RESUMEN
Sex-determining region Y (Sry)-box (Sox)9 is required for chondrogenesis as a transcriptional activator of genes related to chondrocyte proliferation, differentiation, and cartilage-specific extracellular matrix. Although there have been studies investigating the Sox9-dependent transcriptional complexes, not all their components have been identified. In the present study, we demonstrated that thyroid hormone receptor-associated protein (THRAP)3 is a component of a SOX9 transcriptional complex by liquid chromatography mass spectrometric analysis of FLAG-tagged Sox9-binding proteins purified from FLAG-HA-tagged Sox9 knock-in mice. Thrap3 knockdown in ATDC5 chondrogenic cells increased the expression of Collagen type II alpha 1 chain (Col2a1) without affecting Sox9 expression. THRAP3 and SOX9 overexpression reduced Col2a1 levels to a greater degree than overexpression of SOX9 alone. The negative regulation of SOX9 transcriptional activity by THRAP3 was mediated by interaction between the proline-, glutamine-, and serine-rich domain of SOX9 and the innominate domain of THRAP3. These results indicate that THRAP3 negatively regulates SOX9 transcriptional activity as a cofactor of a SOX9 transcriptional complex during chondrogenesis.
Asunto(s)
Condrogénesis , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Núcleo Celular/metabolismo , Condrocitos/citología , Condrocitos/metabolismo , Técnicas de Sustitución del Gen , Placa de Crecimiento/metabolismo , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Unión ProteicaRESUMEN
Mutations in receptors, ion channels, and enzymes are frequently recognized by the cellular quality control system as misfolded and retained in the endoplasmic reticulum (ER) or otherwise misrouted. Retention results in loss of function at the normal site of biological activity and disease. Pharmacoperones are target-specific small molecules that diffuse into cells and serve as folding templates that enable mutant proteins to pass the criteria of the quality control system and route to their physiologic site of action. Pharmacoperones of the gonadotropin releasing hormone receptor (GnRHR) have efficacy in cell culture systems, and their cellular and biochemical mechanisms of action are known. Here, we show the efficacy of a pharmacoperone drug in a small animal model, a knock-in mouse, expressing a mutant GnRHR. This recessive mutation (GnRHR E(90)K) causes hypogonadotropic hypogonadism (failed puberty associated with low or apulsatile luteinizing hormone) in both humans and in the mouse model described. We find that pulsatile pharmacoperone therapy restores E(90)K from ER retention to the plasma membrane, concurrently with responsiveness to the endogenous natural ligand, gonadotropin releasing hormone, and an agonist that is specific for the mutant. Spermatogenesis, proteins associated with steroid transport and steroidogenesis, and androgen levels were restored in mutant male mice following pharmacoperone therapy. These results show the efficacy of pharmacoperone therapy in vivo by using physiological, molecular, genetic, endocrine and biochemical markers and optimization of pulsatile administration. We expect that this newly appreciated approach of protein rescue will benefit other disorders sharing pathologies based on misrouting of misfolded protein mutants.
Asunto(s)
Hipogonadismo/tratamiento farmacológico , Chaperonas Moleculares/farmacología , Pliegue de Proteína/efectos de los fármacos , Deficiencias en la Proteostasis/genética , Receptores LHRH/genética , Testículo/fisiología , Animales , Biomarcadores/metabolismo , Retículo Endoplásmico/metabolismo , Técnicas de Sustitución del Gen , Hipogonadismo/genética , Masculino , Ratones , Chaperonas Moleculares/uso terapéutico , Mutación/genética , Testículo/efectos de los fármacosRESUMEN
Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J×FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.
