Bernard Pessac

Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella

Clonal permanent cell lines with astrocytic properties have been established from explant cultures of 8-day postnatal mouse cerebella after in vitro spontaneous transformation, i.e. without the addition of carcinogens or oncogenic viruses. The cell lines were derived in a multistage process. Slowly proliferating foci with several morphologies appeared 4 months after initiation of the cultures and became progressively enriched by cells with a homogeneous appearance. These cells could be established into permanent cell lines from which many clones were obtained. Some of these cloned cell lines bound anti-GFAP sera and therefore appeared to be astrocytic. According to their morphology, 3 separate types of these GFAP-positive clones could be distinguished. Type I and II cells had small somata; type I had several short processes, while type II had two processes, one of which was very thin and long (greater than 200 microns). Type III cells had large flat somata and no processes. The three types of clonal cell lines were labeled by monoclonal antibodies which bind to astrocytes in vivo. In particular, three monoclonal antibodies (BSP-3, M2 and M3) bound only to type II cells in a distinct pattern. Type I and II astrocytes are pseudodiploid and type III, heteroploid. The properties of these different clonal cell lines are very stable. We have thus obtained permanently established clonal cultures of mouse cerebellum astrocyte-like cells, which might be the in vitro counterparts of fibrous (type I), or velamentous (type III) astrocytes and of Golgi epithelial cells (type II).

Transformation of Chick Embryo Neuroretinal Cells by Rous Sarcoma Virus in vitro: Induction of Cell Proliferation

Neuroretinal cells from 7-day-old chick embryos are transformed and induced to proliferate after infection with Rous sarcoma virus in vitro. Susceptibility of neuroretinal cells to the virus is also dependent on the stage of development since infection of cells from 10-day-old embryos is uneffective.

Choline acetyl transferase activity in chick embryo neuroretinas during development in ovo and in monolayer cultures

Abstract The specific activity of the enzyme choline acetyl transferase (CAT) in chick neuroretinas was investigated during in ovo development and in monolayer cultures. The enzyme activity was barely detectable on the 6th day of incubation but increased markedly between the 7th and 11th days. The activity increased sharply between the 15th and 17th days and then slowly until hatching. When cell suspensions from 6- to 7-day neuroretinas were cultured as monolayers, CAT specific activity increased rapidly. After 4–5 days in culture, the activity of the enzyme was identical to that found in the neuroretina on the 11th day of incubation. Cells from 9-day neuroretinas also differentiate in monolayer cultures, but with a more irregular pattern. These data show that cholinergic neurons from chick embryo neuroretina differentiate in monolayer cultures without a lag and at the same rate as in vivo.

Growth stimulation of chick embryo neuroretinal cells infected with Rous sarcoma virus: Relationship to viral replication and morphological transformation

Abstract Neuroretinal (NR) cells from 7-day-old chick embryos infected with Rous sarcoma virus (RSV) are morphologically transformed, synthesize virus, and are induced to proliferate for several generations. By contrast, uninfected cells have a limited growth capacity and cannot be propagated in vitro . The relationship of induction of cell multiplication to viral replication and morphological transformation was analyzed by infecting NR cells with conditional and nonconditional transformation defective viruses. NR cells infected with a temperature-sensitive mutant of RSV defective for cell transformation, but not for virus replication, were induced to multiply at both nonpermissive and permissive temperatures, although expression of the transformed phenotype, as tested by several parameters, was suppressed in these cells at nonpermissive temperature. Nonconditional, nontransforming viruses replicated normally in NR cells, but failed to induce their multiplication. These results indicate that only transforming viruses can induce NR cell multiplication and that viral replication alone does not account for the growth changes in infected NR cells. These data also suggest that expression of the transformed phenotype and induction of NR cell proliferation may depend on distinct viral informations.

Expression of neuronal markers in chick and quail embryo neuroretina cultures infected with rous sarcoma virus

Cultures of neuroretina (NR) cells from 7-day chick and quail embryos were infected with ts NY-68, a thermosensitive mutant of Rous sarcoma virus (RSV) which transformed NR cells at 36 degrees C. The following differentiation markers for neurones were studied: tetanus toxin-binding sites at the cell surfaces, presence of synapses, and the specific activity of the enzymes choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD). Appearance of synapses and expression of CAT were similar in control and transformed cultures. Tetanus toxin-binding cells were observed in transformed primary cultures and also in quail NR subcultures. GAD-specific activity was markedly stimulated in chick and quail primary cultures transformed by ts NY-68 and further increased in subcultures of ts NY-68-transformed quail NR cells. Stimulation of GAD activity is controlled by the transforming (src) gene of RSV since it was not observed in cultures infected with RAV-1, a leukosis virus which lacks the src gene. These data show that infection of chick and quail NR cultures with RSV results in the transformation of cells with neuronal markers.

