RESUMO
Several reports have shown that doctoral and postdoctoral trainees in biomedical research pursue diverse careers that advance science meaningful to society. Several groups have proposed a three-tier career taxonomy to showcase these outcomes. This three-tier taxonomy will be a valuable resource for institutions committed to greater transparency in reporting outcomes, to not only be transparent in reporting their own institutional data but also to lend greater power to a central repository.
RESUMO
Elevated levels of endogenous angiotensin can cause hypertensive nephrosclerosis as a result of the potent vasopressor action of the peptide. We have produced by gene targeting mice homozygous for a null mutation in the angiotensinogen gene (Atg-1-). Postnatally, Atg-1- animals show a modest delay in glomerular maturation. Although Atg-1- animals are hypotensive by 7 wk of age, they develop, by 3 wk of age, pronounced lesions in the renal cortex, similar to those of hypertensive nephrosclerosis. In addition, the papillae of homozygous mutant kidneys are reduced in size. These lesions are accompanied by local up-regulation of PDGF-B and TGF-beta1 mRNA in the cortex and down-regulation of PDGF-A mRNA in the papilla. The study demonstrates an important requirement for angiotensin in achieving and maintaining the normal morphology of the kidney. The mechanism through which angiotensin maintains the volume homeostasis in mammals includes promotion of the maturational growth of the papilla.
Assuntos
Angiotensina II/metabolismo , Angiotensinogênio/deficiência , Angiotensinogênio/genética , Expressão Gênica , Substâncias de Crescimento/biossíntese , Rim/crescimento & desenvolvimento , Envelhecimento , Angiotensinogênio/sangue , Animais , Pressão Sanguínea , Peso Corporal , Homeostase , Rim/metabolismo , Rim/patologia , Córtex Renal/crescimento & desenvolvimento , Córtex Renal/patologia , Glomérulos Renais/crescimento & desenvolvimento , Glomérulos Renais/patologia , Medula Renal/crescimento & desenvolvimento , Medula Renal/patologia , Camundongos , Camundongos Knockout , Camundongos Mutantes , Tamanho do Órgão , Fator de Crescimento Derivado de Plaquetas/biossíntese , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Mapeamento por Restrição , Fator de Crescimento Transformador beta/biossínteseRESUMO
The embryonic neural crest is a unique group of cells that gives rise to the peripheral nervous system as well as many other cell types. Most of the work describing the behavior and specification of these cells has been done in the avian system, a system amenable to experimental manipulations such as tissue grafting, cell transplantation and lineage tracing. This work has been greatly facilitated by the use of molecular markers of neural crest cells such as HNK-1 and slug, markers that are not yet available for the study of mouse neural crest. Here we demonstrate that Hfh2 (HNF3 forkhead homologue 2), a member of the 'winged helix' or 'forkhead' transcription factor gene family, is expressed in premigratory and migrating neural crest cells in the early mouse embryo and in motorneuron progenitors in the developing spinal cord. Using linkage analysis we have localized the Hfh2 gene to chromosome 4 at 44.91 cM from the centromere.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso , Crista Neural/embriologia , Proteínas Repressoras/genética , Medula Espinal/embriologia , Animais , Sítios de Ligação , Mapeamento Cromossômico , Proteínas de Ligação a DNA/metabolismo , Proteína 2 de Resposta de Crescimento Precoce , Fatores de Transcrição Forkhead , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ/métodos , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Neurônios Motores/metabolismo , Crista Neural/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Medula Espinal/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Activated factor X (FXa) is a vitamin K-dependent serine protease that plays a pivotal role in blood coagulation by converting prothrombin to thrombin. There are no reports of humans with complete deficiency of FX, and knockout of murine F10 is embryonic or perinatal lethal. OBJECTIVE: We sought to generate a viable mouse model of FX deficiency. METHODS: We used a socket-targeting construct to generate F10-knockout mice by eliminating F10 exon 8 (knockout allele termed F10(tm1Ccmt), abbreviated as '-'; wild-type '+'), and a plug-targeting construct to generate mice expressing a FX variant with normal antigen levels but low levels of FX activity [4-9% normal in humans carrying the defect, Pro343-->Ser, termed FX Friuli (mutant allele termed F10(tm2Ccmt), abbreviated as F)]. RESULTS: F10 knockout mice exhibited embryonic or perinatal lethality. In contrast, homozygous Friuli mice [F10 (F/F)] had FX activity levels of approximately 5.5% (sufficient to rescue both embryonic and perinatal lethality), but developed age-dependent iron deposition and cardiac fibrosis. Interestingly, F10 (-/F) mice with FX activity levels of 1-3% also showed complete rescue of lethality. Further study of this model provides evidence supporting a role of maternal FX transfer in the embryonic survival. CONCLUSIONS: We demonstrate that, while complete absence of FX is incompatible with murine survival, minimal FX activity as low as 1-3% is sufficient to rescue the lethal phenotype. This viable low-FX mouse model will facilitate the development of FX-directed therapies as well as investigation of the FX role in embryonic development.