Asunto(s)
Albinismo/genética , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Endonucleasas/genética , Edición Génica/métodos , Monofenol Monooxigenasa/genética , Mosaicismo/embriología , Pigmentación/genética , Alelos , Animales , Secuencia de Bases , Proteína 9 Asociada a CRISPR , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Melaninas/genética , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Mutación , ARN Mensajero/genética , Análisis de Secuencia de ADN , Cigoto/citología , ARN Pequeño no TraducidoRESUMEN
Transcriptional enhancers are genomic sequences bound by transcription factors that act together with basal transcriptional machinery to regulate gene transcription. Several high-throughput methods have generated large datasets of tissue-specific enhancer sequences with putative roles in developmental processes. However, few enhancers have been deleted from the genome to determine their roles in development. To understand the roles of two enhancers active in the mouse embryonic limb bud we deleted them from the genome. Although the genes regulated by these enhancers are unknown, they were selected because they were identified in a screen for putative limb bud-specific enhancers associated with p300, an acetyltransferase that participates in protein complexes that promote active transcription, and because the orthologous human enhancers (H1442 and H280) drive distinct lacZ expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. We show that the orthologous mouse sequences, M1442 and M280, regulate dynamic expression in the developing limb. Although significant transcriptional differences in enhancer-proximal genes in embryonic limb buds accompany the deletion of M1442 and M280 no gross limb malformations during embryonic development were observed, demonstrating that M1442 and M280 are not required for mouse limb development. However, M280 is required for the development and/or maintenance of body size; M280 mice are significantly smaller than controls. M280 also harbors an "ultraconserved" sequence that is identical between human, rat, and mouse. This is the first report of a phenotype resulting from the deletion of an ultraconserved element. These studies highlight the importance of determining enhancer regulatory function by experiments that manipulate them in situ and suggest that some of an enhancer's regulatory capacities may be developmentally tolerated rather than developmentally required.
Asunto(s)
Embrión de Mamíferos/embriología , Extremidades/embriología , Regulación del Desarrollo de la Expresión Génica , Animales , Huesos del Carpo/embriología , Huesos del Carpo/metabolismo , Elementos de Facilitación Genéticos , Humanos , Operón Lac , Esbozos de los Miembros/metabolismo , Ratones , Ratones Transgénicos , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
In sexual species, fertilization of oocytes produces individuals with alleles derived from both parents. Here we use pluripotent stem cells derived from somatic cells to combine the haploid genomes from two males to produce viable sons and daughters. Male (XY) mouse induced pluripotent stem cells (Father #1) were used to isolate subclones that had spontaneously lost the Y chromosome to become genetically female (XO). These male-derived XO stem cells were used to generate female chimeras that were bred with genetically distinct males (Father #2), yielding progeny possessing genetic information that was equally derived from both fathers. Thus, functional oocytes can be generated from male somatic cells after reprogramming and spontaneous sex reversal. These findings have novel implications for mammalian reproduction and assisted reproductive technology.
Asunto(s)
Quimera/embriología , Clonación de Organismos/métodos , Padre , Animales , Supervivencia Celular , Células Cultivadas , Quimera/genética , Embrión de Mamíferos , Femenino , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Pluripotentes Inducidas/trasplante , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Técnicas de Transferencia Nuclear , Caracteres SexualesRESUMEN
The success of postnatal uterine morphogenesis dictates, in part, the embryotrophic potential and functional capacity of the adult uterus. The definitive role of Wnt7a in postnatal uterine development and adult function requires a conditional knockout, because global deletion disrupts müllerian duct patterning, specification, and cell fate in the fetus. The Wnt7a-null uterus appears to be posteriorized because of developmental defects in the embryo, as evidenced by the stratified luminal epithelium that is normally found in the vagina and the presence of short and uncoiled oviducts. To understand the biological role of WNT7A after birth and allow tissue-selective deletion of Wnt7a, we generated loxP-flanked exon 2 mice and conditionally deleted Wnt7a after birth in the uterus by crossing them with Pgr(Cre) mice. Morphological examination revealed no obvious differences in the vagina, cervix, oviduct, or ovary. The uteri of Wnt7a mutant mice contained no endometrial glands, whereas all other uterine cell types appeared to be normal. Postnatal differentiation of endometrial glands was observed in control mice, but not in mutant mice, between Postnatal Days 3 and 12. Expression of morphoregulatory genes, particularly Foxa2, Hoxa10, Hoxa11, Msx1, and Wnt16, was disrupted in the Wnt7a mutant uteri. Conditional Wnt7a mutant mice were not fertile. Although embryos were present in uteri of mutant mice on Day 3.5 of pregnancy, blastocyst implantation was not observed on Day 5.5. Furthermore, expression of several genes (Foxa2, Lif, Msx1, and Wnt16) was reduced or absent in adult Wnt7a-deleted uteri on Day 3.5 postmating. These results indicate that WNT7A plays a critical role in postnatal uterine gland morphogenesis and function, which are important for blastocyst implantation and fertility in the adult uterus.
