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Non‐dividing neuroretina cells from chicken embryos are induced to proliferate after a long latency, following infection with Rous associated virus type 1, an avian retrovirus which does not carry a transforming gene. We have isolated from these proliferating cells an acutely mitogenic retrovirus, designated IC10, which contains a novel oncogene. Nucleotide sequencing showed that the IC10 virus has transduced 1101 nucleotides of cellular origin inserted between the gag and env genes of RAV‐1. This oncogene, designated v‐Rmil, is 70.1% homologous to v‐mil. v‐Rmil encodes a protein of 40,976 daltons sharing 83.8% homology with the catalytic domain of the v‐mil protein. Divergence with the v‐mil gene product is observed at the NH2‐ and COOH‐terminal portions of the v‐Rmil protein. Restriction analysis of normal chicken DNA indicated that v‐Rmil is derived from a cellular gene distinct from c‐mil. The c‐Rmil gene is transcribed through a major mRNA, greater than 10 kb in length, that is detected at much higher levels in neuroretinas, as compared to other embryonic tissues.

Rous sarcoma virus (RSV) stimulates in quail embryo neuro‐retina (NR) cultures the specific activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of gamma‐aminobutyric acid, a major inhibitory neurotransmitter in NR and in central nervous system. In quail embryo NR cultures transformed by ts NY‐68, a thermodependent transformation‐defective mutant of RSV, stimulation of GAD activity is regulated by pp60v‐src, the product of the src gene of RSV. Fibroblasts and myoblasts have a very low GAD activity that is not stimulated after transformation by RSV. Neuronal clones, previously derived from ts NY‐68‐transformed established NR cell lines, have a high GAD activity which is regulated by pp60v‐src, while other clones have a low GAD activity apparently not regulated by pp60v‐src. These data indicate that pp60v‐src selectively activates the expression of GAD in distinct neuronal cells of quail embryo NR cultures transformed by RSV. GAD activity is also stimulated in NR cells infected with viruses containing v‐mil.

The lens-specific proteins alpha and delta crystallins and lentoid bodies, structures that follow a differentiation pathway similar to that of the lens, regularly appear after 4 to 5 weeks in quail embryo neuroretina monolayer cultures. We have investigated the effects of the avian oncogenic retroviruses Mill Hill 2 and Rous sarcoma virus on this process. Quail embryo neuroretina cells transformed by Mill Hill 2 virus were established into permanent cultures that synthesized alpha and delta crystallins and contained stem cells for the production of lentoid bodies. In contrast, transformation with the Rous sarcoma virus mutant tsNY-68 blocked the appearance of mRNA crystallins, but cytoplasmic alpha and delta crystallin mRNA and alpha crystallin appeared 44 h after a shift to the nonpermissive temperature. However, delta crystallins and lentoid bodies were only present after 7 days. The crystallins of transformed quail neuroretina cultures were immunologically indistinguishable from those of quail lenses and of normal quail embryo neuroretina cultures.

Non‐dividing neuroretina cells from chicken embryos are induced to proliferate after a long latency, following infection with Rous associated virus type 1, an avian retrovirus which does not carry a transforming gene. We have isolated from these proliferating cells an acutely mitogenic retrovirus, designated IC10, which contains a novel oncogene. Nucleotide sequencing showed that the IC10 virus has transduced 1101 nucleotides of cellular origin inserted between the gag and env genes of RAV‐1. This oncogene, designated v‐Rmil, is 70.1% homologous to v‐mil. v‐Rmil encodes a protein of 40,976 daltons sharing 83.8% homology with the catalytic domain of the v‐mil protein. Divergence with the v‐mil gene product is observed at the NH2‐ and COOH‐terminal portions of the v‐Rmil protein. Restriction analysis of normal chicken DNA indicated that v‐Rmil is derived from a cellular gene distinct from c‐mil. The c‐Rmil gene is transcribed through a major mRNA, greater than 10 kb in length, that is detected at much higher levels in neuroretinas, as compared to other embryonic tissues.

Neuroretinas from 6–7 day-old chick embryos were cultivated after trypsin dissociation as monolayer cultures in Petri dishes, and examined after various intervals of time with the electron microscope. Soon after plating, cells begin to reaggregate in small clumps, and typical rosettes are formed. During the first week in vitro, cells appear to differentiate as neuroblasts and presumed Müller cells; the latter form a continous sheet on the substrate, upon which neuroblasts migrate and grow their neurites. Differentiated ribbon synapses are found after 8 days in vitro, the time at which they normally appear in situ. After 15 and 21 days in vitro, synapses are still found in large numbers, mimicking their “in vivo” counterparts. Photoreceptor cells were identified on the basis of the presence of typical ribbons in their cytoplasm, but no outer segment was found. It appears then that synaptogenesis in the retina is programmed independently of the tissue environment, which is markedly disturbed in the monolayer culture.

