RESUMO
BACKGROUND: Vasculogenesis in amniotes is often viewed as two spatially and temporally distinct processes, occurring in the yolk sac and in the embryo. However, the spatial origins of the cells that form the primary intra-embryonic vasculature remain uncertain. In particular, do they obtain their haemato-endothelial cell fate in situ, or do they migrate from elsewhere? Recently developed imaging techniques, together with new Tal1 and existing Flk1 reporter mouse lines, have allowed us to investigate this question directly, by visualising cell trajectories live and in three dimensions. RESULTS: We describe the pathways that cells follow to form the primary embryonic circulatory system in the mouse embryo. In particular, we show that Tal1-positive cells migrate from within the yolk sac, at its distal border, to contribute to the endocardium, dorsal aortae and head vasculature. Other Tal1 positive cells, similarly activated within the yolk sac, contribute to the yolk sac vasculature. Using single-cell transcriptomics and our imaging, we identify VEGF and Apela as potential chemo-attractants that may regulate the migration into the embryo. The dorsal aortae and head vasculature are known sites of secondary haematopoiesis; given the common origins that we observe, we investigate whether this is also the case for the endocardium. We discover cells budding from the wall of the endocardium with high Tal1 expression and diminished Flk1 expression, indicative of an endothelial to haematopoietic transition. CONCLUSIONS: In contrast to the view that the yolk sac and embryonic circulatory systems form by two separate processes, our results indicate that Tal1-positive cells from the yolk sac contribute to both vascular systems. It may be that initial Tal1 activation in these cells is through a common mechanism.
Assuntos
Sistema Cardiovascular , Endocárdio , Animais , Embrião de Mamíferos , Hematopoese , Camundongos , Saco Vitelino/diagnóstico por imagemRESUMO
Ovulatory dysfunction is the commonest cause of female infertility. Here we show that the co-repressor nuclear-receptor-interacting protein 1 (Nrip1; encoded by the gene Nrip1) is essential for ovulation. Mice null for this protein are viable, but female mice are infertile because of complete failure of mature follicles to release the oocyte at ovulation. In contrast, luteinization proceeds normally, resulting in a phenotype closely resembling that of luteinized unruptured follicle syndrome, often associated with infertility in women. Therefore, whereas the pre-ovulatory surge of luteinizing hormone induces both ovulation and luteinization, the ability to suppress the action of nuclear receptors is essential for the coordinated control of ovarian function with the essential process of oocyte release dependent on the activity of the transcriptional co-repressor Nrip1 (RIP40).
Assuntos
Anovulação/genética , Infertilidade Feminina/genética , Proteínas Nucleares/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Feminino , Masculino , Camundongos , Camundongos Mutantes , Proteínas Nucleares/isolamento & purificação , Proteína 1 de Interação com Receptor Nuclear , Ovário/fisiologia , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Fatores Sexuais , Distribuição TecidualRESUMO
DNA ligase IV is the most recently identified member of a family of enzymes joining DNA strand breaks in mammalian cell nuclei [1] [2]. The enzyme occurs in a complex with the XRCC4 gene product [3], an interaction mediated via its unique carboxyl terminus [4] [5]. Cells lacking XRCC4 are hypersensitive to ionising radiation and defective in V(D)J recombination [3] [6], implicating DNA ligase IV in the pathway of nonhomologous end-joining (NHEJ) of DNA double-strand breaks mediated by XRCC4, the Ku70/80 heterodimer and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in mammalian cells (reviewed in [7]). The phenotype of a null mutant of the Saccharomyces cerevisiae DNA ligase IV homologue indicates that the enzyme is non-essential and functions in yeast NHEJ [8] [9] [10]. Unlike other mammalian DNA ligases for which cDNAs have been characterised, DNA ligase IV is encoded by an intronless gene (LIG4). Here, we show that targeted disruption of LIG4 in the mouse leads to lethality associated with extensive apoptotic cell death in the embryonic central nervous system. Thus, unlike Ku70/80 and DNA-PKcs [11] [12] [13] [14], DNA ligase IV has an essential function in early mammalian development.