Asunto(s)
Fertilidad/fisiología , Eliminación de Gen , Útero/crecimiento & desarrollo , Proteínas Wnt/metabolismo , Animales , Proliferación Celular , Implantación del Embrión/fisiología , Femenino , Fertilidad/genética , Regulación de la Expresión Génica/fisiología , Ratones , Útero/citología , Útero/metabolismo , Proteínas Wnt/genéticaRESUMEN
Sox9 belongs to the family of Sry-related high-mobility group box transcription factors controlling cell fate, cell proliferation and differentiation in various tissues, including cartilage, testis, the central nervous system, kidney, and gastrointestine. Mice conditionally lacking Sox9 have revealed essential roles for Sox9 in these tissues. To gain further insight into the role of Sox9 in different tissues and at different stages of development, we have generated a transgenic mouse line to express Sox9 in a Cre recombinase-dependent manner. The construct contained the human cytomegalovirus enhancer and chicken ß-actin promoter, and a floxed monomeric red fluorescence protein 1 (mRFP1) cassette to direct ubiquitous expression of mRFP1. Cre expression removed the mRFP1 gene, allowing expression of Sox9 and enhanced green fluorescent protein. Expectedly, overexpression of Sox9 in chondrocytes using Col2a1-Cre mice suppressed chondrocyte hypertrophy, and delayed terminal differentiation and subsequent ossification. Misexpression of Sox9 in hypertrophic chondrocytes using Col10a1-Cre knock-in mice also delayed terminal differentiation. This Sox9 conditional transgenic mouse line will be a valuable tool to uncover tissue-specific and developmental stage-specific functions of Sox9.
Asunto(s)
Factor de Transcripción SOX9/metabolismo , Animales , Diferenciación Celular/fisiología , Condrocitos/citología , Condrocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción SOX9/genéticaRESUMEN
In mice, the Ter mutation causes primordial germ cell (PGC) loss in all genetic backgrounds. Ter is also a potent modifier of spontaneous testicular germ cell tumour (TGCT) susceptibility in the 129 family of inbred strains, and markedly increases TGCT incidence in 129-Ter/Ter males. In 129-Ter/Ter mice, some of the remaining PGCs transform into undifferentiated pluripotent embryonal carcinoma cells, and after birth differentiate into various cells and tissues that compose TGCTs. Here, we report the positional cloning of Ter, revealing a point mutation that introduces a termination codon in the mouse orthologue (Dnd1) of the zebrafish dead end (dnd) gene. PGC deficiency is corrected both with bacterial artificial chromosomes that contain Dnd1 and with a Dnd1-encoding transgene. Dnd1 is expressed in fetal gonads during the critical period when TGCTs originate. DND1 has an RNA recognition motif and is most similar to the apobec complementation factor, a component of the cytidine to uridine RNA-editing complex. These results suggest that Ter may adversely affect essential aspects of RNA biology during PGC development. DND1 is the first protein known to have an RNA recognition motif directly implicated as a heritable cause of spontaneous tumorigenesis. TGCT development in the 129-Ter mouse strain models paediatric TGCT in humans. This work will have important implications for our understanding of the genetic control of TGCT pathogenesis and PGC biology.
