David I. Gottlieb

CYP26, a Novel Mammalian Cytochrome P450, Is Induced by Retinoic Acid and Defines a New Family

A novel member of the cytochrome P450 superfamily, CYP26, which represents a new family of cytochrome P450 enzymes, has been cloned. CYP26 mRNA is up-regulated during the retinoic acid (RA)-induced neural differentiation of mouse embryonic stem cells in vitro and is transiently expressed by embryonic stem cells undergoing predominantly non-neural differentiation. CYP26 transcript is detectable as early as embryonic day 8.5 in mouse embryos, suggesting a function for the gene in early development. CYP26 is expressed in mouse and human liver, as expected for a cytochrome P450, and is also expressed in regions of the brain and the placenta. Acute administration of 100 mg/kg all-trans-RA increases steady-state levels of transcript in the adult liver, but not in the brain. CYP26 is highly homologous to a Zebrafish gene, CYPRA1, which has been proposed to participate in the degradation of RA, but is minimally homologous to other mammalian cytochrome P450 proteins. Thus, we report the cloning of a member of a novel cytochrome P450 family that is expressed in mammalian embryos and in brain and is induced by RA in the liver.

The Effects of Soluble Growth Factors on Embryonic Stem Cell Differentiation Inside of Fibrin Scaffolds

The goal of this research was to determine the effects of different growth factors on the survival and differentiation of murine embryonic stem cell‐derived neural progenitor cells (ESNPCs) seeded inside of fibrin scaffolds. Embryoid bodies were cultured for 8 days in suspension, retinoic acid was applied for the final 4 days to induce ESNPC formation, and then the EBs were seeded inside of three‐dimensional fibrin scaffolds. Scaffolds were cultured in the presence of media containing different doses of the following growth factors: neurotrophin‐3 (NT‐3), basic fibroblast growth factor (bFGF), platelet‐derived growth factor (PDGF)‐AA, ciliary neurotrophic factor, and sonic hedgehog (Shh). The cell phenotypes were characterized using fluorescence‐activated cell sorting and immunohistochemistry after 14 days of culture. Cell viability was also assessed at this time point. Shh (10 ng/ml) and NT‐3 (25 ng/ml) produced the largest fractions of neurons and oligodendrocytes, whereas PDGF (2 and 10 ng/ml) and bFGF (10 ng/ml) produced an increase in cell viability after 14 days of culture. Combinations of growth factors were tested based on the results of the individual growth factor studies to determine their effect on cell differentiation. The incorporation of ESNPCs and growth factors into fibrin scaffolds may serve as potential treatment for spinal cord injury.

On the Distribution of Axonal Terminals Containing Spheroidal and Flattened Synaptic Vesicles in the Hippocampus and Dentate Gyrus of the Rat and Cat

SummaryA quantitative analysis has been made of the distribution of presynaptic profiles containing round (or spheroidal) and flattened (or ellipsoidal) synaptic vesicles in the apical and basal dendritic zones and in the layer of pyramidal cell somata of fields CA1 and CA3 of the hippocampus, and in the molecular and granular layers of the dentate gyrus of the rat and cat.In the apical and basal dendritic zones of fields CA1 and CA3 the overwhelming majority of the synapses are of the asymmetrical variety, the axon terminals ending principally upon dendritic spines, and to a lesser extent upon the shafts and secondary or tertiary branches of the dendrites. Between 1 and 8% of the axon terminals in these zones contained flattened vesicles: all of these formed symmetrical contacts upon medium-sized or large dendritic shafts. In the molecular layer of the dentate gyrus a slightly higher percentage of flattened vesicle containing profiles was observed (∼10%); again these formed symmetrical contacts upon dendritic shafts. In the stratum pyramidale of the hippocampal fields and the stratum granulosum of the dentate gyrus of the rat, flattened vesicle containing synapses are two or three times more numerous than those with spheroidal vesicles. In the cat hippocampus the axosomatic synapses are about equally distributed between those containing round, and those with flattened vesicles.The finding that at the focus of post-synaptic inhibition, at the level of the pyramidal cell somata, the majority of the axon terminals contains flattened synaptic vesicles, whereas in the region of termination of the extrinsic, commissural and long association pathways (all of which are excitatory) virtually all the synapses contain round vesicles, strongly supports the view that endings containing flattened vesicles mediate post-synaptic inhibition in the hippocampal formation.

An in vitro Pathway from Embryonic Stem Cells to Neurons and Glia

Mouse embryonic stem (ES) cells can be induced to differentiate into neurons and glia in vitro. Induction protocols are straightforward and involve culture in the presence of retinoic acid. They result in an efficient conversion of undifferentiated ES cells to neural cells. Mature neurons produced have the key physiological, morphological and molecular properties of primary cultured neurons derived from the central nervous system. Most significantly, they form functional chemical synapses that utilize either glutamate, GABA or glycine as neurotransmitters. ES cell-derived glial cells also correspond well with their normal counterparts. During induction, ES cells undergo a series of developmental steps that resemble key stages in the early mouse embryo. This supports the hypothesis that the in vitro pathway is a valid model of the normal developmental pathway leading to neurons and glia. The in vitro system combines three experimental strengths. It is suitable for genetic manipulation, affords large numbers of cells and allows precise manipulation of the culture environment. It is thus suitable for a wide variety of mechanistic studies in the areas of neural development and cell biology.

A novel assay of neuronal cell adhesion.

