Purification of DNA complementary to nucleotide sequences required for neoplastic transformation of fibroblasts by avian sarcoma viruses.

We have prepared radioactive DNA (cDNA_sarc) complementary to nucleotide sequences which represent at least a portion of the viral gene(s) required for neoplastic transformation of fibroblasts by an avian sarcoma virus. The genetic complexity of cDNA_sarc (~1600 nucleotides) is sufficient to represent an entire cistron. The genomes of three independent isolates of avian sarcoma viruses share nucleotide sequences closely related to cDNA_sarc, whereas the sequences are absent from transformation-defective mutants of avian sarcoma viruses, several avian leukosis viruses, a non-pathogenic endogenous virus of chickens (Rous-associated virus-O), sarcoma-leukosis viruses of mice and cats, and mouse mammary tumor virus. We conclude that the transforming gene(s) of all avian sarcoma viruses have closely related or common genetic lineages distinct from the transforming genes in sarcoma viruses of other species. Our results conform to previous reports that transformation-defective variants of avian sarcoma viruses are mutants with identical regions deleted from each subunit of a polyploid genome.     

The size and genetic composition of virus-specific RNAs in the cytoplasm of cells producing avian sarcoma-leukosis viruses.

The genome of avian sarcoma virus (ASV) contains four known genes: gag, encoding structural proteins of the viral core; pol, encoding the viral RNA-directed DNA polymerase; env, encoding the glycoprotein(s) of the viral envelope; and src, which is responsible for neoplastic transformation of the host cell. We have located these genes on virus-specific RNAs in cells productively infected with both nondefective and defective strains of ASV by using molecular hybridization with DNAs complementary to specific portions of the ASV genome.The cytoplasm of cells producing nondefective ASV contains three species of polyadenylated virus-specific RNA, each of which has chemical polarity identical to that of the viral genome. The largest species has a molecular weight of 3.3 × 10⁶ daltons and a sedimentation coefficient of 38S, encodes all four viral genes, and is probably identical to the viral genome. A second species has a molecular weight of 1.8 × 10⁶ daltons and a sedimentation coefficient of 28S, and encodes the 3′ half of the viral genome, including env, src and a genetically silent region known as “c.” The smallest species has a molecular weight of 1.2 × 10⁶ daltons and a sedimentation coefficient of 21S, and encodes only src and “c.” All three species of virus-specific RNA contain nucleotide sequences at least partially homologous to a sequence of 101 nucleotides found at the extreme 5′ end of the ASV genome. This sequence may not be present in the portions of the ASV genome which encode the 28S and 21S virus-specific RNAs, and hence may be joined to these RNAs during their maturation from precursor molecules.The size and genetic composition of virus-specific RNAs in cells producing defective deletion mutants reflect the nature of the deletion. Deletions of either src or env eliminate the 28S virus-specific RNA, leaving a 21S RNA (which contains either env and “c” in the case of src deletions or src and “c” in the case of env deletions) and a 35S RNA which is probably identical to the viral genome.Based on these and related results, we propose a model for viral gene expression which conforms to previous suggestions that eucaryotic cells initiate translations only at the 5′ termini of messenger RNAs.     

Acquisition of new DNA sequences after infection of chicken cells with avian myeloblastosis virus

DNA-RNA hybridization studies between 70S RNA from avian myeloblastosis virus (AMV) and an excess of DNA from (i) AMV-induced leukemic chicken myeloblasts or (ii) a mixture of normal and of congenitally infected K-137 chicken embryos producing avian leukosis viruses revealed the presence of fast- and slow-hybridizing virus-specific DNA sequences. However, the leukemic cells contained twice the level of AMV-specific DNA sequences observed in normal chicken embryonic cells. The fast-reacting sequences were two to three times more numerous in leukemic DNA than in DNA from the mixed embryos. The slow-reacting sequences had a reiteration frequency of approximately 9 and 6, in the two respective systems. Both the fast- and the slow-reacting DNA sequences in leukemic cells exhibited a higher T_m (2 C) than the respective DNA sequences in normal cells. In normal and leukemic cells the slow hybrid sequences appeared to have a T_m which was 2 C higher than that of the fast hybrid sequences. Individual non-virus-producing chicken embryos, either group-specific antigen positive or negative, contained 40 to 100 copies of the fast sequences and 2 to 6 copies of the slowly hybridizing sequences per cell genome. Normal rat cells did not contain DNA that hybridized with AMV RNA, whereas non-virus-producing rat cells transformed by B-77 avian sarcoma virus contained only the slowly reacting sequences. The results demonstrate that leukemic cells transformed by AMV contain new AMV-specific DNA sequences which were not present before infection.     