Asunto(s)
Células Germinativas/metabolismo , Células Germinativas/patología , Mutación/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Testiculares/genética , Neoplasias Testiculares/patología , Alelos , Animales , Secuencia de Bases , Peso Corporal , Mapeo Cromosómico , Cromosomas de los Mamíferos/genética , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Hibridación in Situ , Masculino , Ratones , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/metabolismo , Neoplasias de Células Germinales y Embrionarias/patología , Tamaño de los Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias Testiculares/metabolismo , Testículo/embriología , Testículo/metabolismo , Testículo/patologíaRESUMEN
Sox9 expression defines cell progenitors in a variety of tissues during mouse embryogenesis. To establish a genetic tool for cell-lineage tracing and gene-function analysis, we generated mice in which the CreERT2 gene was targeted to the endogenous mouse Sox9 locus. In Sox9(CreERT2/+) ;R26R embryos, tamoxifen activated Cre recombinase exclusively in Sox9-expressing tissues. To determine the suitability of this mouse line for developmental stage-specific gene recombination, we investigated the cellular origins of the cruciate ligaments of the knee joint and the limb tendons, in which precursor cells have not been defined. The cells in these tissues were labeled after tamoxifen treatment before or at the stage of chondrogenic mesenchymal condensation, indicating that ligament and tendon cells originated from Sox9-expressing cells and that cell fate determination occurred at mesenchymal condensation. This mouse line is a valuable tool for the temporal genetic tracing of the progeny of, and inducible gene modification in Sox9-expressing cells.
Asunto(s)
Ligamento Cruzado Anterior/embriología , Articulación de la Rodilla/embriología , Ligamento Cruzado Posterior/embriología , Factor de Transcripción SOX9/metabolismo , Células Madre/fisiología , Tendones/embriología , Animales , Ligamento Cruzado Anterior/citología , Ligamento Cruzado Anterior/metabolismo , Ligamento Cruzado Anterior/fisiología , Diferenciación Celular/genética , Células Cultivadas , Embrión de Mamíferos , Extremidades/embriología , Extremidades/fisiología , Femenino , Articulación de la Rodilla/metabolismo , Ratones , Ratones Transgénicos , Organogénesis/genética , Organogénesis/fisiología , Ligamento Cruzado Posterior/citología , Ligamento Cruzado Posterior/metabolismo , Ligamento Cruzado Posterior/fisiología , Embarazo , Factor de Transcripción SOX9/genética , Células Madre/metabolismo , Tendones/metabolismoRESUMEN
The function of cartilage in the adult is dependent on a host of regulatory molecules such as growth factors, extracellular matrix, enzymes, signaling molecules, and transcription factors. However, germline mutations in some genes that are expressed in adult cartilage lead to embryonic or perinatal lethality. To examine the function of these and other genes postnatally, we have generated a targeted mouse by homologous recombination that "knocks in" the inducible Cre recombinase construct, CreERT2, in the 3' untranslated region of the endogenous mouse aggrecan gene (Agc1(tm(IRES-creERT2))). The properties and efficiency of the inducible cre recombinase were tested by examining X-gal staining of tissues from embryos as well as growing and adult Agc1(tm(IRES-creERT2)/+);Rosa 26R mice. These mice were injected with the inducer, tamoxifen, at different time points during embryonic development and postnatally up to 6 months of age. Strong X-gal staining was observed in growth plate and articular cartilage as well as the fibrocartilage of meniscus, trachea, and intervertebral discs reproducing the pattern of endogenous aggrecan gene expression. In conclusion, we have generated a mouse model in which genes implicated in cartilage degenerative diseases can be inactivated in a spatial and temporal fashion in postnatal and adult mice.