Single cell suspensions, prepared from brain stem, cerebellum, and forebrain parenchyma of embryonic and adult mice, were plated on monolayers of an astroglial cell line derived from a spontaneously immortalized mouse cerebellar culture, the D19 clone. A few of the brain cells adhering to the D19 monolayers were immunoreactive to the Mac-1 antibody, which labels all cells of the monocytic and granulocytic lineages. The Mac-1-positive cells proliferated vigorously and later most of them acquired the F4/80 epitope specific for macrophages and microglia cells. Studies in clonal conditions allowed development of large colonies of about 2 x 10(5) cells that expressed typical microglia markers. Bone marrow Mac-1-positive cells cocultured on D19 monolayers were also induced to proliferate, whereas peritoneal macrophages were not. D19 astrocytes express macrophage colony-stimulating factor (CSF-1) activity at a high level, and their conditioned media induced the proliferation of brain and bone marrow Mac-1-positive cells. A specific anti-CSF-1 antiserum completely blocked bone marrow macrophage progenitor proliferation and significantly reduced the multiplication of microglial precursors induced by the D19-conditioned medium. These data indicate that the embryonic and adult mouse brain parenchyma contains potential progenitors for microglial cells.

We report the isolation and characterization of a novel cDNA from quail neuroretina encoding a putative protein named nectinepsin. The nectinepsin cDNA identifies a major 2.2-kilobase mRNA that is detected from ED 5 in neuroretina and is increasingly abundant during embryonic development. A nectinepsin mRNA is also found in quail liver, brain, and intestine and in mouse retina. The deduced nectinepsin amino acid sequence contains the RGD cell binding motif of integrin ligands. Furthermore, nectinepsin shares substantial homologies with vitronectin and structural protein similarities with most of the matricial metalloproteases. However, the presence of a specific sequence and the lack of heparin and collagen binding domains of the vitronectin indicate that nectinepsin is a new extracellular matrix protein. Furthermore, genomic Southern blot studies suggest that nectinepsin and vitronectin are encoded by different genes. Western blot analysis with an anti-human vitronectin antiserum revealed, in addition to the 65- and 70-kDa vitronectin bands, an immunoreactive protein of about 54 kDa in all tissues containing nectinepsin mRNA. It seems likely that the form of vitronectin found in chick egg yolk plasma by Nagano et al. ((1992) J. Biol. Chem. 267, 24863-24870) is the protein that corresponds to the nectinepsin cDNA. This new protein could be an important molecule involved in the early steps of the development.

The avian neuroretina (NR) is part of the central nervous system and is composed of photoreceptors, neuronal cells, and Müller (glial) cells. These cells are derived from proliferating neuroectodermal precursors that differentiate after terminal mitosis and become organized in cell strata. Genes that are specifically expressed at the various stages of retinal development are presently unknown. We have isolated a quail (Coturnix coturnix japonica) cDNA clone, named QR1, encoding a 676-amino acid protein whose carboxyl-terminal portion shows significant similarity to those of the extracellular glycoprotein osteonectin/SPARC/BM40 and of the recently described SC1 protein. The QR1 cDNA identifies a mRNA detected in NR but not in other embryonic tissues examined. The levels of this mRNA are markedly reduced when nondividing NR cells are induced to proliferate by the v-src oncogene. QR1 expression in NR is limited to the middle portion of the inner nuclear layer, a localization that essentially corresponds to that of Müller cells. Transcription of QR1 takes place only during the late phase of retinal development and is shut off sharply at hatching. Signals that regulate this unique pattern of expression appear to originate within the NR, since the QR1 mRNA is transcribed in cultured NR cells and is shut off also in vitro at a time coinciding with hatching.

A major question in central nervous system development, including the neuroretina, is whether migrating cells express cues to find their way and settle at specific locations. We have transplanted quail neuroretinal cell lines QNR/D, a putative amacrine or ganglion cell, and QNR/K2, a putative Müller cell into chicken embryo eyes. Implanted QNR/D cells migrate only to the retinal ganglion and amacrine cell layers and project neurites in the plane of retina; in contrast, QNR/K2 cells migrate through the ganglion and amacrine layers, locate in the inner nuclear layer, and project processes across the retina. These data show that QNR/D and QNR/K2 cell lines represent distinct neural cell types, suggesting that migrating neural cells express distinct address cues. Furthermore, our results raise the possibility that immortalized cell lines can be used for replacement of specific cell types and for the transport of genes to given locations in neuroretina.