Characterization of a normal avian cell protein related to the avian sarcoma virus transforming gene product.

In this paper, we identify and characterize both structurally and functionally a protein from normal uninfected avian cells that is antigenically related to the pp60^src viral protein responsible for transformation by ASV. This protein was detected by immunoprecipitation of radiolabeled normal cell extracts with serum derived from marmosets bearing ASV-induced tumors. The normal avian cell protein, which has been detected in each of the four avian species tested (chicken, duck, quail and pheasant) is a phosphoprotein of 60,000 daltons. This protein is not related to any of the ASV structural proteins; however, its immunoprecipitation is prevented by preadsorption of the antiserum with cell extracts specifically containing pp60^src. Peptide analyses by partial proteolysis using chymotrypsin resulted in a map of the normal cell protein that was very similar to that of pp60^src. When Staphylococcus aureus V8 protease was used, however, one of the major cleavage products of the normal cell protein exhibited an altered migration with respect to the corresponding pp60^src product. Tryptic phosphopeptide analyses demonstrated that phosphorylation of the normal cell protein was also different from that seen in pp60^src. The expression of the normal cell protein did not seem to be affected by cellular growth conditions, maintaining a constant level which was approximately 30–50 fold lower than that of pp60^src in infected cells. The normal cell protein appeared to be functionally dissimilar to pp60^src lacking detectable protein kinase activity in the currently available assay system.     

Translocation of latex beads after laser ablation of the avian neural crest

Previous studies from this laboratory (M. E. Bronner-Fraser, 1982, Dev. Biol.91, 50–63) have demonstrated that latex beads translocate ventrally after injection into avian embryos during the phase of neural crest migration, to settle in the vicinity of neural-crest-derived structures. In order to examine the role of host neural crest cells in the ventral translocation of implanted beads, latex beads have been injected into regions of embryos from which the neural crest cells have been ablated using a laser microbeam. Prior to their migratory phase, neural crest cells reside in the dorsal portion of the neural tube. Laser irradiation of the dorsal neural tube was used to reproducibly achieve either partial or complete ablation of neural crest cells from the irradiated regions. The effectiveness of the ablation was assessed by the degree of reduction in dorsal root ganglia, a neural crest derivative. Because of the rapidity and precision of this technique, it was possible to selectively remove neural crest cells without significantly altering other embryonic structures. The results indicate that, after injection of latex beads into the somites of embryos whose neural crest cells were removed by laser irradiation, the beads translocate ventrally in the absence of the endogenous neural crest.     

Characterisation and germline transmission of cultured avian primordial germ cells

Background

Avian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds.

Principal Findings

We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring.

Conclusions

The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.     

Ammonia elicits a different myogenic response in avian and murine myotubes

Increased myostatin expression, resulting in muscle loss, has been associated with hyperammonemia in mammalian models of cirrhosis. However, there is evidence that hyperammonemia in avian embryos results in a reduction of myostatin expression, suggesting a proliferative myogenic environment. The present in vitro study examines species differences in myotube and liver cell response to ammonia using avian and murine-derived cells. Primary myoblasts and liver cells were isolated from embryonic day 15 and 17 chick embryos to be compared with mouse myoblasts (C2C12) and liver (AML12) cells. Cells were exposed to varying concentrations of ammonium acetate (AA; 2.5, 5, or 10 mM) to determine the effects of ammonia on the cells. Relative expression of myostatin mRNA, determined by quantitative real-time PCR, was significantly increased in AA (10 mM) treated C2C12 myotubes compared to both ages of chick embryonic myotube cultures after 48 h (P < 0.02). Western blot analysis of myostatin protein confirmed an increase in myostatin expression in AA-treated C2C12 myotubes compared to the sodium acetate (SA) controls, while myostatin expression was decreased in the chick embryonic myotube cultures when treated with AA. Myotube diameter was significantly decreased in AA-treated C2C12 myotubes compared to controls, while avian myotube diameter increased with AA treatment (P < 0.001). There were no significant differences between avian and murine liver cell viability, assessed using 2′, 7′- bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein, acetoxymethyl ester, when treated with AA. However, after 24 h, AA-treated avian myotubes showed a significant increase in cell viability compared to the C2C12 myotubes (P < 0.05). Overall, it appears that there is a positive myogenic response to hyperammonemia in avian myotubes compared to murine myotubes, which supports a proliferative myogenic environment.     

