The v-myc oncogene is sufficient to induce growth transformation of chick neuroretina cells

A number of studies have shown that full transformation of non-established rodent fibroblasts can be efficiently achieved in vitro by the concerted action of two oncogenes belonging to different complementation groups. Extension of the two-genes carcinogenesis model to other differentiated cell types, presumably endowed with different controls of growth, is desirable for a better understanding of questions such as the host cell selectivity of oncogene action. A recent report claimed that cooperation between two oncogenes, v-myc and v-mil, is required to achieve transformation of chicken embryo neuroretina cells, which are characterized by a limited growth capacity in monolayer culture. Here we present evidence that the v-myc oncogene alone is sufficient to induce growth transformation of glial and neuronal precursor cell types from chick neuroretina. We also report that induction of transformation by v-myc is accompanied by faithful preservation of some of the differentiated functions of the chick cells.     

Astrocytes induce blood-brain barrier properties in endothelial cells

The highly impermeable tight junctions between endothelial cells forming the capillaries and venules in the central nervous system (CNS) of higher vertebrates are thought to be responsible for the blood-brain barrier that impedes the passive diffusion of solutes from the blood into the extracellular space of the CNS. The ability of CNS endothelial cells to form a blood-brain barrier is not intrinsic to these cells but instead is induced by the CNS environment: Stewart and Wiley demonstrated that when avascular tissue from 3-day-old quail brain is transplanted into the coelomic cavity of chick embryos, the chick endothelial cells that vascularize the quail brain grafts form a competent blood-brain barrier; on the other hand, when avascular embryonic quail coelomic grafts are transplanted into embryonic chick brain, the chick endothelial cells that invade the mesenchymal tissue grafts form leaky capillaries and venules. It is, however, not known which cells in the CNS are responsible for inducing endothelial cells to form the tight junctions characteristic of the blood-brain barrier. Astrocytes are the most likely candidates since their processes form endfeet that collectively surround CNS microvessels. In this report we provide direct evidence that astrocytes are capable of inducing blood-brain barrier properties in non-neural endothelial cells in vivo.     

Inability of Rous sarcoma virus to cause sarcomas in the avian embryo

The injection of Rous sarcoma virus (RSV) into the wing web of newly hatched chicks causes a rapidly growing sarcomatous tumour which is palpable within 1 week of inoculation; and cultures of fibroblasts derived from chick embryos (CEF) and infected with RSV become rapidly transformed. Genetic studies have determined that expression of a single viral gene, designated v-src, is necessary for neoplastic transformation. This gene codes for a 60,000-molecular weight phosphoprotein termed pp60SPC , which possesses a protein kinase activity that phosphorylates polypeptides on tyrosine residues and is constitutively expressed in infected CEF cells. It has been suggested that transformation, and possibly tumorigenesis, may result solely from the consequences of this increase in tyrosine phosphorylations. The pathogenicity of RSV in chick embryos in ovo is less clear. Murphy and Rous suggested that RSV may have caused tumours in "various tissues" of "some embryos", but the subsequent studies of Milford and Duran - Reynals , as well as several other laboratories, failed to find any evidence of intraembryonic tumours in RSV-infected early embryos. The findings of Duran - Reynals , if correct, cannot be explained easily in view of our present understanding of RSV tumorigenicity. Thus, we have re-examined the interaction of RSV with the avian embryo and confirm here that RSV is nontumorigenic and non-teratogenic when microinjected into day 4 chicken embryos. In addition, we found that (1) the virus not only replicates in the embryo, but it also expresses an active src-specific protein kinase and (2) once the cells from the infected limbs are disrupted and placed in culture, they are capable of expressing the transformed phenotype after a 24-h delay.     

Neurones express high levels of a structurally modified, activated form of pp60c-src

Neural tissues contain high levels of the cellular homologue of the transforming protein of Rous sarcoma virus (RSV), but neither the specific cell types expressing high levels of c-src, nor the function of the cellular src (c-src) protein has been determined. Using primary culture methods, we have found that pure neurones and astrocytes derived from the rat central nervous system (CNS) contain 15- to 20-times higher levels of the c-src protein than fibroblasts. However, the specific activity of the c-src protein from the neuronal cultures is 6- to 12-times higher than that from the astrocyte cultures. In addition, the c-src protein expressed in neuronal cultures contains a structural alteration within the amino-terminal region of the molecule that causes a shift in the mobility of the c-src protein on the SDS-polyacrylamide gels. These results indicate that a structurally distinct form of the cellular src protein that possesses an activated tyrosylkinase activity is expressed at very high levels in post-mitotic CNS neurones.     