Assuntos
Sistema Nervoso Central/enzimologia , DNA Ligases/fisiologia , Embrião de Mamíferos/enzimologia , Animais , Apoptose , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/patologia , DNA Ligase Dependente de ATP , DNA Ligases/genética , Embrião de Mamíferos/patologia , Marcação de Genes , Genes Letais , Genótipo , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) zeta, which can replicate past certain types of DNA lesions [1]. Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis [2]. Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol zeta, decreased damage-induced mutagenesis in human cell lines [3]. To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and beta-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc). Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3l expression, traced by beta-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol zeta is essential for cell viability during embryonic development in mammals.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Proteínas Fúngicas/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Proteínas de Saccharomyces cerevisiae , Animais , Humanos , CamundongosRESUMO
The p24 family of type I integral-membrane proteins, which are localised in the endoplasmic reticulum (ER), the intermediate compartment and the Golgi apparatus, are thought to function as receptors for cargo exit from the ER and in transport vesicle formation. Members of the p24 family have been found in a molecular complex and are enriched in COPI-coated vesicles, which are involved in membrane traffic between the ER and Golgi complex. Although expressed abundantly, simultaneous deletion of several family members does not appear to affect cell viability and protein secretion in yeast. In order to gain more insights into the physiological roles of different p24 proteins, we generated mice deficient in the expression of one family member, p23 (also called 24delta1, see for alternative nomenclature). In contrast to yeast genetics, in mice disruption of both p23 alleles resulted in early embryonic lethality. Inactivation of one allele led not only to reduced levels of p23 itself but also to reduced levels of other family members. The reduction in steady-state protein levels also induced structural changes in the Golgi apparatus, such as the formation of dilated saccules. The generation of mice deficient in p23 expression has revealed an essential and non-redundant role for p23 in the earliest stages of mammalian development. It has also provided genetic evidence for the participation of p24 family members in oligomeric complexes and indicates a structural role for these proteins in maintaining the integrity of the early secretory pathway.
Assuntos
Vesículas Revestidas/metabolismo , Desenvolvimento Embrionário e Fetal/fisiologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/fisiologia , Receptores Citoplasmáticos e Nucleares , Alelos , Animais , Transporte Biológico , Northern Blotting , Western Blotting , Proteína Coatomer/metabolismo , Desenvolvimento Embrionário e Fetal/genética , Marcação de Genes , Genes Letais , Genótipo , Complexo de Golgi/ultraestrutura , Substâncias Macromoleculares , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Família Multigênica , Frações Subcelulares/químicaRESUMO
A number of growth factors, growth factor receptors and cell cycle regulatory proteins have been implicated in the genesis of mammary carcinomas both in animal models as well as in human breast tumour samples. Studies on the development of the mammary gland has revealed that several of the proto-oncogenes, or their closely related gene-family members, have a function in the normal growth and differentiation of the gland. In this review the role of fibroblast growth factor signalling and the critical requirement for the cell cycle regulator, cyclin D1 is discussed with respect to their normal function in mammary gland development and abnormal role in mammary carcinogenesis.
Assuntos
Mama/fisiologia , Ciclina D1/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Glândulas Mamárias Animais/fisiologia , Prenhez/fisiologia , Gravidez/fisiologia , Animais , Mama/patologia , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Feminino , Humanos , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/fisiopatologia , Camundongos , Camundongos Transgênicos , Transdução de SinaisRESUMO
We have previously described a strategy for integrating selectable marker genes into yeast artificial chromosomes (YACs) to facilitate their transfer into embryonic stem (ES) cells. Here we apply this technology to create mice carrying the core region of the human immunoglobulin (Ig) kappa light chain locus. A YAC was isolated which contains a 300 kb insert spanning three V kappa segments, the J kappa cluster, the C kappa region and extending downstream of the Kde element. After modification of this YAC to integrate the selectable neo marker gene, the YAC was introduced into ES cells by protoplast fusion. Several ES cell clones were obtained which appeared to harbor one complete copy of the YAC while retaining little or no other yeast DNA. The ES cells were injected into blastocysts and the chimaeric mice were shown to rearrange the introduced human light chain genes with the resultant production of antibodies containing human kappa light chains in the serum.
