- Email: [email protected]
Murine development control genes
183
184
PERMISSIVENESS TO MURINE LEUKEMIA VIRUS EXPRESSION DURING PREIMPLANTATION AND EARLY POSTIMPLANTATION MOUSE DEVELOPMENT Pierre Savatier and Rosa Beddington
MURINE D E V E I O ~ CONTROL GENES Peter Gruss, Dept. Molecular Cell Biology, MaxPlanck Institute of Biophysical Chemistry, Am Fassberg, 3400 G6ttingen, F.R.G. Classical genetic tools are insufficient to reveal mou~e genes involved in the control of pattern formation. As an alternative approach we have employed homologies between sequences conserved in Drosophila developmental control genes and the mouse genome in the hope that structural conservation would be indicative of functional conservation as well. To this end we have isolated three gene families possibly representing murine developmental control genes. More precisely, we have identified murine horacebox (I), "finger"-containing (2), and paired box-contained (3) genes, all encoding putative DNA-binding proteins (4). The analysis of the expression pattern reveals the expression of homeobox genes to be region specific particularly in developing somites, sclerotomes, and embryonal ribs. Additional gene activity can be found in the ectoderm of 8.5 day old embryos, in the neural tube and spinal ganglia of 12 day old embryos. One "finger"containing gene is als expressed in ectodermal tissue - embryonal and adult neurons. Lastly, a paired box-containg gene (Pax-l) was found to be specifically expressed in the developing vertebral column. Another paired-box containing gene Pax-2 shows prominent expression in certain parts of the neural tube, the intermediate zone.
Permissiveness to marine leukemia virus (MLV) expression was examined during mouse development using two different replication defective retroviral vectors: 1) a vector where lac Z is driven off the viral LTR (BAG); 2) a vector where lac Z is driven off an internal rat 13-actin promoter (plRV). Infection of preimplantation cleavage stage embryos, blastocysts and 8th, 9th and 10th day postimplantation embryos was undertaken. The incidence and distribution of cells exhibiting bacterial B-galactosidase activity was compared using the two different vectors. No 13-galactosidase activity was detected after infecting preimplantation embryos with BAG. Postimplantation embryos infected with BAG on the 8th day produced positive cells only in mesoderm, endoderm or extraembryonic mesoderm. Infection on the 9th day generated additional positive cells in the neurectoderm, whereas surface ectoderm cells expressing lac Z only appeared following infection on the 10th day. Therefore, permissiveness to MLV expression, first evident at gastrulation, is not acquired simultaneously by all epiblast derivatives. When plRV was used to infect embryos, cells expressing lac Z were identified in every tissue lineage of the embryo regardless of developmental stage at the time of infection. The incidence of positive cells in different tissues varied, probably reflecting differences in accessibility of different tissues to viral particles, differences in cell division rate and differences in susceptibility of cells to viral infection and integration. These results highlight possible limitations in using retroviral vectors to analyse cell fate in the mouse embryo.
We recently examined a mouse mutant, undulated (un). Un mice exhibit vertebral malformations along the entire rostro-caudal axis. We f o ~ d that un mutants carry a point mutation in a highly conserved part of the paired-box of Pax i leading to a Gly-Ser replacement. The chromosomal location and this mutation in conjunction with the Pax I gene expression (3) implicate a causative role of Pax I in the generation of the vertebral column (5). Current analysis involve the generation of transgenic mice carrying vectors which express individual members of the respective families with the aim of producing phenotypic changes due to dominant gain of dominant loss of function. Similarly, this technique is being used in order to map the elements involved in the control of tissue specific expression. Additionally, in accordance with the concepts on which classical genetic reasoning is based, we are attempting to insertionally inactivate individual members of these gent families via homologous recombinatiorL To this end we have targetted one allel of the Hox I.I gene in ES-cells. Upon introduction of this modified ES-eells into mouse blastocysts we generated chimeric mice (6). Presently we are examining whether the modified ES-cell has passed into the germ line. i. Colberg--Poley, ~M., S.D. Voss, F. Chowdhury, C.L. Stewart, E. W a g n e r and P. Gruss: Clustered homeo boxes are differentially expressed during murine development. Cell 43, 39-45, 1985.
2. Chowdhury, K., U. Deutch and P. Gruss: A multigene family encoding several "finger" structures is present and differentially active in mammalian genomes. Cell 48, 771778, 1987. 3. Deuteh, U., G.R. Dressier, and P. Gruss: Pax I, a member of a paired box homologous murine gent family, is expressed in segmented structures during development. Cell 53, 617625, 1988. 4. Dressler, G.R., and P. Gruss: Pax I, a member of a paired box homologous murine gent family regulate vertebrate development? TIG, Vol.4: 214-219, 1988. 5. Balling, R., U. Deutch, and P. Gruss: undulated, a Mutation Affecting the Development of the Mouse Skeleton, Has a Point Mutation in the Paired Box of Pax I. Cell 55, 531-535, 1988. 6. Zimmer, A., and P. Gruss: Chimeric mice, produced with embryonal stem (ES) cells carrying a homeobox Hox I.i allele mutated by homologous recombination. Nature 338, 150153, 1989.
