Rudolf Jaenisch

Chromosomal position and activation of retroviral genomes inserted into the germ line of mice

The exogenous Moloney leukemia virus (M-MuLV) was inserted into the germ line of mice by exposing embryos to virus at different stages of embryogenesis. Mice derived from exposed embryos were mosaics with respect to integrated virus. Nine new substrains, designated Mov-5 to Mov-13, were derived, each of which carries a single M-MuLV genome at a different chromosomal position in its germ line. Four substrains, Mov-1 to Mov-4, were derived previously. Restriction enzyme analyses demonstrated that, with the exception of Mov-4 and Mov-6 mice, no major rearrangements or deletions have occurred in the integrated proviral genomes. Infectious virus is not activated in the majority of substrains (Mov-4 to Mov-8 and Mov-10 to Mov-12), whereas the other mice develop viremia. A detailed comparison between Mov-1 and Mov-13 mice demonstrated that the time of virus activation is different. Mov-13 mice activate infectious virus during embryogenesis, leading to a distinct pattern of virus expression in all tissues of the adult, but the viral genome in Mov-1 mice is activated only during the first two weeks after birth, leading to virus expression predominantly in lymphatic organs. Together with previous observations, at least four different phenotypes of virus expression-that is, early virus activation during embryogenesis, virus activation after birth, virus activation late in life and no expression of infectious virus at all-can be distinguished among the 13 substrains. Our results suggest that the chromosomal region at which a viral genome is integrated influences its expression during development and differentiation.

Embryonic lethal mutation in mouse collagen I gene causes rupture of blood vessels and is associated with erythropoietic and mesenchymal cell death

The role of collagen I for midgestation development was studied in homozygous Mov 13 embryos, which cannot synthesize alpha 1(1) mRNA as a result of insertional mutagenesis and most of which die between day 12 and 14 of gestation. No type I collagen was detected in mutant embryos, while the distribution of other collagens, laminin, and fibronectin was not affected. Mutant embryos develop normally up to day 12 of gestation, suggesting that collagen I has no essential role in the early phase of morphogenesis. The first pathological events were detected in hemopoietic cells of the liver, followed by necroses of mesenchymal cells in other parts of the embryo. The sudden death is caused by the rupture of a major blood vessel, indicating an important role for collagen I in establishing the mechanical stability of the circulatory system. Our results furthermore suggest that complex cell interactions in embryonic development such as those in early hemopoiesis may depend on the presence of collagen type I.

Germline integration of Moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death

Thirteen mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (M-MuLV) at a single locus (Mov locus) have been derived previously. Experiments were performed to investigate whether homozygosity at the Mov loci would be compatible with normal development. Animals heterozygous at an Mov locus were mated, and the genotype of the offspring was analyzed. From parents heterozygous at the loci Mov1 to Mov12, respectively, homozygous offspring were obtained with the expected Mendelian frequency. In contrast, no homozygous offspring or embryos older than day 15 of gestation were obtained from parents heterozygous at the Mov13 locus. When pregnant Mov13 females at day 13 and day 14 of gestation were analyzed, approximately 25% of the embryos were degenerated. Genotyping revealed that these degenerated embryos were invariably homozygous and the normal appearing embryos were either heterozygous or negative for M-MuLV. These results suggest that integration of M-MuLV at the Mov13 locus leads to insertion mutagenesis, resulting in embryonic arrest between day 12 and day 13 of gestation. It is possible that the Mov13 locus represents a gene or gene complex involved in the early embryonic development of the mouse.

Retroviruses and embryogenesis: Microinjection of Moloney leukemia virus into midgestation mouse embryos