Asunto(s)
Agrecanos/genética , Cartílago/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Integrasas/genética , Agrecanos/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Cartílago/embriología , Cartílago/crecimiento & desarrollo , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Placa de Crecimiento/embriología , Placa de Crecimiento/crecimiento & desarrollo , Placa de Crecimiento/metabolismo , Histocitoquímica , Hibridación in Situ , Integrasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Transgénicos , Embarazo , Tamoxifeno/farmacologíaRESUMEN
Fibroblast growth factor-8 (Fgf8) encodes a secreted protein which was initially identified as the factor responsible for androgen-dependant growth of mouse mammary carcinoma cells (Tanaka et al., 1992). Fgf8 has been subsequently implicated in the patterning and growth of the gastrulating embryo, paraxial mesoderm (somites), limbs, craniofacial tissues, central nervous system and other organ systems during the development of several vertebrate model animals. Consistent with these findings, Fgf8 is expressed in a complex and dynamic pattern during vertebrate embryogenesis. Here we report the isolation and characterization of a bat (Carollia perspicillata) Fgf8 orthologue. Compared with those of other model vertebrates, Carollia Fgf8 is conserved with respect to genomic structure, sequence and many domains of developmental expression pattern. Interestingly, the expression domain marking the apical ectodermal ridge of the developing limb shows a striking difference compared to that of mouse, consistent with evolutionary diversification of bat limb morphology.
Asunto(s)
Quirópteros/embriología , Factor 8 de Crecimiento de Fibroblastos/genética , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Quirópteros/genética , Codón de Terminación , Secuencia Conservada , ADN Complementario , Exones , Hibridación in Situ , Intrones , Modelos Genéticos , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Studying genes involved in organogenesis is often difficult because many of these genes are also essential for early development. The allotetraploid frog, Xenopus laevis, is commonly used to study developmental processes, but because of the presence of two homeologs for many genes, it has been difficult to use as a genetic model. Few studies have successfully used CRISPR in amphibians, and currently there is no tissue-targeted knockout strategy described in Xenopus The goal of this study is to determine whether CRISPR/Cas9-mediated gene knockout can be targeted to the Xenopus kidney without perturbing essential early gene function. We demonstrate that targeting CRISPR gene editing to the kidney and the eye of F0 embryos is feasible. Our study shows that knockout of both homeologs of lhx1 results in the disruption of kidney development and function but does not lead to early developmental defects. Therefore, targeting of CRISPR to the kidney may not be necessary to bypass the early developmental defects reported upon disruption of Lhx1 protein expression or function by morpholinos, antisense RNA, or dominant negative constructs. We also establish a control for CRISPR in Xenopus by editing a gene (slc45a2) that when knocked out results in albinism without altering kidney development. This study establishes the feasibility of tissue-specific gene knockout in Xenopus, providing a cost-effective and efficient method for assessing the roles of genes implicated in developmental abnormalities that is amenable to high-throughput gene or drug screening techniques.
Asunto(s)
Silenciador del Gen , Xenopus laevis/genética , Animales , Sistemas CRISPR-Cas , Edición Génica , Técnicas de Inactivación de Genes , Marcación de Gen , Riñón/metabolismo , Proteínas con Homeodominio LIM/genética , Especificidad de Órganos/genética , Fenotipo , ARN Guía de Kinetoplastida , Factores de Transcripción/genética , Proteínas de Xenopus/genéticaRESUMEN
Spindle poisons represent an important class of anticancer drugs that act by interfering with microtubule polymerization and dynamics and thereby induce mitotic checkpoints and apoptosis. Here we show that mammalian SNM1 functions in an early mitotic stress checkpoint that is distinct from the well-characterized spindle checkpoint that regulates the metaphase-to-anaphase transition. Specifically, we found that compared to wild-type cells, Snm1-deficient mouse embryonic fibroblasts exposed to spindle poisons exhibited elevated levels of micronucleus formation, decreased mitotic delay, a failure to arrest in mitosis prior to chromosome condensation, supernumerary centrosomes, and decreased viability. In addition, we show that both Snm1 and 53BP1, previously shown to interact, coimmunoprecipitate with components of the anaphase-promoting complex (APC)/cyclosome. These findings suggest that Snm1 is a component of a mitotic stress checkpoint that negatively targets the APC prior to chromosome condensation.