Assuntos
Deficiência do Fator X/genética , Fator X/genética , Impressão Genômica/genética , Camundongos Transgênicos/genética , Modelos Animais , Substituição de Aminoácidos , Animais , Cardiomiopatias/etiologia , Éxons/genética , Deficiência do Fator X/complicações , Feminino , Morte Fetal/genética , Fibrose , Marcação de Genes/métodos , Genes Letais , Teste de Complementação Genética , Genótipo , Hemossiderose/etiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos/sangue , Miocárdio/patologiaRESUMO
We wish to identify genes involved in mediating early lineage decisions in the mouse embryo. F9 teratocarcinoma cells treated with retinoic acid (RA) in suspension culture develop into embryoid bodies (EBs) with an outer layer of visceral endoderm. In order to identify genes that are involved in establishing this extraembryonic endoderm lineage we have employed a PCR-based approach using cDNAs from early EBs as templates. PCR reactions were performed with degenerate oligonucleotide primers coding for the highly conserved regions of the homeodomains of the Drosophila Antennapedia, bicoid, and zerknüllt proteins. Among the PCR products were representatives of previously identified mouse genes, including Hox-A5 (1.3), A1 (1.6), A9 (1.7), B8 (2.4), B2 (2.8), C8 (3.1), and D12 (4.7). Whole mount in situ hybridization analysis, performed to examine the temporal and spatial distribution of transcripts, suggests a possible role for the Hox-D12 gene during endoderm differentiation in F9 EBs. Whereas the expression patterns of several other homeobox genes are essentially uniform throughout the aggregates, Hox-D12 expression is restricted to the outer surface of early EBs at a time when lineage decisions may be occurring. In order to establish the relationship between the Hox-D12 expression pattern and the role of RA in inducing F9 EB differentiation, we examined PSA-1 EBs that differentiate in the absence of added RA. PSA-1 EBs show similar temporal and spatial localization of Hox-D12 when compared to F9 EBs. These data suggest that the pattern of Hox-D12 expression correlates with endoderm differentiation and not with RA treatment and point to a possible role for homeobox-containing genes during the early stages of mouse embryogenesis.
Assuntos
Diferenciação Celular/genética , Endoderma/citologia , Genes Homeobox , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Teratoma , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
When primordial germ cells of the mouse are cultured on feeder layers with the addition of the polypeptide signalling molecules leukaemia inhibitory factor, Steel factor and basic fibroblast growth factor they give rise to cells that resemble undifferentiated blastocyst-derived embryonic stem cells. These primordial germ cell-derived embryonic germ cells (EG cells) can be induced to differentiate extensively in culture and also form teratocarcinomas when injected into nude mice. Additionally, they contribute to chimeras when injected into host blastocysts. We have derived multiple EG cell lines from 8.5 days post coitum (dpc) embryos of C57BL/6 inbred mice. Four independent EG cell lines with normal male karyotypes have formed chimeras (up to 70% coat colour chimerism) when injected into BALB/c host blastocysts. Chimeric mice from all four cell lines are fertile, but only those from one line have transmitted coat colour markers through the germline. Studies have also been carried out to determine whether gonadal primordial germ cells can give rise to pluripotent EG cells. Germ cells from gonads of 15.5 dpc C57BL/6 embryos and newborn mice failed to produce EG cell lines. EG cell lines capable of forming teratocarcinomas and coat colour chimeras have been established from primordial germ cells of 12.5 dpc genital ridges. We are currently testing the genomic imprinting status of the insulin-like growth factor type 2 receptor gene (Igf2r) in our different EG cell lines.