Differential expression of the cellular src gene during vertebrate development

Cellular genes that are homologous to the transforming genes of certain RNA tumor viruses are suspected to play a functional role during normal developmental processes. To investigate this further, we are studying the expression of the cellular homolog of the Rous sarcoma virus transforming gene (c-src) during embryogenesis of fish, frog, and chicken by quantitative determination of the activity of the c-src encoded protein kinase (pp60^c-src). The kinase activity from embryos of fish, frog, and chicken displays the same enzymatic characteristics as the kinase from adult animals: It phosphorylates only tyrosine residues in protein substrates, and its activity is relatively insensitive to inhibition by the diadenosine nucleotide Ap4A. During the course of development, the varying kinase activity level reflects differential expression of the c-src gene product. The kinase activity is low during early development, increases dramatically during organogenesis, and decreases thereafter to the level found in adult animals. The kinase activity displays an organ specificity, with brain showing the highest activity in embryos as well as in adults. Muscle, however, shows high activities during organogenesis, but no or barely detectable activity in adult animals. Our data suggest, therefore, that the c-src gene product plays more of a role in differentiation than in proliferation processes during embryogenesis, and that it may act as a pleiotropic effector.     

Changes in amino-terminal sequences of pp60src lead to decreased membrane association and decreased in vivo tumorigenicity

We have suggested previously that the amino-terminal 8 kilodaltons of pp60^src may serve as a structural hydrophobic domain through which pp60^src attaches to plasma membranes. Two isolates of recovered avian sarcoma viruses (rASVs), 1702 and 157, encode pp60^src proteins that have alterations in this amino-terminal region. The rASV 1702 src protein (56 kilodaltons) and the 157 src protein (62.5 kilodaltons) show altered membrane association, and fractionate largely as soluble, cytoplasmic proteins in aqueous buffers, in contrast with the membrane association of more than 80% of the src protein of standard avian sarcoma virus under the identical fractionation procedure. Plasma membranes purified from cells transformed by these rASVs contain less than 10% of the amount of pp60^src found in membranes purified from cells transformed by Rous sarcoma virus or control rASVs. The altered membrane association of these src proteins had little or no effect on the properties of chick embryo fibroblasts transformed in monolayer culture. In contrast, rASV 1702 showed reduced in vivo tumorigenicity compared with Rous sarcoma virus or with other rASVs that encode membrane-associated src proteins. Rous sarcoma virus-induced tumors are malignant, poorly differentiated sarcomas that are lethal to their hosts. rASV 1702 induces a benign, differentiated sarcoma that regresses and is not lethal to its hosts. These data support the role of amino-terminal sequences in the membrane association of pp60^src, and suggest that the amino terminus of pp60^src may have a critical role in the promotion of in vivo tumorigenicity.     

Transformation parameters in chicken fibroblasts transformed by AEV and MC29 avian leukemia viruses.

