RESUMO
During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.
Assuntos
Epilepsia/genética , Proteínas/genética , Espasmos Infantis/genética , Transmissão Sináptica , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Modelos Animais de Doenças , Epilepsia/diagnóstico , Fibroblastos/metabolismo , Técnicas de Genotipagem , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Proteínas/metabolismo , Células de Purkinje/metabolismo , Espasmos Infantis/diagnóstico , Vesículas Sinápticas/metabolismo , Transcriptoma , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
BACKGROUND: The tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3 Tg/Tg homozygotes are not viable and TgTP6.3 Tg/- hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3. RESULTS: Although TgTP6.4 Tg/Tg homozygotes died before weaning, TgTP6.4 Tg/- hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4 Tg/- hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex. CONCLUSIONS: Mosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing.
Assuntos
Linhagem da Célula , Camundongos Transgênicos/genética , Mosaicismo , Transgenes/genética , Proteínas tau/genética , Animais , Expressão Gênica , CamundongosRESUMO
Recent reports of a new generation of ubiquitous transgenic chimaera markers prompted us to consider the criteria used to evaluate new chimaera markers and develop more objective assessment methods. To investigate this experimentally we used several series of fetal and adult chimaeras, carrying an older, multi-copy transgenic marker. We used two additional independent markers and objective, quantitative criteria for cell selection and cell mixing to investigate quantitative and spatial aspects of developmental neutrality. We also suggest how the quantitative analysis we used could be simplified for future use with other markers. As a result, we recommend a five-step procedure for investigators to evaluate new chimaera markers based partly on criteria proposed previously but with a greater emphasis on examining the developmental neutrality of prospective new markers. These five steps comprise (1) review of published information, (2) evaluation of marker detection, (3) genetic crosses to check for effects on viability and growth, (4) comparisons of chimaeras with and without the marker and (5) analysis of chimaeras with both cell populations labelled. Finally, we review a number of different chimaera markers and evaluate them using the extended set of criteria. These comparisons indicate that, although the new generation of ubiquitous fluorescent markers are the best of those currently available and fulfil most of the criteria required of a chimaera marker, further work is required to determine whether they are developmentally neutral.
Assuntos
Biomarcadores/metabolismo , Quimera/genética , Desenvolvimento Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Degeneração Retiniana/genética , Transgenes/fisiologia , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Especificidade de ÓrgãosRESUMO
Genetic approaches that allow lineage tracing are essential to our future understanding of melanocytes and melanoma. To date, the approaches used to label melanocytes in mice have relied on random integration of transgenes driven by the promoters of the Tyrosinase and Dopachrome tautomerase genes, knock-in to the Dopachrome tautomerase locus or knock-in to the Mlana locus in a bacterial artificial chromosome. These strategies result in expression in other tissues such as telencephalon and other cell types such as nerves. Here we used homologous recombination in mouse embryonic stem cells to generate a targeted multicistronic allele of the Pmel locus that drives melanocyte-specific expression of CreERT2, nuclear localised H2B-Cerulean and membrane localised marcks-mKate2 allowing live imaging of melanocytes and activation of other conditional alleles. We combined this allele with R26R-EYFP mice allowing induction of EYFP expression on administration of tamoxifen or its metabolite 4-OHT. The fluorescent proteins H2B-Cerulean and marcks-mKate2 label the cell nucleus and plasma membrane respectively allowing live imaging and FACS isolation of melanoblasts and melanocytes as well as serving to provide an internal control allowing estimation of recombination efficiency after administration of tamoxifen. We demonstrate the utility of the transgene in embryonic and adult tissues.
Assuntos
Melanócitos , Melanoma , Camundongos , Animais , Camundongos Transgênicos , Alelos , Melanócitos/metabolismo , Melanoma/metabolismo , Tamoxifeno/metabolismo , Tamoxifeno/farmacologiaRESUMO
The relative extent of cell mixing in tissues of mouse chimaeras or mosaics can be studied by comparing the distributions of the two cell populations in the tissues. However, the mean patch size is misleading because it is affected by both the extent of cell mixing and the relative contributions of the two cell populations. Previous work suggested that effects attributable to differences in tissue composition among chimaeras can be factored out either by correcting the mean patch size or by using the median patch size for the minority cell population and restricting the analysis to grossly unbalanced chimaeras. In the present study, computer simulations of two-dimensional mosaic arrays of black and white squares (representing cells) were used to simulate chimaeric tissues. Random arrays simulated tissues with extensive cell mixing, arrays of cell clumps (representing coherent clones) simulated less mixed tissues, and striped arrays simulated tissues with elongated but fragmented descendent clones. The computer simulations predicted that (i) the median patch length (minority cell population) and the corrected mean patch length would both distinguish between random and clumped patterns and (ii) differences in the variation of the composition of two perpendicular series of one-dimensional transects would distinguished between stripes and randomly orientated patches. Both predictions were confirmed by analysis of histological sections of the retinal pigment epithelium from fetal and adult mouse chimaeras. This study demonstrates that two types of non-random two-dimensional variegated patterns (clumps and stripes) can be identified in chimaeras without two-dimensional reconstruction of serial sections.
