induction by these steroids in vitro. The significance of GS localization in the ectostriatum is discussed in terms of the possible normal roles for this enzyme in nervous tissue.