CEPU-1 expression in the early embryonic chick brain

We describe the expression pattern of CEPU-1, a cell adhesion molecule of the immunoglobulin superfamily, in the early chick embryo brain. An initially broad domain of expression, encompassing forebrain, midbrain and anterior hindbrain, is subsequently narrowed down to a ring-shaped domain at the midbrain-hindbrain boundary, co-localizing precisely with the expression of Wnt1 at the isthmus. In addition, CEPU-1 is expressed in the dorsal aspect of rhombomere 4 and its emigrating neural crest cells. Later in development, we also find CEPU-1 expression in other parts of the developing nervous system such as sensory ganglia and in the ventral aspect of forebrain, midbrain and hindbrain.     

Acrylamide alters neurofilament protein gene expression in rat brain

Acrylamide, a prototype neurotoxin, alters neurofilament protein (NF) gene expression in rat brain. Levels of mRNA coding for neurofilament protein subunits NF-L, NF-M, and NF-H have been determined by Northern blot analysis using³²P-labeled cDNA probes. Acrylamide given acutely (100 mg/kg, single intraperitoneal injection) causes a selective increase in NF-M mRNA (approximately 50%) compared to controls. The expression of NF-L or NF-H mRNA is not affected by acrylamide. In contrast, chronic treatment with acrylamide [0.03% (w/v) in drinking water for 4 weeks] induces a modest but significant increase (approximately 22%) in NF-L mRNA compared to controls. Levels of NF-M, and NF-H mRNA are not altered by acrylamide treatment. The expression of -actin mRNA, an ubiquitous protein, is not affected by either treatment regimen of acrylamide. The results of this study show that acrylamide increases the expression of mRNA for NF protein subunits in rat brain. The increase of specific mRNA for NF subunits depends on the dose, duration and route of acrylamide administration.     

FGF2 plays a key role in embryonic cerebrospinal fluid trophic properties over chick embryo neuroepithelial stem cells

During early stages of brain development, neuroepithelial stem cells undergo intense proliferation as neurogenesis begins. Fibroblast growth factor 2 (FGF2) has been involved in the regulation of these processes, and although it has been suggested that they work in an autocrine-paracrine mode, there is no general agreement on this because the behavior of neuroepithelial cells is not self-sufficient in explants cultured in vitro. In this work, we show that during early stages of development in chick embryos there is another source of FGF2, besides that of the neuroepithelium, which affects the brain primordium, since the cerebrospinal fluid (E-CSF) contains several isoforms of this factor. We also demonstrate, both in vitro and in vivo, that the FGF2 from the E-CSF has an effect on the regulation of neuroepithelial cell behavior, including cell proliferation and neurogenesis. In order to clarify putative sources of FGF2 in embryonic tissues, we detected by in situ hybridization high levels of mRNA expression in notochord, mesonephros and hepatic primordia, and low levels in brain neuroectoderm, corroborated by semiquantitative PCR analysis. Furthermore, we show that the notochord segregates several FGF2 isoforms which modify the behavior of the neuroepithelial cells in vitro. In addition, we show that the FGF2 ligand is present in the embryonic serum; and, by means of labeled FGF2, we prove that this factor passes via the neuroepithelium from the embryonic serum to the E-CSF in vivo. Considering all these results, we propose that, in chick embryos, the behavior of brain neuroepithelial stem cells at the earliest stages of development is influenced by the action of the FGF2 contained within the E-CSF which could have an extraneural origin, thus suggesting a new and complementary way of regulating brain development.     

Developmental changes in phosphorylation state of neurofilament proteins in the chick embryonic optic nerve