Assuntos
Células Germinativas/citologia , Camundongos/embriologia , Animais , Blastocisto , Diferenciação Celular , Linhagem Celular , Transplante de Células , Quimera , Feminino , Impressão Genômica , Substâncias de Crescimento/fisiologia , Cor de Cabelo/genética , Masculino , Camundongos Endogâmicos , Camundongos Nus , Receptor IGF Tipo 2/genética , Células-Tronco/citologia , Teratocarcinoma/etiologia , Teratocarcinoma/patologiaRESUMO
Primordial germ cells of the mouse cultured on feeder layers with leukemia inhibitory factor, Steel factor and basic fibroblast growth factor give rise to cells that resemble undifferentiated blastocyst-derived embryonic stem cells. These primordial germ cell-derived embryonic germ cells can be induced to differentiate extensively in culture, form teratocarcinomas when injected into nude mice and contribute to chimeras when injected into host blastocysts. Here, we report the derivation of multiple embryonic germ cell lines from 8.5 days post coitum embryos of C57BL/6 inbred mice. Four independent embryonic germ cell lines with normal male karyotypes have formed chimeras when injected into BALB/c host blastocysts and two of these lines have transmitted coat color markers through the germline. We also show that pluripotent cell lines capable of forming teratocarcinomas and coat color chimeras can be established from primordial germ cells of 8.0 days p.c. embryos and 12.5 days p.c. genital ridges. We have examined the methylation status of the putative imprinting box of the insulin-like growth factor type 2 receptor gene (Igf2r) in these embryonic germ cell lines. No correlation was found between methylation pattern and germline competence. A significant difference was observed between embryonic stem cell and embryonic germ cell lines in their ability to maintain the methylation imprint of the Igf2r gene in culture. This may illustrate a fundamental difference between these two cell types.
Assuntos
Genes , Impressão Genômica , Células Germinativas/fisiologia , Receptor IGF Tipo 2/genética , Células-Tronco/fisiologia , Animais , Sequência de Bases , Southern Blotting , Diferenciação Celular/genética , Linhagem Celular , Primers do DNA/genética , Células Germinativas/citologia , Metilação , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Células-Tronco/citologiaRESUMO
The neural crest is a migratory cell population that gives rise to multiple cell types in the vertebrate embryo. The intrinsic determinants that segregate neural crest cells from multipotential dorsal progenitors within the neural tube are poorly defined. In this study, we show that the winged helix transcription factor Foxd3 is expressed in both premigratory and migratory neural crest cells. Foxd3 is genetically downstream of Pax3 and is not expressed in regions of Pax3 mutant mice that lack neural crest, implying that Foxd3 may regulate aspects of the neural crest differentiation program. We show that misexpression of Foxd3 in the chick neural tube promotes a neural crest-like phenotype and suppresses interneuron differentiation. Cells that ectopically express Foxd3 upregulate HNK1 and Cad7, delaminate and emigrate from the neural tube at multiple dorsoventral levels. Foxd3 does not induce Slug and RhoB, nor is its ability to promote a neural crest-like phenotype enhanced by co-expression of Slug. Together these results suggest Foxd3 can function independently of Slug and RhoB to promote the development of neural crest cells from neural tube progenitors.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/citologia , Neurônios/citologia , Proteínas Repressoras/genética , Animais , Proteínas Aviárias , Biomarcadores , Antígenos CD57/genética , Caderinas/genética , Diferenciação Celular/genética , Movimento Celular , Embrião de Galinha , Proteínas de Ligação a DNA/metabolismo , Indução Embrionária/genética , Fatores de Transcrição Forkhead , Sequências Hélice-Volta-Hélice , Camundongos , Camundongos Mutantes , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Proteínas Repressoras/metabolismo , Fatores de Transcrição da Família Snail , Medula Espinal/embriologia , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína rhoB de Ligação ao GTP/genéticaRESUMO
TGFbeta signals play important roles in establishing the body axes and germ layers in the vertebrate embryo. Vg1 is a TGFbeta-related gene that, due to its maternal expression and vegetal localization in Xenopus, has received close examination as a potential regulator of development in Xenopus, zebrafish, and chick. However, a mammalian Vg1 ortholog has not been identified. To isolate mammalian Vg1 we screened a mouse expression library with a Vg1-specific monoclonal antibody and identified a single cross-reactive clone encoding mouse Gdf1. Gdf1 is expressed uniformly throughout the embryonic region at 5.5-6.5 days postcoitum and later in the node, midbrain, spinal cord, paraxial mesoderm, lateral plate mesoderm, and limb bud. When expressed in Xenopus embryos, native GDF1 is not processed, similar to Vg1. In contrast, a chimeric protein containing the prodomain of Xenopus BMP2 fused to the GDF1 mature domain is efficiently processed and signals via Smad2 to induce mesendoderm and axial duplication. Finally, right-sided expression of chimeric GDF1, but not native GDF1, reverses laterality and results in right-sided Xnr1 expression and reversal of intestinal and heart looping. Therefore, GDF1 can regulate left-right patterning, consistent with the Gdf1 loss-of-function analysis in the mouse (C. T. Rankin, T. Bunton, A. M. Lawler, and S. J. Lee, 2000, Nature Genet. 24, 262-265) and a proposed role for Vg1 in Xenopus. Our results establish that Gdf1 is posttranslationally regulated, that mature GDF1 activates a Smad2-dependent signaling pathway, and that mature GDF1 is sufficient to reverse the left-right axis. Moreover, these findings demonstrate that GDF1 and Vg1 are equivalent in biochemical and functional assays, suggesting that Gdf1 provides a Vg1-like function in the mammalian embryo.