Uptake of γ-aminobutyric acid and glutamic acid decar☐ylase activity in chick embryo neuroretinas in monolayer cultures

We have previously reported that cells from 7-day chick embryo neuroretina (NR) can differentiate in monolayer cultures as shown by the appearance of mature neurones with characteristic synapses 2 and by the expression of choline-acetyltransferase specific activity a that reaches a level similar to that found in NR at an equivalent stage during in ovo development. Among several possible neurotransmitters, the presence of GABA has been demonstrated in the retina of various species including birds6,7,9,10,16. The cellular localization of GABA formation and uptake has been studied mainly by autoradiography of [3H]GABA in intact retina or in retina preparations. In rat retina, [aH]GABA was taken up only by Miiller cells 15 while in rabbit both neuronal and glial cells were labeled4, ~. On the other hand, in lower vertebrates and particularly in birds, [aH]GABA appeared incorporated into neuronesl0,13,20. Monolayer cultures of chick embryo NR may help elucidating the cellular localization of GABA synthesis and uptake. These cultures are made of neuronal cells that migrate and grow their processes on a bottom layer of flattened cells which probably represent glial, Mi~ller cells 2. After 2 weeks, neuronal cells begin to detach and after 3 or 4 weeks, only flat cells remained. Thus, monolayer cultures may allow to determine the role of neuronal and non-neuronal cells in GABA metabolism. We report here that differentiation of the GABA system occurs in chick embryo neuroretina in cell culture as measured by the glutamic acid decarboxylase (GAD) activity and by the GABA uptake. Furthermore, our data suggest that neuronal cells are mainly responsible for synthesis and uptake of GABA. Neuroretinas from 7-day White-Leghorn chick embryos were dissected, dissociated and cultured as described2, a. At this stage of embryonic life, differentiation is starting only in the ganglion cell layer while other NR cells are still unorganized and undifferentiated. The uptake of GABA in these cultures (Fig. 1) was measured at various intervals. Preliminary experiments done in cultures 4 days after plating showed that uptake of 5 × 10 -7 M [~H]GABA was rapid and linear during the first 10 min and maximal at 37 °C. In addition, the effect of GABA concentration on the

A novel cDNA corresponding to transcripts expressed in retina post-mitotic neurons

The long term objective of this study is to isolate genes specifically expressed at the onset of neuronal cell cycle withdrawal. As an experimental paradigm we have used a quail neuroretinal cell clone (clone K2) immortalized by a thermosensitive mutant of Rous Sarcoma Virus. K2 cells proliferate at 36 degrees C but stop synthesizing DNA after a shift to 41.5 degrees C. We have constructed a cDNA library from K2 cells transferred to 41.5 degrees C and autosubtracted with RNAs from K2 cells maintained at 36 degrees C. This strategy has led to the isolation of cDNAs which recognize mRNAs expressed in quail neuroretina (NR) during development. We report here one of these cDNAs, cDNA QN1, that hybridizes with transcripts expressed in retina neurons, in parallel with their withdrawal from the cell cycle. QN1 ORF codes for a 138 kDa polypeptide corresponding to the protein observed in Western blot analysis. A role of QN1 product(s) on neuronal quiescence is suggested by the positive effect of an antisense oligonucleotide on DNA synthesis of K2 cells.

Free Amino Acid Content of Astroglial Cell Clones Derived from 8‐Day Postnatal Mouse Cerebella

Abstract: We have measured the free amino acid content of three distinct astroglial cell clones derived from permanent lines obtained after “spontaneous immortalization” of 8‐day postnatal mouse cerebellar cultures; these clones show characteristics similar to the Golgi Bergmann glia cells, the fibrous astrocytes, and the velate protoplasmic astrocytes, i.e., the three main types of cerebellar astrocytes. The relative concentrations of amino acids that are thought to act as neurotransmitters were compared in confluent cultures of the different astroglial clones. The most striking result was a high concentration of glycine (20% of free amino acids), even in astroglial cells cultured in a glycine‐free medium, a finding suggesting that glycine is synthesized by the astroglial clones. Furthermore, no γ‐aminobutyric acid (GABA) was detected. In contrast, a “neuron‐like” clone derived from the same cerebellar culture contained GABA, whereas its glycine content was much lower than that of the astroglial clones. The present results, together with our previous finding of glycine synthesis in an astrocytic clone derived from 14‐day postnatal mouse cerebella transformed by simian virus 40, indicate that a high glycine content may be characteristic of many cerebellar astroglial types.

[5] Avian neuroretina cells in oncogene studies

Publisher Summary This chapter presents the two main reasons to use cells from the avian embryonic neuroretina (NR) as an experimental system to study the effects of oncogenes on regulation of cell growth and differentiation. First, NR cultures are exclusively composed of neuroectodermal cells: this tissue is not vascularized and does not contain cells of mesenchymal origin. Second, when these cells are maintained in culture, they rapidly stop dividing and express several markers of neural differentiation. The avian NR development proceeds through essentially three phases: proliferation of neuroectodermal precursor cells, lamination of cell strata, and differentiation of postmitotic cells. The chapter discusses that the NR cultures obtained from 7-day-old chicken embryos can be maintained as monolayers and are able to differentiate into neuronal and nonneuronal (glial) cells. This differentiation proceeds to the stage at which morphologically mature synapses are found. It, therefore, appears that synaptogenesis primarily depends on the genetic programs of the cells and that complex interactions, found in tridimensional structures, may not be required for the formation of synapses. The chapter also highlights that the changing culture conditions may influence the growth pattern of NR cells. Nr cells also provide a convenient model for in vitro studies on activation and transduction of protooncogenes by retroviruses.

Differentiation of retrovirus-infected avian neuroretina cells☆

The effects of oncogenic retroviruses on the expression of differentiation markers were studied in monolayer cultures of chick and quail embryo neuroretinas. Transformation by Rous sarcoma virus (RSV) did not affect the appearance of synapses, and the expression of glutamic acid decarboxylase was stimulated by pp60v-src, the product of the src gene. Quail embryo neuroretina cells transformed by Mill Hill 2 (which contains the two oncogenes v-mil and v-myc) were induced to proliferate into permanent cultures that synthesized crystallins and produced lentoid bodies. In contrast, transformation with a temperature-sensitive mutant of RSV reversibly blocked the production of crystallins and lentoid bodies. These data show that given cellular genes can respond differently to distinct oncogenes.