Assuntos
Quimera , Simulação por Computador , Epitélio Pigmentado da Retina/citologia , Animais , Simulação por Computador/normas , Camundongos , Camundongos Endogâmicos BALB C , Epitélio Pigmentado da Retina/embriologiaRESUMO
Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits and demonstrate an accumulation of 'coat-less' vesicles that fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.
Most human cells have at least one small hair-like structure on their surface called a cilium. These structures can act as antennae and allow the cell to sense signals from the rest of the body. To do this, they contain proteins that differ from the rest of the cell. The content of cilia depends on regulated delivery of these proteins in and out of cilia by a process called the intraflagellar transport or IFT, which involves a large complex made of several proteins. This complex shuttles the cargo proteins back and forth between the base and the tip of the cilia. However, ciliary proteins are not produced in the cilia; instead, they are made in a different part of the cell and then they are transported to the ciliary base. At the point where they enter the cilia, they were thought to bind to the assembling IFT 'trains' and be transported across the ciliary gate to the positions where they are needed in cilia. One of the components of the IFT machinery is a protein called WDR35, also known as IFT121. If the gene that codes for this protein is faulty or missing, it results in severe disorders in both humans and mice including a range of potentially lethal skeletal dysplasias. Interestingly, without WDR35, cells cannot build functional cilia. The absence of this protein not only disrupts IFT, stopping certain ciliary proteins and their associated membranes from entering cilia; it also causes a 'traffic jam' with a pile-up of transport intermediates from the place in cell where they are made to the cilia. It is unclear why a mutation in one of the components of the IFT would have this effect, raising the question of whether WDR35, or IFTs a whole, has another role in bringing the cargo proteins into the cilia. To understand this phenomenon, Quidwai et al. analysed the structure of WDR35 and other IFT proteins and found that they are very similar to a protein complex called COPI, which is involved in transporting membrane proteins around the cell. When certain proteins are newly made, they are stored in small lipid bubbles called vesicles that then selectively move to where the proteins are needed. COPI coats these vesicles, helping them get to where they need to go in a process called vesicular transport. Quidwai et al. found that WDR35 and other IFT proteins are able to bind to specific types of lipid molecules, suggesting that they might be assisting in a form of vesicle transport too. Indeed, when mouse cells grown in the lab were genetically engineered so they could not produce WDR35, coatless vesicles accumulated around the base of the cilia. Adding back WDR35 to these mutant cells rescued these defects in vesicle transport to cilia as well as allowed functional cilia to be formed. These results provide evidence that WDR35, likely with other IFT proteins, acts as a COPI-like complex to deliver proteins to growing cilia. Further research will investigate the composition of these vesicles that transport proteins to cilia, and help pinpoint where they originate. Quidwai et al.'s findings not only shed light on how different genetic mutations found in patients with cilia dysfunction affect different steps of transporting proteins to and within cilia. They also increase our understanding of the cellular roadmap by which cells shuttle building blocks around in order to assemble these important 'antennae'.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Cílios/metabolismo , Proteínas do Citoesqueleto/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Animais , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Ligação Proteica , Transporte ProteicoRESUMO
The composition of adult mouse aggregation chimaeras is much more variable than X-inactivation mosaics. An early theoretical model proposed that almost all the extra variation in chimaeras arises, before X-inactivation occurs, by spatially constrained, geometrical allocation of inner cell mass (ICM) cells to the epiblast and primitive endoderm (PrE). However, this is inconsistent with more recent embryological evidence. Analysis of published results for chimaeric blastocysts and mid-gestation chimaeras suggested that some variation exists among chimaeric morulae and more variation arises both when morula cells are allocated to the ICM versus the trophectoderm (TE) and when ICM cells are allocated to the epiblast versus the PrE. Computer simulation results were also consistent with the conclusion that stochastic allocation of cells to blastocyst lineages in two steps, without the type of geometrical sampling that was originally proposed, could cause a wide variation in chimaeric epiblast composition. Later allocation events will cause additional variation among both chimaeras and X-inactivation mosaics. We also suggest that previously published U-shaped frequency distributions for chimaeric placenta composition might be explained by how TE cells are allocated to the polar TE and/or the subsequent movement of cells from polar TE to mural TE.