Assuntos
Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/genética , Glicoproteínas/genética , Sequência de Aminoácidos , Animais , Antígenos/genética , Proteínas Morfogenéticas Ósseas/imunologia , Proteínas Morfogenéticas Ósseas/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/imunologia , Glicoproteínas/fisiologia , Fatores de Diferenciação de Crescimento , Hibridização In Situ , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteína Smad2 , Transativadores/genética , Transativadores/fisiologia , Fator de Crescimento Transformador beta , Xenopus , Proteínas de XenopusRESUMO
BMP signaling is essential for a wide variety of developmental processes. To evaluate the role of Bmp2/4 in cranial neural crest (CNC) formation or differentiation after its migration into the branchial arches, we used Xnoggin to block their activities in specific areas of the CNC in transgenic mice. This resulted in depletion of CNC cells from the targeted areas. As a consequence, the branchial arches normally populated by the affected neural crest cells were hypomorphic and their skeletal and neural derivatives failed to develop. In further analyses, we have identified Bmp2 as the factor required for production of migratory cranial neural crest. Its spatial and temporal expression patterns mirror CNC emergence and Bmp2 mutant embryos lack both branchial arches and detectable migratory CNC cells. Our results provide functional evidence for an essential role of BMP signaling in CNC development.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/fisiologia , Osteogênese , Fator de Crescimento Transformador beta , Animais , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/genética , Osso e Ossos/embriologia , Proteínas de Transporte , Desenvolvimento Embrionário e Fetal , Camundongos , Camundongos Transgênicos , Proteínas/genética , Proteínas/fisiologia , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
Bone morphogenetic protein-4 (BMP-4) is a member of the TGF-beta superfamily of polypeptide signaling molecules, closely related to BMP-2 and to Drosophila decapentaplegic (DPP). To elucidate the role of BMP-4 in mouse development the gene has been inactivated by homologous recombination in ES cells. Homozygous mutant Bmp-4tm1blh embryos die between 6.5 and 9.5 days p.c., with a variable phenotype. Most Bmp-4tm1blh embryos do not proceed beyond the egg cylinder stage, do not express the mesodermal marker T(Brachyury), and show little or no mesodermal differentiation. Some homozygous mutants develop to the head fold or beating heart/early somite stage or beyond. However, they are developmentally retarded and have truncated or disorganized posterior structures and a reduction in extraembryonic mesoderm, including blood islands. These results provide direct genetic evidence that BMP-4 is essential for several different processes in early mouse development, beginning with gastrulation and mesoderm formation. Moreover, in the presumed absence of zygotic ligand, it appears that homozygous mutants can be rescued partially by related proteins or by maternal BMP-4.
Assuntos
Desenvolvimento Embrionário e Fetal , Gástrula/fisiologia , Mesoderma/fisiologia , Proteínas/fisiologia , Animais , Sequência de Bases , Proteínas Morfogenéticas Ósseas , Linhagem Celular , Quimera , Cruzamentos Genéticos , Feminino , Marcação de Genes , Heterozigoto , Homozigoto , Masculino , Mesoderma/citologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fenótipo , Proteínas/genética , Células-TroncoRESUMO
Members of the winged helix family of transcription factors are required for the normal embryonic development of the mouse. Using the interspecific backcross panel from The Jackson Laboratory, we have determined the chromosomal locations of four genes that encode winged helix containing proteins. Mf1 was assigned to mouse Chromosome 8, Mf2 to Chromosome 4, Mf3 to Chromosome 9, and Mf4 to Chromosome 13. Since Mf3 is located in a region of Chromosome 9 containing many well-characterized mouse mutations such as short ear (se), ashen (ash), and dilute (d), we have analyzed deletion mutants to determine the location of Mf3 more precisely.