Assuntos
Cromossomos Fúngicos , Genoma Humano , Cadeias kappa de Imunoglobulina/genética , Animais , Sequência de Bases , Blastocisto , Quimera , Biblioteca Gênica , Rearranjo Gênico , Marcadores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Protoplastos , TransfecçãoRESUMO
The highly conserved AAA ATPase p97 (VCP/CDC48) has well-established roles in cell cycle progression, proteasome degradation and membrane dynamics. Gene disruption in Saccromyces cerevisiae, Drosophila melanogaster and Trypanosoma brucei demonstrated that p97 is essential in unicellular and multicellular organisms. To explore the requirement for p97 in mammalian cell function and embryogenesis, we disrupted the p97 locus by gene targeting. Heterozygous p97+/- mice were indistinguishable from their wild-type littermates, whereas homozygous mutants did not survive to birth and died at a peri-implantation stage. These results show that p97 is an essential gene for early mouse development.
Assuntos
Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Perda do Embrião/genética , Deleção de Genes , Marcação de Genes , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Adenosina Trifosfatases/fisiologia , Animais , Cruzamento , Proteínas de Ciclo Celular/fisiologia , Implantação do Embrião/genética , Células-Tronco Embrionárias , Feminino , Heterozigoto , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Proteínas Nucleares/fisiologia , Proteína com ValosinaRESUMO
In order to facilitate alterations of large DNA molecules for their introduction into mammalian cells we have characterised the mechanism of site-specific modifications in yeast artificial chromosomes (YACs). Newly developed yeast integration vectors with dominant selectable marker genes allow targeted integration into left (centromeric) and right (non-centromeric) YAC arms as well as alterations to the human derived insert DNA. In transformation experiments, integration proceeds exclusively by homologous recombination although yeast prefers linear ends of homology for predefined insertions. Targeted regions can be rescued which expedite the cloning of internal human sequences and the identification of 5' and 3' YAC/insert borders. Integration of the neomycin resistance gene into various parts of the YAC allowed the transfer and stable integration of large DNA molecules into a variety of mammalian cells including embryonic stem cells.
Assuntos
Clonagem Molecular/métodos , Vetores Genéticos , Transfecção , Animais , Biblioteca Genômica , Humanos , Mutagênese Insercional , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/genética , Especificidade da Espécie , Transformação GenéticaRESUMO
We have used the mouse mammary tumor virus promoter to express two dominant negative (DN) fibroblast growth factor receptor (FGFR) isoforms in the mammary epithelium of transgenic mice. While expression of DN-FGFR1(IIIc) showed no discernible phenotype, a similar kinase negative form of FGFR2(IIIb) caused a marked impairment of lobuloalveolar development. The growth retardation was apparent by mid-pregnancy and persisted in the post-partum glands. Despite the substantial underdevelopment of the mammary gland there was a measurable lactational response, but it was insufficient to properly sustain the new-born pups. These findings demonstrate that fibroblast growth factor signalling is necessary for pregnancy dependent lobuloalveolar development of the mammary gland.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais , Animais , Caseínas/genética , Feminino , Expressão Gênica , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Transgênicos , Proteínas do Leite/química , Fenótipo , Gravidez , Regiões Promotoras Genéticas , Proteínas/análise , RNA/análise , RNA Mensageiro/análise , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genética , TransgenesRESUMO
Using homologous recombination, mice lacking cyclin D1 were generated by replacing most of the first exon of the Cyl-1 gene with sequences encoding neomycin resistance. Cyl-1(-1-) mice were viable and fertile but consistently smaller than their heterozygous or wild-type littermates. The nullizygous animals also showed two distinctive abnormalities: a severe retinopathy caused by impaired development of all layers of the retina and, in the mammary gland during pregnancy, a marked reduction in acinar development accompanied by a failure to lactate. Approximately 50% of animals also had a malformation of the jaw that manifested itself as a misalignment of the incisor teeth. Mouse embryo fibroblasts isolated from 14 day nullizygous, heterozygous, or wild-type embryos and grown under standard conditions showed similar cell-cycle and growth characteristics. Thus although cyclin D1 kinase activity may facilitate G1 progression, it is not essential for the development of most tissues and organs, and only a few specialized cell lineages are demonstrably sensitive to its absence.