Developmental changes in the phosphorylation state of neurofilament proteins (NFPs) in the chick embryonic optic nerve were histochemically and biochemically studied using monoclonal antibody (MAb) 82E10 specific to the highly phosphorylated components of high (180K)- and middle (160K)-molecular-weight subunits of neurofilament (NF) in the chicken. Cross sections of developing embryonic optic nerve were studied by enzyme immunohistochemistry using this MAb. The staining pattern showed marked changes with the developmental stage. In 6-day embryos (E6) the entire cross section was stained, whereas in E10 only about a ventroposterior half of the cross section was stained. In E14 nearly the entire area of the cross section became unstained. Thereafter, the immunoreactivity reappeared and gradually increased, such that in E20 the entire cross section became immunopositive again. Electrophoretic and immunoblot analyses were made on optic nerves dissected out of embryos of various stages. The 82E10 immunoreactivity at the position of NF-M underwent a transient loss in E14 in parallel with the time course of histochemical change. Two-dimensional gels stained for protein further showed that the highly phosphorylated form of NF-M is transiently lost from embryonic optic nerve in E14, while the less phosphorylated form persists throughout the embryonic developmental stages. In order to understand the orderly loss of the 82E10 immunoreactivity in relation to retinotopic and chronotopic organizations of the fibers in the embryonic optic nerve, retinal injection of a fluorescent dye DiI as an anterograde tracing marker for selected fibers was utilized. An ordered arrangement of the fibers was present within the embryonic optic pathway, suggesting that the orderly loss of the 82E10 immunoreactivity in the embryonic optic nerve reflects the chronological order of the optic axons. These changes in the phosphorylation state of NFPs in the embryonic optic nerve presumably reflect dynamic changes of the neuronal cytoskeleton at certain stages during development.     

Synthesis of sterols by chick brain and liver during embryonic development

The evolution throughout embryonic development of the rate at which acetate was converted into sterols was studied in chick brain and liver. Acetate incorporation (nmol/h/g tissue) was clearly higher in brain than in liver and sharply decreased with the age of embryo. Cholesterol and desmosterol were the major sterols formed from acetate by chick embryo brain, followed by lanosterol and squalene. No desmosterol was found in chick embryo liver, organ where cholesterol was the major sterol synthesized. In brain, the relative percentage of cholesterol increased throughout embryonic development reaching more than 50% at hatching, while the percentage of desmosterol decreased during the same period and represented at hatching only about 10–15% of the total nonsaponifiable fraction. The relative percentages of lanosterol and squalene did not change significantly throughout the period assayed. In liver, the percentage of cholesterol increased until 19 days but sharply decreased at hatching.     

Transient expression of GAP-43 in nonneuronal cells of the embryonic chicken limb.

Growth associated protein (GAP)-43 is a membrane-bound phosphoprotein expressed in neurons and is particularly abundant during periods of axonal outgrowth in development and regeneration of the nervous system. In previous work, we cloned a full-length chicken GAP-43 cDNA and described the expression of its corresponding mRNA during early development of the chicken nervous system. We report here that the GAP-43 mRNA is also expressed transiently in developing limbs of chicken embryos, which contain axons of spinal cord and dorsal root ganglion neurons, but do not contain neuronal cell bodies. GAP-43 mRNA was first detectable by RNA blot analysis in limbs from Embryonic Day 5 (E5) embryos, reached maximal levels between E6 and E8, and diminished by E10. In situ hybridization analysis showed that the GAP-43 mRNA was localized in distal regions of developing limbs and was particularly abundant in the mesenchyme surrounding the digital cartilage. In some regions of the limb, GAP-43 immunoreactivity colocalized in cells that were also immunoreactive for meromyosin, a muscle-specific marker. These data suggest that both GAP-43 mRNA and the protein are expressed in nonneuronal cells of the developing limb, some of which may be part of the muscle cell lineage.     

pp60c-src expression in transdifferentiating cultures of embryonic chick neural retina cells

Chick embryo neural retinal cells transdifferentiate extensively into lens cells when cultured in Eagle's MEM containing horse and fetal calf sera (FHMEM). Such cultures express elevated levels of pp60c-src-associated tyrosine kinase activity relative to parallel cultures prevented from transdifferentiating by the addition of supplementary glucose (FHGMEM) or replacement of MEM by medium 199 (F199). Northern blotting and in vitro translation studies suggest that c-src mRNA levels are only slightly higher in late transdifferentiating (FHMEM) cultures as compared to parallel blocked (FHGMEM or F199) cultures. By immunocytochemical staining, we show that pp60c-src protein is largely localized in cell groups undergoing conversion into lens (i.e. expressing delta crystallin) in late FHMEM cultures. Initial studies of pp60c-src in chick lens tissues during development indicate that higher kinase activity is found in the epithelial cells relative to mature lens fibres. Thus pp60c-src may be expressed both during the differentiation of lens cells in vivo and during the transdifferentiation of neural retina cells into lens in vitro.     