RESUMO
It has been shown previously that BALB/c strain embryos tend to contribute poorly to mouse aggregation chimaeras. In the present study we showed that BALB/c cells were not preferentially allocated to any extraembryonic lineages of mouse aggregation chimaeras, but their contribution decreased during the early postimplantation period and they were significantly depleted by E8.5. The development of BALB/c strain preimplantation embryos lagged behind embryos from some other strains and the contribution that BALB/c and other embryos made to chimaeras correlated with their developmental stage at E2.5. This relationship suggests that the poor contribution of BALB/c embryos to aggregation chimaeras is at least partly a consequence of generalised selection related to slow or delayed preimplantation development. The suitability of BALB/c embryos for maximising the ES cell contribution to mouse ES cell chimaeras is also discussed.
RESUMO
Isocitrate dehydrogenase (IDH) is an enzyme required for the production of α-ketoglutarate from isocitrate. IDH3 generates the NADH used in the mitochondria for ATP production, and is a tetramer made up of two α, one ß and one γ subunit. Loss-of-function and missense mutations in both IDH3A and IDH3B have previously been implicated in families exhibiting retinal degeneration. Using mouse models, we investigated the role of IDH3 in retinal disease and mitochondrial function. We identified mice with late-onset retinal degeneration in a screen of ageing mice carrying an ENU-induced mutation, E229K, in Idh3a Mice homozygous for this mutation exhibit signs of retinal stress, indicated by GFAP staining, as early as 3â months, but no other tissues appear to be affected. We produced a knockout of Idh3a and found that homozygous mice do not survive past early embryogenesis. Idh3a-/E229K compound heterozygous mutants exhibit a more severe retinal degeneration compared with Idh3aE229K/E229K homozygous mutants. Analysis of mitochondrial function in mutant cell lines highlighted a reduction in mitochondrial maximal respiration and reserve capacity levels in both Idh3aE229K/E229K and Idh3a-/E229K cells. Loss-of-function Idh3b mutants do not exhibit the same retinal degeneration phenotype, with no signs of retinal stress or reduction in mitochondrial respiration. It has previously been reported that the retina operates with a limited mitochondrial reserve capacity and we suggest that this, in combination with the reduced reserve capacity in mutants, explains the degenerative phenotype observed in Idh3a mutant mice.This article has an associated First Person interview with the first author of the paper.
Assuntos
Isocitrato Desidrogenase/genética , Mitocôndrias/patologia , Mutação/genética , Degeneração Retiniana/genética , Degeneração Retiniana/fisiopatologia , Animais , Fibroblastos/metabolismo , Genótipo , Isocitrato Desidrogenase/metabolismo , Mutação com Perda de Função/genética , Camundongos , Mutação de Sentido Incorreto/genética , Fenótipo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Retina/patologia , Retina/fisiopatologiaRESUMO
Molecular chaperones promote the folding and macromolecular assembly of a diverse set of 'client' proteins. How ubiquitous chaperone machineries direct their activities towards specific sets of substrates is unclear. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone that confers specificity for the FKBP8-HSP90 chaperone complex towards axonemal dynein clients required for cilia motility. Loss of ZMYND10 perturbs the chaperoning of axonemal dynein heavy chains, triggering broader degradation of dynein motor subunits. We show that pharmacological inhibition of FKBP8 phenocopies dynein motor instability associated with the loss of ZMYND10 in airway cells and that human disease-causing variants of ZMYND10 disrupt its ability to act as an FKBP8-HSP90 co-chaperone. Our study indicates that primary ciliary dyskinesia (PCD), caused by mutations in dynein assembly factors disrupting cytoplasmic pre-assembly of axonemal dynein motors, should be considered a cell-type specific protein-misfolding disease.