Assuntos
Constituição Corporal/genética , Ciclinas/fisiologia , Glândulas Mamárias Animais/anormalidades , Proteínas Oncogênicas/fisiologia , Retina/anormalidades , Animais , Sequência de Bases , Ciclo Celular , Diferenciação Celular , Células Cultivadas , Ciclina D1 , Ciclinas/deficiência , Ciclinas/genética , Primers do DNA , Feminino , Fibroblastos/metabolismo , Expressão Gênica , Marcação de Genes , Heterozigoto , Homozigoto , Anormalidades Maxilomandibulares/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Gravidez , Recombinação Genética/genética , Células-Tronco/metabolismoRESUMO
Analysis of lambda light chain use in normal mice is made difficult by the dominant chi light chain repertoire. We produced mice rendered deficient in chi light chain expression by gene targeting and focused on questions concerned with the generation of lambda light chain diversity. Whilst these mice compensate the chi deficiency with increased lambda liters, and their Ig level is therefore not significantly reduced, they show major differences in immunization titers, germinal center (GC) development and somatic hypermutation. After immunization, using antigens that elicit a restricted IgL response in normal mice, we obtained in the chi-/- mice elevated primary antibody titers but a subsequent lack in titer increase after repeated antigen challenge. Analysis of the Peyer's patches (PP) revealed a dramatically reduced cell content with rather small but highly active GC. Flow cytometric analysis showed different cell populations in the PP with enriched peanut agglutinin (PNA)hi/CD45R(B220)+ B cells, implying that the apparent compensation for the lack of lambda light chain expression involves the GC microenvironment in cell selection, the initiation of hypermutation and high affinity expansion. The three V lambda genes, V1, V2 and Vx, are mutated in the GC B cells, but show no junctional diversity. In contrast, a reduced rate of V lambda hypermutation is found in the hybridoma antibodies, which appears to reflect a selection bias rather than structural constraints. However, mechanisms of somatic mutation and specificity selection can operate with equal efficiency on the few V lambda genes.
Assuntos
Formação de Anticorpos/imunologia , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/metabolismo , Cadeias lambda de Imunoglobulina/genética , Mutação/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Sequência de Bases , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Mutantes , Dados de Sequência Molecular , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologiaRESUMO
Mice deficient for FgfR2-IIIb were generated by placing translational stop codons and an IRES-LacZ cassette into exon IIIb of FgfR2. Expression of the alternatively spliced receptor isoform, FgfR2-IIIc, was not affected in mice deficient for the IIIb isoform. FgfR2-IIIb(-/-) (lac)(Z) mice survive to term but show dysgenesis of the kidneys, salivary glands, adrenal glands, thymus, pancreas, skin, otic vesicles, glandular stomach, and hair follicles, and agenesis of the lungs, anterior pituitary, thyroid, teeth, and limbs. Detailed analysis of limb development revealed an essential role for FgfR2-IIIb in maintaining the AER. Its absence did not prevent expression of Fgf8, Fgf10, Bmp4, and Msx1, but did prevent induction of Shh and Fgf4, indicating that they are downstream targets of FgfR2-IIIb activation. In the absence of FgfR2-IIIb, extensive apoptosis of the limb bud ectoderm and mesenchyme occurs between E10 and E10.5, providing evidence that Fgfs act primarily as survival factors. We propose that FgfR2-IIIb is not required for limb bud initiation, but is essential for its maintenance and growth.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Fatores de Crescimento de Fibroblastos/genética , Proteínas de Homeodomínio/genética , Botões de Extremidades/fisiologia , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transativadores , Fatores de Transcrição , Animais , Apoptose , Proteína Morfogenética Óssea 4 , Divisão Celular , Fator 10 de Crescimento de Fibroblastos , Fator 4 de Crescimento de Fibroblastos , Fator 8 de Crescimento de Fibroblasto , Regulação da Expressão Gênica , Proteínas Hedgehog , Fator de Transcrição MSX1 , Camundongos , Receptor Tipo 2 de Fator de Crescimento de FibroblastosRESUMO
In order to study the requirements for a human antibody repertoire to be engineered in transgenic animals, we have created mouse strains that express human immunoglobulin genes from 100 kb of cosmid DNA. Undisrupted integration of the human DNA in the mouse germ line, encoding germ-line V,D,J and the Cmu constant region, was achieved, and proved sufficient for the production of human IgM in the mouse serum. Co-integration of one cosmid (containing the VH6 gene and a 36-kb region of the J-proximal D cluster) with a second cosmid (containing VH26, a further 38-kb of the D cluster, JH and Cmu) results in V-D-J rearrangements in the transgenic mice, similar to those seen in human DNA, were found only in spleen but not in thymus. Random hybridomas made from these transgenic mice show heterogeneous rearrangements of the human transgenes. Sequences of V-D-J units derived from transgene rearrangements reveal extensive N-region and apparent D segment diversity. These results show that utilisation of human Ig genomic segments does occur in transgenic mice, paving the way for the derivation of a mouse strain that makes authentic human antibodies from inserted heavy and light chain gene loci.