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Mitosis , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Huso Acromático , Anafase , Animales , Antineoplásicos/farmacología , Apoptosis , Quinasas CDC2-CDC28/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Cromosomas/ultraestructura , ADN Complementario/metabolismo , Endodesoxirribonucleasas , Citometría de Flujo , Células HeLa , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metafase , Ratones , Microscopía Fluorescente , Microscopía por Video , Microtúbulos/metabolismo , Mutación , Nocodazol/farmacología , Fosfoproteínas/metabolismo , ARN Interferente Pequeño/metabolismo , Estrés Fisiológico , Factores de Tiempo , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Renal failure is a frequent and costly complication of many chronic diseases, including diabetes and hypertension. One common feature of renal failure is glomerulosclerosis, the pathobiology of which is unclear. To help elucidate this, we generated a mouse strain carrying the missense mutation Wt1 R394W, which predisposes humans to glomerulosclerosis and early-onset renal failure (Denys-Drash syndrome [DDS]). Kidney development was normal in Wt1(+/R394W) heterozygotes. However, by 4 months of age 100% of male heterozygotes displayed proteinuria and glomerulosclerosis characteristic of DDS patients. This phenotype was observed in an MF1 background but not in a mixed B6/129 background, suggestive of the action of a strain-specific modifying gene(s). WT1 encodes a nuclear transcription factor, and the R394W mutation is known to impair this function. Therefore, to investigate the mechanism of Wt1 R394W-induced renal failure, the expression of genes whose deletion leads to glomerulosclerosis (NPHS1, NPHS2, and CD2AP) was quantitated. In mutant kidneys, NPHS1 and NPHS2 were only moderately downregulated (25 to 30%) at birth but not at 2 or 4 months. Expression of CD2AP was not changed at birth but was significantly upregulated at 2 and 4 months. Podocalyxin was downregulated by 20% in newborn kidneys but not in kidneys at later ages. Two other genes implicated in glomerulosclerosis, TGFB1 and IGF1, were upregulated at 2 months and at 2 and 4 months, respectively. It is not clear whether the significant alterations in gene expression are a cause or a consequence of the disease process. However, the data do suggest that Wt1 R394W-induced glomerulosclerosis may be independent of downregulation of the genes for NPHS1, NPHS2, CD2AP, and podocalyxin and may involve other genes yet to be implicated in renal failure. The Wt1(R394W) mouse recapitulates the pathology and disease progression observed in patients carrying the same mutation, and the mutation is completely penetrant in male animals. Thus, it will be a powerful and biologically relevant model for investigating the pathobiology of the earliest events in glomerulosclerosis.
Asunto(s)
Síndrome de Denys-Drash/genética , Genes del Tumor de Wilms , Proteínas Adaptadoras Transductoras de Señales , Animales , Secuencia de Bases , División Celular , Proteínas del Citoesqueleto , ADN/genética , Síndrome de Denys-Drash/patología , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Electrónica , Fenotipo , Mutación Puntual , Proteínas/genética , Insuficiencia Renal/genética , Insuficiencia Renal/patología , Especificidad de la EspecieRESUMEN
Peutz-Jeghers syndrome (PJS) is a dominantly inherited disorder characterized by gastrointestinal hamartomatous polyps and mucocutaneous melanin pigmentation. Germ line mutations in LKB1 cause PJS. We have generated mice carrying an Lkb1 exon 2 to 8 deletion by gene targeting in embryonic stem cells. Heterozygotes develop gastric hamartomas that are histologically similar to those found in humans with PJS. LKB1 is also reportedly a mediator of p53-dependent apoptosis. To explore the potential combined effects of p53 and Lkb1 alterations on tumorigenesis, we carried out a series of matings with Lkb1(+/-) and p53 null mice to generate Lkb1(+/-)/p53(+/-) and Lkb1(+/-)/p53(-/-) mice. Similar to the Lkb1(+/-) mice, gastrointestinal hamartomas have also been detected in the mice with these two genotypes. The Lkb1(+/-)/p53(+/-) mice displayed a dramatically reduced life span and increased tumor incidence compared to the mice with either Lkb1 or p53 single gene knockout. The time to onset of polyposis in Lkb1(+/-)/p53(-/-) mice is approximately 2 months earlier than Lkb1(+/-)/p53(+/-) and Lkb1(+/-) mice, whereas the latter two show a similar time to onset which is at approximately 6 months of age. These results strongly suggested that mutations of p53 and Lkb1 gene cooperate in the acceleration of tumorigenesis.