Assuntos
Axonema/metabolismo , Cílios/metabolismo , Proteínas de Ligação a DNA/genética , Dineínas/química , Proteínas de Choque Térmico HSP90/genética , Chaperonas Moleculares/genética , Proteínas de Ligação a Tacrolimo/genética , Animais , Animais Recém-Nascidos , Axonema/ultraestrutura , Sequência de Bases , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular , Cílios/ultraestrutura , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/metabolismo , Dineínas/genética , Dineínas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Cultura Primária de Células , Proteínas de Ligação a Tacrolimo/metabolismo , Traqueia/citologia , Traqueia/metabolismoRESUMO
The cilia and cell cycles are inextricably linked. Centrioles in the basal body of cilia nucleate the ciliary axoneme and sequester pericentriolar matrix (PCM) at the centrosome to organize the mitotic spindle. Cilia themselves respond to growth signals, prompting cilia resorption and cell cycle re-entry. We describe a fluorescent cilia and cell cycle biosensor allowing live imaging of cell cycle progression and cilia assembly and disassembly kinetics in cells and inducible mice. We define assembly and disassembly in relation to cell cycle stage with single-cell resolution and explore the intercellular heterogeneity in cilia kinetics. In all cells and tissues analyzed, we observed cilia that persist through the G1/S transition and into S/G2/M-phase. We conclude that persistence of cilia after the G1/S transition is a general property. This resource will shed light at an individual cell level on the interplay between the cilia and cell cycles in development, regeneration, and disease.
Assuntos
Ciclo Celular/fisiologia , Centríolos/metabolismo , Centrossomo/metabolismo , Cílios/metabolismo , Animais , Corpos Basais/metabolismo , Técnicas Biossensoriais/métodos , Proteínas de Ciclo Celular/metabolismo , Cinética , Camundongos , Microtúbulos/metabolismoRESUMO
The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1(-/-) null mouse embryos die but a previous study showed that some homozygous Gpi1(-/-) null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1(-/-)âGpi1(c/c) chimaera with functional Gpi1(-/-) null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1(-/-) null cells in adult Gpi1(-/-)âGpi1(c/c) chimaeras and determine if Gpi1(-/-) null germ cells are functional. Analysis of adult Gpi1(-/-)âGpi1(c/c) chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1(-/-) null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1(-/-) null oocytes in one female Gpi1(-/-)âGpi1(c/c) chimaera were functional and provided preliminary evidence that one male putative Gpi1(-/-)âGpi1(c/c) chimaera produced functional spermatozoa from homozygous Gpi1(-/-) null germ cells. Although the male chimaera was almost certainly Gpi1(-/-)âGpi1(c/c), this part of the study is considered preliminary because only blood was typed for GPI. Gpi1(-/-) null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1(-/-) null germ cells, it successfully identified functional Gpi1(-/-) null oocytes and revealed that some Gpi1(-/-) null cells could survive in many adult tissues.
RESUMO
Bands of colour extending laterally from the dorsal to ventral trunk are a common feature of mouse chimeras. These stripes were originally taken as evidence of the directed dorsoventral migration of melanoblasts (the embryonic precursors of melanocytes) as they colonize the developing skin. Depigmented 'belly spots' in mice with mutations in the receptor tyrosine kinase Kit are thought to represent a failure of this colonization, either due to impaired migration or proliferation. Tracing of single melanoblast clones, however, has revealed a diffuse distribution with high levels of axial mixing--hard to reconcile with directed migration. Here we construct an agent-based stochastic model calibrated by experimental measurements to investigate the formation of diffuse clones, chimeric stripes and belly spots. Our observations indicate that melanoblast colonization likely proceeds through a process of undirected migration, proliferation and tissue expansion, and that reduced proliferation is the cause of the belly spots in Kit mutants.
Assuntos
Pigmentos Biológicos/fisiologia , Animais , Embrião de Mamíferos/fisiologia , Camundongos , Modelos Biológicos , Pele/metabolismo , Técnicas de Cultura de TecidosRESUMO
Human confined placental mosaicism (CPM), where the placental trophoblast is mosaic for a chromosome abnormality but the fetus is chromosomally normal, can cause problems for prenatal diagnosis, but its causes are poorly understood. Tetraploid<-->diploid chimeras provide a model for the development of one type of CPM, but animal models for other types of restricted mosaicism are needed. The objective of the present study was to evaluate triploid<-->diploid and trisomy-3<-->diploid chimeric mouse conceptuses as new models for investigating the development of restricted mosaicism. Novel stocks of mice were generated to produce triploid and trisomy-3 embryos that could be identified by DNA in situ hybridisation to a chromosome 3 transgenic marker. Triploid<-->diploid and trisomy-3<-->diploid mouse chimeras were produced by embryo aggregation, and the contribution of triploid or trisomy-3 cells was analysed in the fetus and extraembryonic tissues. Only two trisomy-3<-->diploid chimeras were analysed but trisomy-3 cells contributed well to all lineages, so these chimeras did not show restricted mosaicism. In contrast, triploid cells usually contributed poorly to all lineages in the ten 3n<-->2n chimeras analysed. They contributed more to the primitive endoderm derivatives than other lineages and were present in the primitive endoderm derivatives of all ten chimeras, but excluded from fetuses and trophectoderm derivatives in some cases. This pattern of restricted mosaicism differs from that reported for tetraploid cells in tetraploid<-->diploid chimeras, and triploid<-->diploid chimeras may provide a useful model for the development of some types of restricted mosaicism in human conceptuses.