Assuntos
Formação de Anticorpos , Mapeamento Cromossômico , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Sequência de Bases , Rearranjo Gênico , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Two B-type cyclins, B1 and B2, have been identified in mammals. Proliferating cells express both cyclins, which bind to and activate p34(cdc2). To test whether the two B-type cyclins have distinct roles, we generated lines of transgenic mice, one lacking cyclin B1 and the other lacking cyclin B2. Cyclin B1 proved to be an essential gene; no homozygous B1-null pups were born. In contrast, nullizygous B2 mice developed normally and did not display any obvious abnormalities. Both male and female cyclin B2-null mice were fertile, which was unexpected in view of the high levels and distinct patterns of expression of cyclin B2 during spermatogenesis. We show that the expression of cyclin B1 overlaps the expression of cyclin B2 in the mature testis, but not vice versa. Cyclin B1 can be found both on intracellular membranes and free in the cytoplasm, in contrast to cyclin B2, which is membrane-associated. These observations suggest that cyclin B1 may compensate for the loss of cyclin B2 in the mutant mice, and implies that cyclin B1 is capable of targeting the p34(cdc2) kinase to the essential substrates of cyclin B2.
Assuntos
Ciclina B/deficiência , Fertilidade/genética , Morte Fetal , Regulação da Expressão Gênica no Desenvolvimento , Células 3T3 , Animais , Membrana Celular/fisiologia , Clonagem de Organismos , Ciclina B/biossíntese , Ciclina B/genética , Ciclina B1 , Evolução Molecular , Feminino , Variação Genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Filogenia , Gravidez , Recombinação Genética , Testículo/metabolismoRESUMO
T cell development is regulated by extracellular signals that mediate cellular proliferation and differentiation via specific signal transduction pathways. To determine the importance of the mitogen-activated protein kinase (MAP kinase) pathway in thymocyte development, we analyzed transgenic mice expressing dominant negative Raf (DN Raf) and a constitutively active v-Raf under the control of the p56lck proximal promoter. DN Raf had a profound effect on T cell receptor (TCR)-mediated signaling events as assessed by the inhibition of mitogen-induced proliferation of thymocytes in vitro. Overall thymocyte numbers were decreased by at most twofold from nontransgenic littermates. Positive selection was inhibited in DN Raf transgenic mice, as evidenced by both reduced numbers of mature thymocytes and a decrease in CD8+ thymocytes in female mice doubly transgenic for DN-Raf and a class I-restricted H-Y TCR. In contrast, the differentiation of double-positive thymocytes to single-positive thymocytes was enhanced in H-YTCR transgenic mice expressing constitutively active Raf (v-Raf). Thus, Raf regulates positive selection in the thymus.
Assuntos
Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Subpopulações de Linfócitos T/citologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Diferenciação Celular , Antígeno H-Y/imunologia , Lectinas Tipo C , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas v-raf , Proteínas Proto-Oncogênicas c-raf , Receptores de Antígenos de Linfócitos T/fisiologia , Proteínas Oncogênicas de Retroviridae , Transdução de Sinais , Timo/citologia , Timo/crescimento & desenvolvimentoRESUMO
The fibroblast growth factor receptor 2 gene is differentially spliced to encode two transmembrane tyrosine kinase receptor proteins that have different ligand-binding specificities and exclusive tissue distributions. We have used Cre-mediated excision to generate mice lacking the IIIb form of fibroblast growth factor receptor 2 whilst retaining expression of the IIIc form. Fibroblast growth factor receptor 2(IIIb) null mice are viable until birth, but have severe defects of the limbs, lung and anterior pituitary gland. The development of these structures appears to initiate, but then fails with the tissues undergoing extensive apoptosis. There are also developmental abnormalities of the salivary glands, inner ear, teeth and skin, as well as minor defects in skull formation. Our findings point to a key role for fibroblast growth factor receptor 2(IIIb) in mesenchymal-epithelial signalling during early organogenesis.