Intact cells can be made to adhere to a glass surface which is derivitized with γ-aminopropyl-triethoxysilane, and then treated with glutaraldehyde. Such cells remain fully viable and a monolayer of such cells can be formed within 1 hour after the cells are removed from an embryo. The rate of binding of radioactively labeled single cells to such a monolayer can be used as a measure of intercellular adhesion (Walther, Ohman and Roseman, Proc. Natl. Acad. Sci. USA 70, 1569–1573 (1973). The lack of toxicity of this surface is shown by the fact that it supports normal growth of C-6 rat glial cells.

Localization of the NGFI-A protein in the rat brain

Antibodies are used to localize the NGFI-A protein in the rat brain. The protein is found in a wide variety of neurons. However, not all neurons are stained. The protein is either absent or present at undetectable levels in glial cells. Neuronal nuclei stain intensely, cytoplasmic staining is lighter. Seizures cause a detectable increase in the intensity of staining.

A subset of ES-cell-derived neural cells marked by gene targeting.

Embryonic stem cells differentiate efficiently in culture into neural progenitors, neurons, oligodendrocytes, and astrocytes. An embryonic stem (ES) cell line with green fluorescent protein (GFP) inserted into the gene for Olig2, a lineage‐specific transcription factor, permits visualization and physical separation of a subset of living ES‐cell‐derived neural cells. GFP‐expressing cells have morphological and antigenic properties of the oligodendrocyte lineage. The differentiation of living GFP‐expressing cells can be followed in cultures, and they can be separated by fluorescence‐activated cell sorting and cultured as pure populations. This system will allow detailed biochemical and molecular analysis of a neural differentiation pathway at a level not previously feasible. The strategy may have general applicability, since other neural lineages can be marked in an analogous manner.

A monoclonal antibody which binds to the surface of chick brain cells and myotubes: cell selectivity and properties of the antigen.

A monoclonal antibody has been obtained which binds to the cell surface of cultured chick myotubes and retinal and tectal neurons but not fibroblasts, myoblasts and embryonic liver cells. Indirect immunocytochemistry reveals that antigen is present in all layers of the chick neural retina. The antibody therefore recognizes an antigen common to most, if not all, chick neurons. The antigen has been identified by staining SDS gels with [125I]monoclonal antibody and appears to be a polydisperse collection of polypeptides with molecular weights centered about 250 kdalton.

Expression of the genes coding for glutamic acid decarboxylase in pluripotent cell lines

The expression of glutamic acid decarboxylase (GAD) is a basic characteristic of a wide array of inhibitory neurons the use gamma-aminobutyric acid as a neurotransmitter. Clonal cell models will be essential for investigating the mechanisms which are responsible for the selective expression of GAD. P19 embryonal carcinoma cells are an important model for the analysis of neuronal gene expression. Depending on culture conditions, undifferentiated cells can be induced to form cells as widely divergent as cardiac muscle-like cells and neuron-like and glial-like cells. P19 cells are amendable to a number of powerful genetic manipulations including transformation with foreign DNA and selection of mutants. In this study we used nuclease protection assays and Northern blot analysis to determine if P19 cells express the GAD1 and GAD2 genes. The results show that uninduced P19 cells express these genes at very low but easily detectable levels. When the cells are induced to differentiate along the neuronal pathway with retinoic acid, the levels of transcripts for both GAD genes rise dramatically. At least some RNA transcripts of both genes from induced cells comigrate with the corresponding mRNA from the brain and thus probably represent processed mRNA. The expression of GAD genes in undifferentiated cultures of embryonal stem (ES) cells was also investigated. These cultures express levels of GAD1 transcripts that are higher than uninduced P19 cells. In contrast, expression of the GAD2 gene was barely detectable. These results indicate that P19 EC cells and ES cells will be useful for the investigation of the mechanisms that regulate the expression of the GAD1 and GAD2 genes.

Neurons derived from embryonic stem (ES) cells resemble normal neurons in their vulnerability to excitotoxic death

We determined whether embryonic stem (ES) cells could provide a model system for examining neuronal death mediated by glutamate receptors. Although limited evidence indicates that normal neurons can be derived from mouse ES cells, there have been no studies examining pathophysiological responses in mouse ES cell systems. Mouse ES cells, induced down a neural lineage by retinoic acid (RA), were found to have enhanced long-term survival when plated onto a layer of cultured mouse cortical glial cells. In these conditions, the ES cells differentiated into neural cells that appeared normal morphologically and displayed normal features of immunoreactivity when tested for neuron-specific elements. Varying the culture medium generated cultures of mixed neuronal/glial cells or enriched in oligodendrocytes. These cultures were viable for at least four weeks. Real-time PCR analysis of N-methyl-D-aspartate (NMDA) receptor subunits revealed an appropriate age-in-vitro dependent pattern of expression. Neurons derived from ES cells were vulnerable to death induced by a 24-h exposure to the selective glutamate receptor agonists NMDA, kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). This vulnerability to agonist-induced death increased with age in vitro, and related closely to expression of receptor subunits, as it does in cultured primary neurons. Experiments with selective receptor antagonists showed that glutamate receptors mediated the NMDA- and kainate-induced death. Neuronal differentiated ES cells therefore exhibited an excitotoxic response resembling that displayed by central nervous system (CNS) neurons. Thus, ES cells, which are very amenable to genetic manipulation, provide a valid system for studying glutamate receptor-mediated toxicity at the molecular level.