Assuntos
Modelos Animais , Mosaicismo/embriologia , Ploidias , Trofoblastos/fisiologia , Animais , Cruzamento , Linhagem da Célula/genética , Quimera , Diploide , Membranas Extraembrionárias/fisiologia , Feminino , Feto/fisiologia , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos , Placenta/fisiologia , Gravidez , TrissomiaRESUMO
The mouse TgTP6.3 transgene, encoding a tauGFP fusion protein, is becoming widely used but has yet to be fully characterized and evaluated as suitable lineage marker. The aim of the present study was to investigate the phenotype of TgTP6.3(+/+) homozygotes and TgTP6.3(+/-) hemizygotes, characterize the expression of the TgTP6.3 transgene in different tissues and critically evaluate its use as a lineage marker. TgTP6.3(+/+) homozygotes died between embryonic day 14.5 and weaning, whereas TgTP6.3(+/-) hemizygotes were mostly viable and fertile but smaller than non-transgenic siblings. TgTP6.3 expression began in the late two-cell stage, persisted in most fetal and adult tissues and was uniformly expressed in many (but not all) tissues. TgTP6.3(+/-) cells were readily identified in many chimeric tissues and their contribution appeared to be quantitatively and spatially normal. Overall, tauGFP expression in hemizygous TgTP6.3(+/-) cells fulfils the main criteria of a good lineage marker for many tissues. It provides a useful lineage marker, which should be particularly suitable for axons, blood vessels and pre-implantation embryos.
Assuntos
Quimera , Marcadores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas tau/genética , Animais , Axônios/metabolismo , Vasos Sanguíneos/metabolismo , Linhagem da Célula , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Heterozigoto , Homozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Microscopia Confocal , Proteínas Recombinantes de Fusão/genética , Sensibilidade e Especificidade , Transgenes , Proteínas tau/metabolismoRESUMO
Cell ploidy in the ovarian follicle and corpus luteum was investigated by DNA in situ hybridization to a reiterated, chromosome 3 transgene in mice that were hemizygous for the transgene. This approach was first validated by analysis of mouse kidney, pancreas and liver control tissues, which contain different frequencies of polyploid nuclei. Polyploid nuclei (with multiple hybridization signals) were seen in histological sections of both ovarian follicles and corpora lutea. The frequency of polyploid nuclei in follicles showed no consistent relationship with age (between 6 weeks and 10 months) but polyploid nuclei were significantly more abundant in corpora lutea than follicles (6.3% vs. 2.5%). This implies that production of polyploid cells is more closely associated with differentiation of ovarian follicles into corpora lutea than with the age of the female. Polyploidy tended to be more frequent in corpora lutea of mice that had mated even if they did not become pregnant. This study has highlighted the presence of polyploid cells in the mouse ovarian follicle and corpus luteum and has identified mating as a possible trigger for polyploidy in the corpus luteum. Further work is required to determine the physiological role of polyploid ovarian cells in reproduction.
Assuntos
Cromossomos Humanos Par 3 , Ovário/fisiologia , Poliploidia , Animais , Copulação/fisiologia , Corpo Lúteo/citologia , Corpo Lúteo/fisiologia , Feminino , Humanos , Hibridização In Situ/métodos , Masculino , Camundongos , Camundongos Transgênicos , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Ovário/citologia , GravidezRESUMO
Patterns of growth and cell movement in the developing and adult corneal epithelium were investigated by analysing clonal patches of LacZ-expressing cells in chimeric and X-inactivation mosaic mice. It was found that cell proliferation throughout the basal corneal epithelium during embryogenesis and early postnatal life creates a disordered mosaic pattern of LacZ(+) clones that contrasts with patterns of proliferation and striping produced during the later embryonic stages of retinal pigmented epithelium development. The early mosaic pattern in the corneal epithelium is replaced in the first 12 postnatal weeks by an ordered pattern of radial stripes or sectors that reflects migration without mixing of the progeny of clones of limbal stem cells. In contrast to previous assumptions, it was found that maturation of the activity of limbal stem cells and the pattern of migration of their progeny are delayed for several weeks postnatally. No evidence was found for immigration of the progeny of stem cells until the 5th postnatal week. There are approximately 100 clones of limbal stem cells initially, and clones are lost during postnatal life. Our studies provide a new assay for limbal and corneal defects in mutant mice.