Asunto(s)
Transformación Celular Neoplásica , Hamartoma/genética , Mutación , Síndrome de Peutz-Jeghers/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Quinasas Activadas por AMP , Animales , Hamartoma/patología , Heterocigoto , Incidencia , Pérdida de Heterocigocidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Síndrome de Peutz-Jeghers/patología , Tasa de SupervivenciaRESUMEN
To understand the physiological function of the mammalian heterotrimeric CCAAT binding factor CBF, also known as NF-Y, we have generated a conditional Cbf-b mouse mutant by introducing loxP sites in the murine Cbf-b/Nf-ya gene. Controlled expression of Cre recombinase deletes the gene in vivo, which leads to a loss of DNA binding by the CBF complex and hence CBF-mediated transcription. Deletion of both Cbf-b alleles causes early embryo lethality, indicating that CBF activity is essential for early mouse development. In primary cultures of mouse embryonic fibroblasts, conditional inactivation of CBF results in a block in cell proliferation and inhibition of S phase or DNA synthesis, which is followed by induction of apoptosis. We conclude that the CBF transcription factor complex is essential for cell proliferation and viability.
Asunto(s)
Factor de Unión a CCAAT/fisiología , Ratones/embriología , Alelos , Animales , Apoptosis/genética , Apoptosis/fisiología , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , División Celular/genética , División Celular/fisiología , Eliminación de Gen , Ratones/genética , Ratones Endogámicos C57BL , Fase S/genética , Fase S/fisiología , Transcripción GenéticaRESUMEN
Our previous studies indicated that the Jumonji C (JmjC)-domain-containing NO66 is a histone demethylase with specificity for methylated histone H3K4 and H3K36. NO66 binds to the transcription factor Osterix (Osx) and inhibits its transcriptional activity in promoter assays. However, the physiological role of NO66 in formation of mammalian bones is unknown. Here, using a genetically engineered mouse model, we show that during early skeletal development, Prx1-Cre-dependent mesenchymal deletion of NO66 promotes osteogenesis and formation of both endochondral as well as intramembranous skeletal elements, leading to a larger skeleton and a high bone mass phenotype in adult mice. The excess bone formation in mice where NO66 was deleted in cells of mesenchymal origin is associated with an increase in the number of preosteoblasts and osteoblasts. Further analysis revealed that in the embryonic limbs and adult calvaria of mice with deletion of NO66 in cells of mesenchymal origin, expression of several genes including bone morphogenetic protein 2 (Bmp2), insulin-like growth factor 1 (Igf1), and osteoclast inhibitor osteoprotegerin was increased, concurrent with an increase in expression of bone formation markers such as osterix (Osx), type I collagen, and bone sialoprotein (Bsp). Taken together, our results provide the first in vivo evidence that NO66 histone demethylase plays an important role in mammalian osteogenesis during early development as well as in adult bone homeostasis. We postulate that NO66 regulates bone formation, at least in part, via regulating the number of bone-forming cells and expression of multiple genes that are critical for these processes.
Asunto(s)
Huesos/metabolismo , Eliminación de Gen , Histona Demetilasas con Dominio de Jumonji/metabolismo , Mesodermo/metabolismo , Osteogénesis/genética , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Colágeno Tipo I/metabolismo , Femenino , Fluoresceínas/química , Regulación de la Expresión Génica , Genotipo , Histonas/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Sialoproteína de Unión a Integrina/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Masculino , Ratones , Ratones Noqueados , Osteoblastos/metabolismo , Fenotipo , Factor de Transcripción Sp7 , Microtomografía por Rayos XRESUMEN
Sox9 is a direct transcriptional activator of cartilage-specific extracellular matrix genes and has essential roles in chondrogenesis. Mutations in or around the SOX9 gene cause campomelic dysplasia or Pierre Robin Sequence. However, Sox9-dependent transcriptional control in chondrogenesis remains largely unknown. Here we identify Wwp2 as a direct target of Sox9. Wwp2 interacts physically with Sox9 and is associated with Sox9 transcriptional activity via its nuclear translocation. A yeast two-hybrid screen using a cDNA library reveals that Wwp2 interacts with Med25, a component of the Mediator complex. The positive regulation of Sox9 transcriptional activity by Wwp2 is mediated by the binding between Sox9 and Med25. In zebrafish, morpholino-mediated knockdown of either wwp2 or med25 induces palatal malformation, which is comparable to that in sox9 mutants. These results provide evidence that the regulatory interaction between Sox9, Wwp2 and Med25 defines the Sox9 transcriptional mechanisms of chondrogenesis in the forming palate.