Assuntos
Anormalidades Múltiplas/genética , Desenvolvimento Embrionário e Fetal/fisiologia , Mesoderma/fisiologia , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Proteínas Virais , Anormalidades Múltiplas/embriologia , Animais , Osso e Ossos/anormalidades , Anormalidades Craniofaciais/genética , Epitélio/embriologia , Éxons , Heterozigoto , Integrases/metabolismo , Camundongos , Camundongos Knockout , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/deficiência , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
A major issue is whether surface expression of the pre-TCR is necessary for signaling the development of immature thymocytes. To address this question, we generated transgenic mice expressing a TCRbeta chain that had a strong endoplasmic reticulum (ER) retrieval signal (TCRbetaER) and that was expressed intracellularly but failed to reach the cell surface. In TCRbetaER transgenic mice, there was a failure of allelic exclusion. Also, the transgene failed to rescue the developmental defects observed in TCRbeta-null mice. In contrast, TCRbeta transgenes with a mutant ER retrieval sequence or lacking this sequence signaled efficient allelic exclusion and suppressed the TCRbeta-/- defect. These data show that exit of the pre-TCR from the ER/cis-Golgi is required for progression through the double-negative thymocyte checkpoint.
Assuntos
Retículo Endoplasmático Rugoso/metabolismo , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Complexo de Golgi/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/citologia , Timo/citologia , Alelos , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Diferenciação Celular , Divisão Celular , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Aggregation chimeras were formed between C57BL/6 mice heterozygous for the Apc(min) (Min) mutation and wild-type SWR mice, that differ in their Pla2g2a status, a modifier of Apc(min), and also in their resistance to intestinal polyp formation. Variation in the dolichos biflorus agglutinin-staining patterns of the intestines of these mouse strains was used to determine the chimeric composition of the intestine in individual mice and to examine the clonal composition of adenomas. Macroscopic adenoma numbers in chimeric mice were compared with the expected adenoma numbers based on the percentage of C57BL/6J-Apc(min/+) epithelium in individual mice. These results unexpectedly show that there was no apparent inhibitory effect of the SWR-derived (Pla2g2a wild-type) tissue on adenoma formation in the C57BL/6J-Apc(min/+) epithelium. This suggests that the main genetic modifiers of the Min phenotype act at a cellular or crypt-restricted level with no discernable systemic effect. All adenomas were seen to contain C57BL/6J-Apc(min/+)-derived epithelium, confirming that the germ-line mutation of the mApc gene is necessary to initiate tumorigenesis in this model system, and that the mApc gene acts in a cell autonomous fashion.
Assuntos
Adenoma/patologia , Quimera , Neoplasias Intestinais/patologia , Neoplasias Primárias Múltiplas/patologia , Adenoma/genética , Animais , Sequência de Bases , Primers do DNA , Feminino , Neoplasias Intestinais/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Primárias Múltiplas/genética , Reação em Cadeia da PolimeraseRESUMO
The mixed lineage leukemia gene (MLL) was originally identified through its involvement in reciprocal translocations in leukemias. MLL codes for a large multidomain protein and bears homology to the Drosophila developmental control gene trithorax in two small domains in the amino terminal region, the central zinc finger domain and the carboxy SET domain. Like the Drosophila trx, MLL has also been shown to be a positive regulator of Hox gene expression. We have targeted Mll (the murine homologue of MLL) in exon 5 causing expression of three truncated in-frame Mll transcripts. These transcripts retain all or some of the AT hook motifs and the DMT domain. This mutant allele causes early in vivo preimplantation lethality of homozygous embryos prior to the 2-cell stage. Embryos cultured in vitro progress to the 2-cell stage, but further development is arrested. The heterozygotes exhibit mild skeletal defects as well as defects in some neuroectodermal derivatives.