Abstract The interaction of Moloney leukemia virus (M-MuLV) with developing post-implantation mouse embryos was studied. First, the frequency at which embryos in utero are infected by transplacental transmission with maternal virus was explored. To exclude milk transmission from the viremic mother, embryos were delivered by cesarean section prior to birth and given to normal foster mothers. None of 72 mice raised this way developed viremia. This indicates that the placenta is an efficient barrier protecting the developing embryo against infection with exogenous retroviruses. To overcome the placental barrier and to introduce virus into embryos at defined stages of differentiation, Moloney leukemia virus was microinjected directly into embryos in utero at day 8 or 9 of gestation. Between 60 and 70% of the injected embryos survived to birth and were tested for viremia at 4 weeks of age. M-MuLVspecific sequences were quantitated in organs of viremic animals derived from midgestation embryos microinjected with virus. Molecular hybridization experiments with nucleic acids extracted from different organs of these animals indicated that every cell type carried M-MuLV-specific DNA sequences and that high concentrations of M-MuLV-specific RNA sequences were present in every organ. In contrast, M-MuLV infection and expression is restricted to lymphatic tissues when animals are exposed to virus after birth or in BALB/Mo mice. These results indicate that the most important parameter determining the target tropism of Moloney leukemia virus infection and expression is the stage of embryogenesis and cellular differentiation at which virus infection takes place. In viremic C57BL animals derived from microinoculated embryos, the hair color changed beginning at age 6 weeks. This was not observed in animals exposed to virus after birth. All animals succumbed to MMuLV-induced leukemia at a later age. The results suggest that expression of M-MuLV may also lead to cellular dysfunctions other than leukemic transformation.

Infectivity and Methylation of Retroviral Genomes Is Correlated with Expression in the Animal

We studied mechanisms controlling gene expression during animal development using retroviruses as model genes. For this, substrains of mice have been previously derived carrying the Moloney leukemia virus (M-MuLV) in their germ line. Virus activation occurs in some of these substrains at different stages of development, resulting in two classes of viral genomes. The genetically transmitted (endogenous) copy is present in every cell, whereas somatically acquired (exogenous) copies are carried only in cells that were superinfected. We compared these two classes of M-MuLV genomes using two parameters. DNA sequences of the endogenous M-MuLV genome in all mouse substrains were highly methylated in GCGC, the recognition sequence of the restriction enzyme Hha I, and were not infectious (specific infectivity less than 10(-7) pfu per proviral genome) in a DNA transfection assay. In contrast, the exogenous copies were hypomethylated and infectious. These parameters are strongly correlated to genome activity in the animal: only tissues carrying exogenous copies express virus-specific RNA. With the assumption that gene expression of transfected DNA is controlled by mechanisms that are relevant for gene expression in the animal, our results suggest that DNA methylation plays a causative role in gene regulation during development and differentiation.

Retrovirus-induced lethal mutation in collagen I gene of mice is associated with an altered chromatin structure

The chromatin structure of the collagen alpha 1(I) gene, which has been mutated by retrovirus insertion in Mov13 mice, was compared with that of the wildtype allele. Limited digestions with DNAase I revealed the presence of two hypersensitive sites in all normal cells analyzed, while a third site at 100 to 200 bp 5 of the transcription start was detected only in cells synthesizing collagen alpha 1(I) mRNA. This transcription-associated site was not present in chromatin of the mutant allele, while the two other hypersensitive sites, one of which is located close to the provirus, were not changed by the virus integration. Our results suggest that the virus insertion in Mov13 mice may prevent the developmentally regulated appearance of a transcription-associated hypersensitive site, thereby interfering with proper activation of the gene during embryonic development.

DNA Methylation in Early Mammalian Development

The genomic DNAs of vertebrates and many invertebrates contain 5-methylcytosine (5-meCyt) as the only modified base (Table 10.1). Considerable interest in DNA methylation has been created by increasing evidence that links methylation patterns to patterns of gene expression in differentiation. An inverse correlation between methylation and transcriptional activity has been found for a number of developmentally regulated genes; it will be reviewed elsewhere (see Chapter 8).

Moloney leukemia virus gene expression and gene amplification in preleukemic and leukemic BALB/Mo mice