Assuntos
Mapeamento Cromossômico , Regulação da Expressão Gênica no Desenvolvimento , Camundongos/genética , Fatores de Transcrição/genética , Animais , Cruzamentos Genéticos , Sondas de DNA , DNA Complementar , Marcadores Genéticos , Biblioteca Genômica , Camundongos Mutantes , Família Multigênica , Muridae , Mapeamento por Restrição , Fatores de Transcrição/biossínteseRESUMO
Bone morphogenetic protein 8B (BMP8B) is a member of the TGFbeta superfamily of growth factors. In the mouse, Bmp8b is expressed in male germ cells of the testis and trophoblast cells of the placenta, suggesting that it has a role in spermatogenesis and reproduction. To investigate these possibilities, we have generated mice with a targeted mutation in Bmp8b. Here, we show that homozygous Bmp8b(tm1blh) mutant males exhibit variable degrees of germ-cell deficiency and infertility. Detailed analysis reveals two separable defects in the homozygous mutant testes. First, during early puberty (2 weeks old or younger) the germ cells of all homozygous mutants either fail to proliferate or show a marked reduction in proliferation and a delayed differentiation. Second, in adults, there is a significant increase in programmed cell death (apoptosis) of spermatocytes, leading to germ-cell depletion and sterility. Sertoli cells and Leydig cells appear relatively unaffected in mutants. This study therefore provides the first genetic evidence that a murine germ cell-produced factor, BMP8B, is required for the resumption of male germ-cell proliferation in early puberty, and for germ-cell survival and fertility in the adult.
Assuntos
Substâncias de Crescimento/fisiologia , Proteínas/fisiologia , Espermatogênese/genética , Animais , Apoptose , Proteínas Morfogenéticas Ósseas , Diferenciação Celular , Divisão Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Genes/fisiologia , Substâncias de Crescimento/genética , Homozigoto , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas/genética , RNA Mensageiro/análise , Células de Sertoli , Maturidade Sexual/genética , Espermatozoides/citologia , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/patologiaRESUMO
The ability to lactate is a process restricted to mammals and is necessary for the survival of nonhuman mammals. Female mice carrying a null mutation in the winged helix transcription factor Foxb1 (previously Mf3/Fkh5/TWH) have lactation defects on inbred genetic backgrounds. To determine the cellular basis of the Foxb1 lactation defect we have inserted a tau-lacZ lineage marker into the locus to follow the fate of Foxb1 expressing cells. This approach has revealed that Foxb1 is expressed in epithelial cells of developing and adult mammary glands as well as previously described regions of the central nervous system. Mammary glands from C57BL/6 Foxb1-/- mice have incomplete lobuloalveolar development. In addition, the tau-lacZ lineage label was used to determine that the mammillothalamic tract was lost in all Foxb1-/- mice. Finally, morphological defects in the inferior colliculi of the midbrain in Foxb1-/- mice correlate with the inability to lactate, suggesting that the midbrain defect, but not the loss of the mammillothalamic tract, may be responsible for the lactation defect.
Assuntos
Encéfalo/crescimento & desenvolvimento , Proteínas de Ligação a DNA/genética , Marcação de Genes/métodos , Glândulas Mamárias Animais/crescimento & desenvolvimento , Corpos Mamilares/crescimento & desenvolvimento , Ejeção Láctea/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/genética , Animais , Encéfalo/embriologia , Cruzamentos Genéticos , Feminino , Fatores de Transcrição Forkhead , Heterozigoto , Homozigoto , Técnicas Imunoenzimáticas , Óperon Lac/fisiologia , Masculino , Glândulas Mamárias Animais/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fenótipo , Reflexo/genética , Proteínas tau/metabolismoRESUMO
Matrix metalloproteinases (MMPs) classically have been implicated in basement membrane destruction associated with late-stage tumor cell invasion and metastasis. However, recent studies have demonstrated that one MMP family member, matrilysin, is expressed in a high percentage of early-stage human colorectal tumors. We analyzed matrilysin expression in benign intestinal tumors from mice heterozygous for the ApcMin allele (Min/+) and found that the mRNA was induced in the majority (88%) of these adenomas. Protein was detected in the tumor cells, where, surprisingly, it was predominantly immunolocalized to the lumenal surface of dysplastic glands rather than the basement membrane or extracellular matrix. To address the role of matrilysin in Min intestinal tumorigenesis, we generated Min/+ mice deficient in this MMP by gene targeting and homologous recombination. The absence of matrilysin resulted in a reduction in mean tumor multiplicity in Min/+ animals of approximately 60% and a significant decrease in the average tumor diameter. Based on these findings, we conclude that matrilysin is a suppressor of the Min phenotype, possibly by functioning in a capacity independent of matrix degradation. These results argue for the use of MMP inhibitors in the treatment and prevention of early-stage colon cancer.