$58
184
PERMISSIVENESS TO MURINE LEUKEMIA VIRUS EXPRESSION DURING PREIMPLANTATION AND EARLY POSTIMPLANTATION MOUSE DEVELOPMENT Pierre Savatier and Rosa Beddington
MURINE D E V E I O ~ CONTROL GENES Peter Gruss, Dept. Molecular Cell Biology, MaxPlanck Institute of Biophysical Chemistry, Am Fassberg, 3400 G6ttingen, F.R.G. Classical genetic tools are insufficient to reveal mou~e genes involved in the control of pattern formation. As an alternative approach we have employed homologies between sequences conserved in Drosophila developmental control genes and the mouse genome in the hope that structural conservation would be indicative of functional conservation as well. To this end we have isolated three gene families possibly representing murine developmental control genes. More precisely, we have identified murine horacebox (I), "finger"-containing (2), and paired box-contained (3) genes, all encoding putative DNA-binding proteins (4). The analysis of the expression pattern reveals the expression of homeobox genes to be region specific particularly in developing somites, sclerotomes, and embryonal ribs. Additional gene activity can be found in the ectoderm of 8.5 day old embryos, in the neural tube and spinal ganglia of 12 day old embryos. One "finger"containing gene is als expressed in ectodermal tissue - embryonal and adult neurons. Lastly, a paired box-containg gene (Pax-l) was found to be specifically expressed in the developing vertebral column. Another paired-box containing gene Pax-2 shows prominent expression in certain parts of the neural tube, the intermediate zone.
Permissiveness to marine leukemia virus (MLV) expression was examined during mouse development using two different replication defective retroviral vectors: 1) a vector where lac Z is driven off the viral LTR (BAG); 2) a vector where lac Z is driven off an internal rat 13-actin promoter (plRV). Infection of preimplantation cleavage stage embryos, blastocysts and 8th, 9th and 10th day postimplantation embryos was undertaken. The incidence and distribution of cells exhibiting bacterial B-galactosidase activity was compared using the two different vectors. No 13-galactosidase activity was detected after infecting preimplantation embryos with BAG. Postimplantation embryos infected with BAG on the 8th day produced positive cells only in mesoderm, endoderm or extraembryonic mesoderm. Infection on the 9th day generated additional positive cells in the neurectoderm, whereas surface ectoderm cells expressing lac Z only appeared following infection on the 10th day. Therefore, permissiveness to MLV expression, first evident at gastrulation, is not acquired simultaneously by all epiblast derivatives. When plRV was used to infect embryos, cells expressing lac Z were identified in every tissue lineage of the embryo regardless of developmental stage at the time of infection. The incidence of positive cells in different tissues varied, probably reflecting differences in accessibility of different tissues to viral particles, differences in cell division rate and differences in susceptibility of cells to viral infection and integration. These results highlight possible limitations in using retroviral vectors to analyse cell fate in the mouse embryo.
We recently examined a mouse mutant, undulated (un). Un mice exhibit vertebral malformations along the entire rostro-caudal axis. We f o ~ d that un mutants carry a point mutation in a highly conserved part of the paired-box of Pax i leading to a Gly-Ser replacement. The chromosomal location and this mutation in conjunction with the Pax I gene expression (3) implicate a causative role of Pax I in the generation of the vertebral column (5). Current analysis involve the generation of transgenic mice carrying vectors which express individual members of the respective families with the aim of producing phenotypic changes due to dominant gain of dominant loss of function. Similarly, this technique is being used in order to map the elements involved in the control of tissue specific expression. Additionally, in accordance with the concepts on which classical genetic reasoning is based, we are attempting to insertionally inactivate individual members of these gent families via homologous recombinatiorL To this end we have targetted one allel of the Hox I.I gene in ES-cells. Upon introduction of this modified ES-eells into mouse blastocysts we generated chimeric mice (6). Presently we are examining whether the modified ES-cell has passed into the germ line. i. Colberg--Poley, ~M., S.D. Voss, F. Chowdhury, C.L. Stewart, E. W a g n e r and P. Gruss: Clustered homeo boxes are differentially expressed during murine development. Cell 43, 39-45, 1985.
2. Chowdhury, K., U. Deutch and P. Gruss: A multigene family encoding several "finger" structures is present and differentially active in mammalian genomes. Cell 48, 771778, 1987. 3. Deuteh, U., G.R. Dressier, and P. Gruss: Pax I, a member of a paired box homologous murine gent family, is expressed in segmented structures during development. Cell 53, 617625, 1988. 4. Dressler, G.R., and P. Gruss: Pax I, a member of a paired box homologous murine gent family regulate vertebrate development? TIG, Vol.4: 214-219, 1988. 5. Balling, R., U. Deutch, and P. Gruss: undulated, a Mutation Affecting the Development of the Mouse Skeleton, Has a Point Mutation in the Paired Box of Pax I. Cell 55, 531-535, 1988. 6. Zimmer, A., and P. Gruss: Chimeric mice, produced with embryonal stem (ES) cells carrying a homeobox Hox I.i allele mutated by homologous recombination. Nature 338, 150153, 1989.
$58