Asunto(s)
Complejo Mediador/deficiencia , Hueso Paladar/metabolismo , Proteínas Recombinantes/metabolismo , Factor de Transcripción SOX9/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Animales , Displasia Campomélica/embriología , Displasia Campomélica/genética , Displasia Campomélica/metabolismo , Displasia Campomélica/patología , Cartílago/embriología , Cartílago/metabolismo , Cartílago/patología , Línea Celular , Condrogénesis/efectos de los fármacos , Condrogénesis/genética , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Complejo Mediador/genética , Ratones , Ratones Transgénicos , Morfolinas/farmacología , Mutación , Hueso Paladar/efectos de los fármacos , Hueso Paladar/embriología , Hueso Paladar/patología , Unión Proteica , ARN Interferente Pequeño , Proteínas Recombinantes/genética , Factor de Transcripción SOX9/genética , Transcripción Genética , Activación Transcripcional , Transfección , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/genética , Pez CebraRESUMEN
The liver and exocrine pancreas share a common structure, with functioning units (hepatic plates and pancreatic acini) connected to the ductal tree. Here we show that Sox9 is expressed throughout the biliary and pancreatic ductal epithelia, which are connected to the intestinal stem-cell zone. Cre-based lineage tracing showed that adult intestinal cells, hepatocytes and pancreatic acinar cells are supplied physiologically from Sox9-expressing progenitors. Combination of lineage analysis and hepatic injury experiments showed involvement of Sox9-positive precursors in liver regeneration. Embryonic pancreatic Sox9-expressing cells differentiate into all types of mature cells, but their capacity for endocrine differentiation diminishes shortly after birth, when endocrine cells detach from the epithelial lining of the ducts and form the islets of Langerhans. We observed a developmental switch in the hepatic progenitor cell type from Sox9-negative to Sox9-positive progenitors as the biliary tree develops. These results suggest interdependence between the structure and homeostasis of endodermal organs, with Sox9 expression being linked to progenitor status.
Asunto(s)
Mucosa Intestinal/metabolismo , Hígado/metabolismo , Páncreas/metabolismo , Factor de Transcripción SOX9/metabolismo , Células Madre/metabolismo , Animales , Diferenciación Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Intestinos/citología , Hígado/citología , Ratones , Ratones Noqueados , Páncreas/citología , Factor de Transcripción SOX9/genética , Células Madre/citologíaRESUMEN
Permanent and reversible genetic modifications are important approaches to study gene function in different cell types. They are also important for stem cell researchers to explore and test the therapeutic potential of stem cells. The piggyBac transposon from insects is a rising nonviral system that efficiently mutagenizes and mediates gene transfer into the mammalian genome. It is also characterized by its precise excision, leaving no trace sequence behind so that the genomic integrity of the mutated cell can be restored. Here, we use an optimized piggyBac transposon system to mediate gene transfer and expression of a bifunctional fluorescent reporter in human embryonic stem (ES) cells. We provide molecular evidence for transposase-mediated piggyBac integration events and functional evidence for successful expression of a transferred fluorescent protein genes in human ES cells and their in vitro differentiated derivatives. We also demonstrate that the integrated piggyBac transposon can be removed and an undisrupted insertion site can be restored, which implies potential applications for its use in gene therapy and genetics studies.