Infection of chicken fibroblasts with avian erythroblastosis virus (AEV) strain ES4 or with avian myelocytomatosis virus strain MC29 leads to a rapid morphological transformation of most cells. AEV-transformed fibroblasts are similar to Rous sarcoma virus (RSV)-transformed fibroblasts in that they exhibit microvilli at their surface, show a disappearance of actin cables, are agglutinable by lectins, and show a decrease in LETS protein and an increase in the rate of hexose uptake. They also elicit slightly increased levels of cell-associated proteolytic activity, but show no increase in the fibrinolytic activity of the harvest fluids. In addition, as shown previously, they are capable of anchorage-independent growth and of sarcoma induction.In contrast, MC29-transformed fibroblasts express a different pattern of transformation parameters. They are similar to both RSV- and AEV-transformed fibroblasts in that they are morphologically transformed, show a disappearance of actin cables and are agglutinable by lectins. They also elicit surface alterations which consist of bleb-like protrusions rather than of microvilli, and are capable of anchorage-independent growth. They are strikingly different from RSV- and AEV-transformed cells, however, in that they express normal levels of LETS protein and elicit no increase in the rate of hexose uptake or in proteolytic activity. They are not sarcomagenic although they show an accelerated growth rate in culture.In conjunction with the finding that MC29 and AEV do not contain sequences related to the fibroblast-transforming src gene of RSV, these results raise the possibility that MC29 and perhaps also AEV transform fibroblasts by a mechanism different from RSV.     

The src gene product (pp60 src) of avian sarcoma virus rapidly induces DNA synthesis and proliferation of calcium-deprived rat cells

A general characteristic of neoplastic cells, but not their non-neoplastic counterparts, is the ability to proliferate in calcium-deficient medium. NRK cells infected with the transformation-defective, temperature-sensitive, ASV mutant, tsLA23, were unable to proliferate in calcium-deficient medium at the non-permissive 40°C, but they very rapidly initiated DNA synthesis (within 1 hour) and resumed proliferation in this medium after being shifted to 36°C, a temperature permissive for the production of active pp60^src and for neoplastic transformation. These observations suggest that activated pp60^src acts near the $\text{G1}\text{S}$ transition point in the cell cycle to bypass or stimulate a calcium-dependent mechanism required for the initiation of DNA synthesis, which enables the cells to display the neoplastic property of proliferating in calcium-deficient medium.     

Robust and ubiquitous GFP expression in a single generation of chicken embryos using the avian retroviral vector,RCASBP

Functional genomics in avian models has lagged behind that of mammals, and the production of transgenic birds has proven to be challenging and time-consuming. All current methods rely upon breeding chimeric birds through at least one generation. Here, we report a rapid method for the ubiquitous expression of GFP in chicken embryos in a single generation (G-0), using the avian retroviral vector, Replication-Competent Avian sarcoma-leukosis virus, with a Splice acceptor, Bryan RSV Pol (RCASBP). High-titre RCASBP retrovirus carrying eGFP was injected into unincubated (stage X) blastoderms in ovo. This resulted in stable and widespread expression of eGFP throughout development in a very high proportion of embryos. Transgenic tissues were identified by fluorescence and immunohistochemistry. These results indicate that chicken blastodermal cells are permissive for infection by the RCASBP virus. This system represents a rapid and efficient method of producing global gene expression in the chicken embryo. The method can be used to generate avian cells with a stable genetic marker, or to induce global expression of a gene of choice. Interestingly, in day 8.5 embryos, somatic cells the embryonic gonads were predominantly GFP positive but primordial germ cells were GFP negative, indicating viral silencing in the embryonic germline. This dichotomy in the gonads allows the isolation or enrichment of the germ cells through negative selection during embryonic stages. This transgenic chicken model is of value in developmental studies, and for the isolation and study of avian primordial germ cells.     

Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo

Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.     

The erbB gene of avian erythroblastosis virus is a member of the src gene family

The erbB gene of an avian erythroblastosis virus, AEV-H, was determined to be 1812 nucleotides long and was predicted to code for a protein of 67,638 daltons. Unexpectedly, a sequence of 285 amino acids in the middle of the protein showed a significant homology (38%) with the sequence in the carboxy terminus of p60^src. The nucleotide sequence of a mutant of AEV-H, td-130, which induces sarcomas but not erythroblastosis in chicken, was also analyzed. A deletion of 169 nucleotides was identified in the 3′ half of the erbB gene, indicating that the gene codes for a truncated protein with the predicted molecular weight of 46,667. These findings suggest that the homologous domain of erbB protein with its N-terminal portion is sufficient for the transformation of fibroblasts and that one-third of the carboxy-terminal domain has a key role for the transformation of erythroid cells.