Assuntos
Adenoma/genética , Polipose Adenomatosa do Colo/genética , Genes APC , Genes Supressores de Tumor , Metaloendopeptidases/deficiência , Animais , Células Clonais , Modelos Animais de Doenças , Imuno-Histoquímica , Hibridização In Situ , Metaloproteinase 7 da Matriz , Metaloendopeptidases/genética , Metaloendopeptidases/isolamento & purificação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutagênese Sítio-Dirigida , Fenótipo , Células-Tronco , Distribuição TecidualRESUMO
We previously reported the isolation of a cDNA clone for a homeobox-containing gene designated Pem, shown by Northern analysis of Day 7 through Day 16 mouse embryos to be expressed in extraembryonic tissues. In this study, Pem gene expression was further examined using in situ hybridization and immunocytochemistry to determine the spatial distribution of Pem transcripts and protein in peri-implantation embryos and in embryoid bodies (EBs). Low amounts of Pem mRNA were detected in undifferentiated EBs. When EBs were induced to differentiate, the outer cell layer of visceral or parietal endoderm expressed both Pem mRNA and protein. In developing embryos, no Pem protein was detectable in the uncompacted morula; 12% of the nuclei in compacted morulae were Pem positive, while 25% of the blastocyst trophectoderm and 15% of inner cell mass cells expressed Pem protein. Shortly after implantation, in 5.5 and 6.5 d.p.c. embryos, Pem expression was limited to extraembryonic tissues and was present in distal and proximal visceral endoderm, parietal endoderm, and ectoplacental cone. By 7.5-8.5 d.p.c. neither Pem RNA nor protein was found in the distal squamous visceral endoderm, which surrounds the embryonic region of the egg cylinder, nor in the parietal endoderm. Expression was retained in the proximal columnar epithelium of the visceral endoderm. Prominent Pem expression was observed in the chorion, in trophoblast-derived cells of the ectoplacental cone, and in secondary giant cells, localized in the nuclear compartment. Pem was localized to the X chromosome and found to be expressed in cell lineages where only the maternal X chromosome is active. The data indicate a possible role for Pem in regulating genes involved in the differentiation of extraembryonic tissues.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Embrião de Mamíferos/fisiologia , Expressão Gênica , Genes Homeobox , Proteínas de Homeodomínio , Fatores de Transcrição/biossíntese , Cromossomo X , Animais , Diferenciação Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário e Fetal , Células Híbridas , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Sondas de Oligonucleotídeos , Oligonucleotídeos Antissenso , Recombinação Genética , Teratoma , Fatores de Transcrição/genética , Transcrição Gênica , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
The mouse Mf3 gene, also known as Fkh5 and HFH-e5.1, encodes a winged helix/forkhead transcription factor. In the early embryo, transcripts for Mf3 are restricted to the presomitic mesoderm and anterior neurectoderm and mesoderm. By 9.5 days post coitum, expression in the nervous system is predominantly in the diencephalon, midbrain and neural tube. After midgestation, the highest level of mRNA is in the mammillary bodies, the posterior-most part of the hypothalamus. Mice homozygous for a deletion of the mf3 locus on a [129 x Black Swiss] background display variable phenotypes consistent with a requirement for the gene at several stages of embryonic and postnatal development. Approximately six percent of the mf3-/- embryos show an open neural tube in the diencephalon and midbrain region, and another five percent show a severe reduction of the posterior body axis; both these classes of affected embryos die in utero. Surviving homozygotes have an apparently normal phenotype at birth. Postnatally, however, mf3-/- pups are severely growth retarded and approximately one third die before weaning. This growth defect is not a direct result of lack of circulating growth hormone or thyrotropin. Mice that survive to weaning are healthy, but they show an abnormal clasping of the hindfeet when suspended by the tail. Although much smaller than normal, the mice are fertile. However, mf3-/- females cannot eject their milk supply to feed their pups. This nursing defect can be corrected with interperitoneal injections of oxytocin. These results provide evidence that Mf3 is required for normal hypothalamus development and suggest that Mf3 may play a role in postnatal growth and lactation.