Abstract Mice carrying the exogenous Moloney leukemia virus (M-MuLV) as an endogenous virus have been derived previously. This mouse strain (BALB/Mo) transmits the virus as a single Mendelian gene (Mov-1 locus) from one generation to the next. Molecular hybridization experiments were performed to identify the organs in which the M-MuLV gene is activated during postnatal life of BALB/Mo mice and to determine the age-dependent onset of M-MuLV gene expression. Spleen and thymus cells of BALB/Mo mice synthesize M-MuLV-specific RNA soon after birth and virus gene expression reaches high levels at 3–4 weeks of age. A substantial further increase in virus gene expression is not observed in leukemic tissues of older animals. Other organs, such as liver, brain, and kidneys, do not express M-MuLV-specific RNA throughout the animals life. These observations define lymphatic tissues (spleen and thymus) as the target organs of M-MuLV expression in BALB/Mo mice. Virus gene expression was correlated with somatic amplification of M-MuLV-specific DNA sequences during the preleukemic and leukemic phase of the animals life. DNA amplification occurs in two steps in target tissues of BALB/Mo mice. A first step to approximately two copies per haploid genome equivalent is observed in preleukemic mice and a second step to three to four copies is observed in leukemic tissues. Nontarget tissues carry one copy of M-MuLV-specific DNA sequences regardless of the age of the animal. These results suggest that M-MuLV-specific gene expression is not sufficient for leukemic transformation and is related to virus-specific DNA amplification in preleukemic animals. A second amplification of M-MuLV DNA sequences appears to be related to leukemic transformation.

The integration sites of endogenous and exogenous Moloney murine leukemia virus

Specific cDNA probes of Moloney and AKR murine leukemia viruses have been prepared to characterize the proviral integration sites of these viruses in the genomes of Balb/Mo and Balb/c mice. The genetically transmitted Moloney provirus of Balb/Mo mice was detected in a characteristic Eco RI DNA fragment of 16 x 10(6) daltons. No fragment of this size was detected in tissue DNAs from Balb/c mice infected as newborns with Moloney virus. We conclude that a viral integration site, occupied in preimplantation mouse embryos, is not necessarily occupied when virus infects cells in post-natal animals. Balb/Mo and Balb/c mice do carry the AkR structural gene in an Eco RI DNA fragment of 12 x 10(6) daltons. Further restriction analysis of this fragment indicated that both mouse lines carry one AKR-type provirus. Leukemogenesis in Balb/Mo and newborn infected Balb/c mice is accompanied by reintegration of Moloney viral sequences in new chromosomal sites of tumor tissues. Part of the reintegrated Moloney viral sequences are of subgenomic size. The AKR viral sequences, however, are not found in new sites. Further restriction analysis revealed that the development of Moloney virus-induced leukemia in Balb/Mo mice does not lead to detectable structural alteration of the genetically transmitted Moloney and AKR structural genes. Possible mechanisms of the reintegration process are also discussed.

Conformation of free and of integrated Moloney leukemia virus proviral DNA in preleukemic and leukemic BALB/Mo mice

Abstract Restriction enzyme analysis has been used to characterize the structure of Moloney leukemia virus (M-MuLV) DNA in preleukemic and leukemic BALB/Mo mice. Leukemogenesis in these mice is accompanied by a somatic amplification of M-MuLV-specific DNA sequences which are derived from the germ line-transmitted Moloney leukemia virus genome. Quantitation by DNA-DNA annealing in solution has shown that this increase of M-MuLV-specific DNA sequences occurs in two steps, a first step from 1 to approximately 2 copies in spleen and thymus of preleukemic mice and a second increase to 3–4 copies following leukemic transformation. Using a specific cDNA probe for restriction enzyme analysis, no somatically acquired M-MuLV copies are detectable in tissues of preleukemic BALB/Mo mice. This indicates that virus integrated at many different chromosomal sites in individual cells. In contrast, restriction enzyme analysis of DNA from leukemic tissues shows 5–8 integrated copies in addition to the endogenous M-MuLV genome. Identical integration patterns are observed in leukemic tumors arising at different anatomical sites in individual animals. Tumors from different animals, however, have distinct integration patterns and no specific integration site common to all tumors can be identified. These results suggest that in BALB/Mo mice transformed cells originate among a population of preleukemic target cells which is heterogenous with respect to integration sites of newly acquired proviral copies. Selection of one transformed cell clone leads to monoclonal disease. Similar results are obtained with BALB/c mice infected with virus as newborns. Gel analysis indicated that unintegrated forms of M-MuLV viral DNA appear in target tissues of preleukemic BALB/Mo mice. Quantitation by quantitative hybridization in solution showed that up to 0.5 copy per cell can be present in thymic DNA from 1− to 5-month-old mice but not in spleens of the same animals. Small amounts of unintegrated proviral DNA are occasionally detected in thymus, spleen, and bone marrow of animals younger than 4 weeks of age. These results provide the first evidence for occurrence of free proviral DNA in animals and suggest that superinfection of target cells with endogenous virus is involved in the process of leukemic transformation.