Effect of cytochalasin B on the surface of neuroepithelial cells of the chick embryo

Neuroepithelial cells of young chick embryos, exposed in vitro for 1.5 and 13hr to cytochalasin B (5μg/ml) and examined by scanning electron microscopy, show changes in their surface morphology. The cell surface loses its cytoplasmic projections, its ruffle appearance and acquires a great number of cavities. These effects of the drug are not reversible after 4hr incubation on control media. Our results show that cytochalasin B does affect the cell surface morphology in our specimens but they give no indication as to manner in which the drug inhibits interkinetic nuclear migration.     

Distinct patterns of gene expression induced by viral oncogenes in human embryonic brain cells

1. The limited lifespan of human embryonic brain (HEB) cells hampers their therapeutic use for the treatment of neurodegenerative diseases.2. Stable expression of SV40 large T antigen (LTA) or E6E7 genes of human papillomavirus type 16 significantly increased the lifespan of HEB cells, but did not induce transformation.3. The extended lifespan was triggered by changes in the expression of antiproliferative genes. We found that changes in the expression of p16 (INK4a), p21 (WAF1), p14ARF, and p53 tumor suppressor gene, but not p27 (Kip1), differed between the LTA- and E6E7-HEB cells.4. Despite the induction of p53 RNA, p53 protein was undetectable in HEB-E6E7 cells. In contrast, p53 protein was increased in HEB-LTA cells as compared with the parental cells. Expression of p21 was, however, reduced in both cell lines.5. While p16 was decreased in HEB-E6E7 cells, its expression was increased in HEB-LTA cells.6. Despite these changes, HEB cell lines showed neuron-like morphological differentiation when the intracellular level of cAMP was elevated.7. This suggests that the mechanisms for inducing neuronal differentiation are still intact in HEB-E6E7 and HEB-LTA cells. More importantly, differentiation signals can override the effects of viral oncogenes in HEB cells.     

Characterization and regulation of insulin binding by embryonic chick heart cells

We have compared insulin binding by heart cells at 7 and 14 days of development. Species specificity, optimum pH, temperature relationships, and time to equilibrium for binding of insulin were the same in both 7 and 14-day systems. Curvilinear Scatchard plots for chicken insulin binding were demonstrated. Binding affinities and capacities were calculated based on a two-receptor model including a specific high-affinity receptor and a less specific low-affinity receptor that bound insulin and other growth peptides. Apparent association constants (K_A) were 4.0 and 0.05 nM^?1 and binding capacities were 600 and 9000 sites per cell for high- and low affinity receptors, respectively. We have also investigated the ability of insulin to regulate binding to its own receptors. Chick heart cells from 7- and 14-day embryos, cultured for 44 hr in insulin-enriched medium (3.4 μM), bound 50% less insulin (down-regulated) than control cells. At both developmental stages, down regulation was primarily a reduction in binding to the high-affinity receptor. The low-affinity receptor was less susceptible to down regulation and retained its ability to mediate maximal insulin stimulation of amino acid transport.     

Stereological study of the early ultrastructural differentiation of chick embryo neuroepithelial cells during neurulation

The neuroectodermal cells of chick embryos have been analyzed during neurulation by stereological and morphometrical ultrastructural methods in an attempt to describe their cytometric evolution. A profound change of cellular form coefficient was observed which is related to the typical process of columnarization of these cells. At stages 7 and 8, the nucleus appeared round in shape, probably due to a loss of pressure of the vitelline inclusions. In this sense, the volume density of these inclusions falls during this period. There was also a significant increase of the nuclear surface density, the significance of which is discussed on the basis of the nucleo-cytoplasmic interchanges and the differentiation process. At the same time, an increase in the number of mitochondria was observed, which is related to the neural folding process. Simultaneously, the amount of rough endoplasmic reticulum increases, presumably related to the remarkable changes of the embryonic extracellular matrix.