We report that nondividing neuroretina cells from chicken embryos can be induced to proliferate following infection with Rous-associated virus type 1 (RAV-1), an avian lymphomatosis retrovirus lacking transforming genes. Multiplication of RAV-1-infected neuroretina cells is observed after a long latency period and takes place initially in a small number of cells. We also show that serial virus passaging onto fresh neuroretina cultures leads to the generation of novel mitogenic viruses containing the mil oncogene. DNA analysis indicated that RAV-1 is the only provirus detected in cells infected at virus passage 1, whereas neuroretina cells infected at subsequent virus passages harbor mil-containing proviruses. Three viruses, designated IC1, IC2, and IC3, were molecularly cloned. Restriction mapping indicated that in each virus, truncated c-mil sequences were inserted within different portions of the RAV-1 genome. In addition, IC1 and IC2 viruses have transduced novel sequences that belong to the 3' noncoding portion of the c-mil locus. All three viruses induce neuroretina cell multiplication and direct the synthesis of mil-specific proteins. Proliferation of neuroretina cells infected at passage 1 of RAV-1 was not associated with any detectable rearrangement of c-mil, when a v-mil probe was used. However, these cells expressed high levels of an aberrant 2.8-kilobase mRNA hybridizing to mil but not to a long terminal repeat probe. Therefore, transcriptional activation of a portion of c-mil could represent the initial events induced by RAV-1 infection and lead to retroviral transduction of activated c-mil sequences.

Rous sarcoma virus (RSV) stimulates in quail embryo neuro‐retina (NR) cultures the specific activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of gamma‐aminobutyric acid, a major inhibitory neurotransmitter in NR and in central nervous system. In quail embryo NR cultures transformed by ts NY‐68, a thermodependent transformation‐defective mutant of RSV, stimulation of GAD activity is regulated by pp60v‐src, the product of the src gene of RSV. Fibroblasts and myoblasts have a very low GAD activity that is not stimulated after transformation by RSV. Neuronal clones, previously derived from ts NY‐68‐transformed established NR cell lines, have a high GAD activity which is regulated by pp60v‐src, while other clones have a low GAD activity apparently not regulated by pp60v‐src. These data indicate that pp60v‐src selectively activates the expression of GAD in distinct neuronal cells of quail embryo NR cultures transformed by RSV. GAD activity is also stimulated in NR cells infected with viruses containing v‐mil.

The genome of the avian retro virus MH2 contains, in addition to the v-myconcogene shared with three other avian retroviruses (MC29, CMII and OK-10), a second cell-derived oncogene, v-mil(refs 1–3). Like the three other viruses, which contain only v-myc, MH2 induces mainly liver and kidney carcinomas in fowl and transforms fibroblasts and macrophages in vitro4. However, MH2 and MC29 differ in their biological properties when assayed on cultures of chicken embryo neuroretina (NR) cells. Indeed, NR cells, which normally do not multiply in vitro, are induced to proliferate and become transformed upon infection with MH2, whereas infection with MC29 has no apparent effect on these cells5,6. To analyse the functions of the two oncogenes of MH2, we isolated spontaneous and in vitro-constructed mutants of this virus and investigated their effects on NR cell multiplication and transformation. We report here that expression of v-milis sufficient to induce NR cell proliferation, although it does not result in cell transformation. In addition, viruses expressing only the v-myconcogene fail to induce any detectable change in NR cells. However, cooperation of the two oncogenes is required to achieve transformation of NR cells by MH2.

The binding of125I-insulin to uncloned and cloned cultures of mouse retinal cells has been investigated. At 15° C, binding of the hormone reached a steady state by 60 min, while at 37° C equilibrium was reached earlier but at a lower level than at 15° C. Porcine insulin, porcine proinsulin and guinea pig insulin displaced labelled insulin in proportion to their known biological potency. A sharp pH dependence of the hormone binding was observed with an optimum at pH 7.8. The dissociation rate of the125I-insulin was increased in the presence of unlabelled hormone, suggesting the existence of negative cooperativity in the insulin-receptor interaction. The availability of established retinal cell lines with insulin receptors should facilitate the study of the insulin-retina interactions in a controlled in vitro system.