Assuntos
Padronização Corporal/genética , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Proteínas de Ligação a DNA/genética , Ejeção Láctea/fisiologia , Fatores de Transcrição/genética , Animais , Comportamento Animal/fisiologia , Quimera , Cruzamentos Genéticos , Diencéfalo/embriologia , Diencéfalo/crescimento & desenvolvimento , Feminino , Fatores de Transcrição Forkhead , Heterozigoto , Membro Posterior/fisiologia , Homozigoto , Hipotálamo/anatomia & histologia , Imuno-Histoquímica , Masculino , Glândulas Mamárias Animais/anormalidades , Mesencéfalo/embriologia , Mesencéfalo/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Mutantes , Fenótipo , Hipófise/fisiologia , Radioimunoensaio , Reflexo/genéticaRESUMO
Hedgehog genes in Drosophila and vertebrates control patterning of a number of different structures during embryogenesis. They code for secreted signaling proteins that are cleaved into an active aminopeptide and a carboxypeptide. The aminopeptide can mediate local and long range events and can act as a morphogen, inducing differentiation of distinct cell types in a concentration-dependent manner. We demonstrate here that the expression of Indian hedgehog mRNA and protein is upregulated dramatically as F9 cells differentiate in response to retinoic acid, into either parietal endoderm or embryoid bodies, containing an outer visceral endoderm layer. The ES cell line D3 forms embryoid bodies in suspension culture without addition of retinoic acid and also upregulates Indian hedgehog expression. RT-PCR analysis of blastocyst outgrowth cultures demonstrates that whereas little or no Indian hedgehog message is present in blastocysts, significant levels appear upon subsequent days of culture, coincident with the emergence of parietal endoderm cells. In situ hybridization analysis for Indian hedgehog mRNA expression demonstrates the presence of elevated levels of message in the outer visceral endoderm cells relative to the core cells in mature embryoid bodies and in the visceral endoderm of Day 6.5 embryos. Whole-mount in situ hybridization analysis of Day 7.5 and 8.5 embryos indicates that Indian hedgehog expression is highest in the visceral yolk sac at this stage. F9 cell lines expressing a full length Indian hedgehog cDNA express a number of characteristics of differentiated cells, in the absence of retinoic acid. Taken together, these data suggest that Indian hedgehog is involved in mediating differentiation of extraembryonic endoderm during early mouse embryogenesis.
Assuntos
Embrião de Mamíferos , Endoderma , Regulação da Expressão Gênica no Desenvolvimento , Biossíntese de Proteínas , Transativadores , Animais , Blastocisto , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Clonagem Molecular , DNA Complementar/genética , Proteínas Hedgehog , Hibridização In Situ , Camundongos , Camundongos Endogâmicos ICR , Ativadores de Plasminogênio/análise , Reação em Cadeia da Polimerase , Proteínas/genética , RNA Mensageiro/análise , Análise de Sequência de DNA , Distribuição Tecidual , Transfecção , Tretinoína/farmacologia , Vísceras/embriologiaRESUMO
There are two major angiotensin II receptor isoforms, AT1 and AT2. AT1 mediates the well-known pressor and mitogenic effects of angiotensin II, but the signalling mechanism and physiological role of AT2 has not been established. Its abundant expression in fetal tissues and certain brain nuclei suggest possible roles in growth, development and neuronal functions. Here we report the unexpected finding that the targeted disruption of the mouse AT2 gene resulted in a significant increase in blood pressure and increased sensitivity to the pressor action of angiotensin II. Thus AT2 mediates a depressor effect and antagonizes the AT1-mediated pressor action of angiotensin II. In addition, disruption of the AT2 gene attenuated exploratory behaviour and lowered body temperature. Our results show that angiotensin II activates AT1 and AT2, which have mutually counteracting haemodynamic effects, and that AT2 regulates central nervous system functions, including behaviour.