鸟类胚胎消化道原基细胞分化的研究

本文用不同发育时期鹌鹑胚与鸡胚的种间移植实验结果表明,相当于鸡胚H.＆ H.17—23期的鹌鹑胚后肠原基尚不具备自主分化的潜能,只能并入寄主大肠发育,但不同区域的消化道中胚层间质细胞对内胚层细胞具有不同的诱导能力.相当于鸡胚H.＆ H.28—23期的鹌鹑胚后肠原基已具有自主分化的潜能,在寄主鸡胚体腔内能定向发育成鹌鹑大肠.本文还对移植系统中消化道原基细胞的分化进行了分析.     

Segmentation in the chick embryo hindbrain is defined by cell lineage restrictions

In the chick embryo hindbrain, morphological segmentation into rhombomeres is matched by metameric patterns of early neuronal differentiation and axonogenesis. Boundaries between rhombomeres coincide with boundaries of expression of murine regulatory genes. By clonal analysis using intracellular marking, we show here that the rhombomere boundaries are partitions across which cells do not move. When a parent cell is marked before the appearance of rhombomere boundaries, the resulting clone is able to spread into the neighbouring rhombomere. When marked after boundary appearance, the clone still expands freely within the rhombomere of origin, but it is now restricted at the boundaries. Rhombomeres in the chick embryo thus behave like polyclonal units, raising the possibility that they are analogous to the compartments of insects.     

Role of src gene in growth regulation of Rous sarcoma virus-infected chicken embryo fibroblasts

We report here a study of the mechanisms leading to loss of growth control in chicken embryo fibroblasts transformed by Rous sarcoma virus (RSV). We have been particularly concerned with the role of the src gene in this process, and have used RSV mutants temperature sensitive (ts) for transformation to investigate the nature of the growth regulatory lesion. The two principal findings were (1) the stationary phase of the cell cycle (G1) in chick embryo fibroblasts seems to have two distinct regulatory compartments (using the terminology of Brooks et al. we refer to these as 'Q' and 'A' states). When rendered stationary at 41.5 degrees C by serum deprivation, normal cells enter a Q state, but cells infected with the ts-mutant occupy an A state. (2) Whereas normal cells can occupy either state depending on culture conditions, the ts-infected cells, at 41.5 degrees C, do not seem to enter Q even though a known src gene product, a kinase, is reported to be inactive at this temperature. We discuss the possibility that viral factors other than the active src protein kinase influence growth control in infected cultures.     

Polarizing activity and retinoid synthesis in the floor plate of the neural tube

In many developing organisms the establishment of axial polarity and the patterning of cells depend on local signals that derive from restricted regions of the embryo. In vertebrate embryos, the origins of tissue polarity have been examined extensively in the developing limb. The anteroposterior pattern of the chick limb seems to be controlled by a morphogen, possibly retinoic acid, that is enriched in a region of the limb known as the zone of polarizing activity (ZPA). Certain tissues other than the ZPA have also shown polarizing activity experimentally in the chick limb, raising the possibility that signalling molecules involved in pattern formation in different embryonic tissues are conserved. Here we provide evidence that a similar polarizing activity is also present in a restricted region of the developing central nervous system (CNS). We show that a specialized group of neural cells termed the floor plate, but not other regions of the CNS, mimics the ZPA in respecifying the digit pattern in the developing chick limb. In addition, using an in vitro biochemical assay, we show that the floor plate can synthesize retinoic acid and 3,4-didehydroretinol, the precursor of a second morphogenetically active retinoid, 3,4-didehydroretinoic acid. These results show that the floor plate is a local source of a ZPA-like polarizing signal, possibly a retinoid, which may regulate the pattern of cell differentiation in the developing CNS.     

Inhibitory neurones of a motor pattern generator in Xenopus revealed by antibodies to glycine

Glycine and gamma-aminobutyric acid (GABA) are inhibitory transmitters of major importance. Whereas neurones using GABA as the transmitter can be visualized by immunocytochemical methods for glutamate decarboxylase (GAD) or GABA, no comparable techniques have been available for the selective visualization of glycinergic neurones. We have now produced polyclonal antibodies which specifically recognize glycine in glutaraldehyde-fixed tissue. We used these antibodies to investigate the distribution of glycine in the simple central nervous system (CNS) of the Xenopus embryo, which contains an anatomically and physiologically defined class of reciprocal inhibitory interneurones, the commissural interneurones. These interneurones have an important role in the generation of the swimming motor pattern and are thought to be glycinergic. The glycine antibodies specifically stain these interneurones, revealing their distribution and number in the embryo CNS. This is the first demonstration of the selective localization of glycine-like immunoreactivity in a putative glycinergic class of neurone that has been characterized physiologically, pharmacologically and anatomically.     

Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina

In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.     

Molecular cloning, expression and regional distribution of rat ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) was originally characterized as a survival factor for chick ciliary neurons in vitro. More recently, it was shown to promote the survival of a variety of other neuronal cell types and to affect the differentiation of E7 chick sympathetic neurons by inhibiting their proliferation and by inducing the expression of vasoactive intestinal peptide immunoreactivity (VIP-IR). In cultures of dissociated sympathetic neurons from newborn rats, CNTF induces cholinergic differentiation as shown by increased levels of choline acetyltransferase (ChAT). This increase is paralleled by a reduction of tyrosine hydroxylase (TH) activity. Moreover, CNTF promotes the differentiation of bipotential 02A progenitor cells to type-2-astrocytes in vitro. To help establish which, if any, of these functions CNTF exerts in vivo, it is necessary to determine its primary structure, cellular expression, developmental regulation and localization. The complementary DNA-deduced amino-acid sequence and subsequent expression of cDNA clones covering the entire coding region in HeLa-cells indicate that CNTF is a cytosolic protein. This, together with its regional distribution and its developmental expression, show that CNTF is not a target-derived neurotrophic factor. CNTF thus seems to exhibit neurotrophic and differentiation properties only after becoming available either by cellular lesion or by an unknown release mechanism.     

Retrograde axonal transport of target tissue-derived macromolecules

Neurones depend on contact with their target tissues for survival and subsequent development. The protein, nerve growth factor (NGF), can be selectively taken up by sympathetic nerve terminals and reaches the neuronal perikaryon by a process of retrograde intra-axonal transport, suggesting that its role in vivo is to act as a target tissue-derived trophic factor. The development of the neurones of the chick ciliary ganglion requires the presence of structures derived from the optic cup. Several studies in vitro have shown that media conditioned by non-neuronal cells contain factors that result in the survival of neurones from ciliary ganglia. In particular, chick embryo iris, ciliary body and choroid contained large amounts of these factors indicating the presence of a target tissue-derived trophic factor for the cholinergic ciliary ganglion. This study demonstrates that neurones of the ciliary ganglion accumulate, by retrograde intra-axonal transport, proteins synthesized and released by optic tissues in culture.     

Cell determination and regulation during development of neuroblasts and neurones in grasshopper embryo

The embryonic development of the central nervous system (CNS) involves the generation of an enormous diversity of cellular types arranged and interconnected in a remarkably precise pattern. In each hemisegment of the grasshopper embryo, the ectoderm generates a stereotyped pattern of 30 neuronal precursor cells, called neuroblasts (Fig. 1). Each of these stem cells makes a stereotyped contribution of 6-100 progeny to the approximately 1,000 different neurones, each cell identifiable according to its unique morphology, physiology and biochemistry. What are the contributions of cell interactions and cell lineage to the generation of this diversity and specificity of identified neurones in the grasshopper CNS? Here we report on cell ablations with a laser microbeam at different stages of development. Our results suggest the importance of cell-cell interactions in the determination of ectodermal cells to become identified neuroblasts. However, once a neuroblast begins to divide, then cell lineage appears to play an important role in the determination of its stereotyped family of neuronal progeny. Furthermore, cell-specific interactions continue to play an important role as neurones, according to their mitotic ancestry, recognize and interact with other differentiating neurones in their environment.     

Prevention of natural motoneurone cell death by dibutyryl cyclic GMP

Natural neuronal cell death is a well-described developmental phenomenon common to many nerve centres in a variety of animal species. Neuronal survival has been shown to depend on the presence and size of the available target tissue and it has been suggested that neuronal survival is dependent on successful competition for either a limited number of synaptic sites or a limited amount of trophic factor(s). In the lateral motor column of the lumbar spinal cord in the chick embryo, the period of axon elongation and innervation of the periphery has been shown to precede that of natural motoneurone cell death. While muscle contractile activity appears to regulate the extent of motoneurone death, to date the intracellular molecular events that initiate and regulate the developmental process of natural neuronal cell death or, more importantly, neuronal survival are unknown. Our earlier studies suggested that either contact or association between spinal cord processes and muscle cells during neuromuscular junction formation in vivo leads to an increase in cyclic GMP in whole spinal cord. We now show that treatment of chick embryos with the membrane-permeable cyclic GMP analogue, dibutyryl cyclic GMP during the period of natural motoneurone cell death prevents greater than 58% of natural motoneurone cell death in the lumbar lateral motor column.     

Induction of proliferation or transformation of neuroretina cells by the mil and myc viral oncogenes

The genome of the avian retrovirus MH2 contains, in addition to the v-myc oncogene 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 vitro. 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 cells. 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-mil is sufficient to induce NR cell proliferation, although it does not result in cell transformation. In addition, viruses expressing only the v-myc oncogene 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.     

Neuropeptides modulate the beta-adrenergic response of purified astrocytes in vitro

Neuropeptides may have functions in the central nervous system (CNS) other than altering neuronal excitability. For example, they may act as regulators of brain metabolism by affecting glycogenolysis. Since it has been suggested that glial cells might provide metabolic support for neuronal activity, they may well be one of the targets for neuropeptide regulation of metabolism. Consistent with this view are reports that peptide-containing nerve terminals have been seen apposed to astrocytes, but it is also quite possible that peptides could act at sites lacking morphological specialization. Primary cultures containing CNS glial cells have been shown to respond to beta-adrenergic agonists with an increase in cyclic AMP and, as a result, with an increase in glycogenolysis and have also been shown to respond to a variety of peptides with changes in cyclic AMP. In the study reported here, we have examined the effects of several peptides on relatively pure cultures of rat astrocytes. We demonstrate that the increase in intracellular cyclic AMP induced by noradrenaline is markedly enhanced by somatostatin and substance P and is inhibited by enkephalin, even though these peptides on their own have little or no effect on the basal levels of cyclic AMP. Vasoactive intestinal peptide (VIP) on the other hand increases cyclic AMP in the absence of noradrenaline. These results suggest that neuropeptides influence glial cells as well as neurones in the CNS and, in the case of somatostatin and substance P, provide further examples of neuropeptides modulating the response to another chemical signal without having a detectable action on their own.     

东亚飞蝗（Locusta migratoria manilensis）卵巢和胚胎组织的原代培养

用东亚飞蝗４～５龄蝗蝻卵巢组织及孵化１２ｄ的胚胎组织，首次在我们改良的ＴＣ－１９９－ＭＫ和ＴＣ－１００培养基中，２８℃恒温培养，第１０ｄ可见大量透明的梭形细胞从卵巢块周围逸出，第１６０ｄ可见大量圆球状的胚胎细胞分裂成团，来自两种组织的细胞分别培养９０ｄ和３３０ｄ，均贴壁生长，但胚胎细胞贴壁不牢。从培养结果看，它们发展成连续细胞系的潜力很大。     

Origin of cells giving rise to mesoderm and endoderm in chick embryo.

In amniotes, all of the tissues of the adult arise from the epiblast, one of the two layers of cells present in the early embryo; the mesoderm and gut endoderm arise from an epiblast-derived structure known as the primitive streak. The monoclonal antibody HNK-1 recognizes the cells of the primitive streak in the chick embryo. Before streak formation, HNK-1 identifies cells that are randomly distributed within the epiblast. We have now used two novel ways to study cell lineage and commitment to show that the epiblast of the early chick embryo contains two distinct populations of cells with different developmental fates at a stage during which 'mesodermal induction' is believed to occur. One cell population, recognized by monoclonal antibody HNK-1, is destined to form mesoderm and endoderm; the rest of the epiblast is unable to give rise to mesoderm if this population of cells is removed.     

Retinal ganglion cells lose response to laminin with maturation

The decisive role played by adhesive interactions between neuronal processes and the culture substrate in determining the form and extent of neurite outgrowth in vitro has greatly influenced ideas about the mechanisms of axonal growth and guidance in the vertebrate nervous system. These studies have also helped to identify adhesive molecules that might be involved in guiding axonal growth in vivo. One candidate molecule is laminin, a major glycoprotein of basal laminae which has been shown to induce a wide variety of embryonic neurones to extend neurites in culture. Moreover, laminin is found in large amounts in injured nerves that can successfully regenerate but is absent from nerves where regeneration fails. However, it is unclear to what extent the mechanisms that regulate axonal regeneration also operate in the embryo when axon outgrowth is initiated. Here we have examined the substrate requirements for neurite outgrowth in vitro by chick embryo retinal ganglion cells, the only cells in the retina to send axons to the brain. We show that while retinal ganglion cells from embryonic day 6 (E6) chicks extend profuse neurites on laminin, those from E11 do not, although they retain the ability to extend neurites on astrocytes via a laminin-independent mechanism. This represents the first evidence that central nervous system neurones may undergo a change in their substrate requirements for neurite outgrowth as they mature.