RESUMEN
BACKGROUND: Desmin is a major cytoskeletal protein considered ubiquitous in mature muscle fibers. However, we earlier reported that a subgroup of muscle fibers in the soft palate of healthy subjects and obstructive sleep apnea patients (OSA) lacked immunoexpression for desmin. This raised the question of whether these fibers also lack messenger ribonucleic acid (mRNA) for desmin and can be considered a novel fiber phenotype. Moreover, some fibers in the OSA patients had an abnormal distribution and aggregates of desmin. Thus, the aim of the study was to investigate if these desmin protein abnormalities are also reflected in the expression of desmin mRNA in an upper airway muscle of healthy subjects and OSA patients. METHODS: Muscle biopsies from the musculus uvulae in the soft palate were obtained from ten healthy male subjects and six male patients with OSA. Overnight sleep apnea registrations were done for all participants. Immunohistochemistry, in-situ hybridization, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) techniques were used to evaluate the presence of desmin protein and its mRNA. RESULTS: Our findings demonstrated that a group of muscle fibers lacked expression for desmin mRNA and desmin protein in healthy individuals and OSA patients (12.0 ± 5.6% vs. 23.1 ± 10.8%, p = 0.03). A subpopulation of these fibers displayed a weak subsarcolemmal rim of desmin accompanied by a few scattered mRNA dots in the cytoplasm. The muscles of OSA patients also differed from healthy subjects by exhibiting muscle fibers with reorganized or accumulated aggregates of desmin protein (14.5 ± 6.5%). In these abnormal fibers, the density of mRNA was generally low or concentrated in specific regions. The overall quantification of desmin mRNA by RT-qPCR was significantly upregulated in OSA patients compared to healthy subjects (p = 0.01). CONCLUSIONS: Our study shows evidence that muscle fibers in the human soft palate lack both mRNA and protein for desmin. This indicates a novel cytoskeletal structure and challenges the ubiquity of desmin in muscle fibers. Moreover, the observation of reorganized or accumulated aggregates of desmin mRNA and desmin protein in OSA patients suggests a disturbance in the transcription and translation process in the fibers of the patients.
Asunto(s)
Síndromes de la Apnea del Sueño , Apnea Obstructiva del Sueño , Humanos , Masculino , Desmina/genética , Apnea Obstructiva del Sueño/genética , ARN Mensajero/genética , Expresión GénicaRESUMEN
In muscular dystrophies, muscle fibers loose integrity and die, causing significant suffering and premature death. Strikingly, the extraocular muscles (EOMs) are spared, functioning well despite the disease progression. Although EOMs have been shown to differ from body musculature, the mechanisms underlying this inherent resistance to muscle dystrophies remain unknown. Here, we demonstrate important differences in gene expression as a response to muscle dystrophies between the EOMs and trunk muscles in zebrafish via transcriptomic profiling. We show that the LIM-protein Fhl2 is increased in response to the knockout of desmin, plectin and obscurin, cytoskeletal proteins whose knockout causes different muscle dystrophies, and contributes to disease protection of the EOMs. Moreover, we show that ectopic expression of fhl2b can partially rescue the muscle phenotype in the zebrafish Duchenne muscular dystrophy model sapje, significantly improving their survival. Therefore, Fhl2 is a protective agent and a candidate target gene for therapy of muscular dystrophies.
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Proteínas con Dominio LIM , Proteínas Musculares , Distrofia Muscular de Duchenne , Músculos Oculomotores , Animales , Proteínas del Citoesqueleto/metabolismo , Distrofina/genética , Expresión Génica Ectópica , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Músculos Oculomotores/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas Musculares/metabolismo , Proteínas con Dominio LIM/metabolismoRESUMEN
Purpose: The cytoskeleton of the extraocular muscles (EOMs) is significantly different from that of other muscles. We aimed to investigate the role of obscurin, a fundamental cytoskeletal protein, in the EOMs. Methods: The distribution of obscurin in human and zebrafish EOMs was compared using immunohistochemistry. The two obscurin genes in zebrafish, obscna and obscnb, were knocked out using CRISPR/Cas9, and the EOMs were investigated using immunohistochemistry, qPCR, and in situ hybridization. The optokinetic reflex (OKR) in five-day-old larvae and adult obscna-/-;obscnb-/- and sibling control zebrafish was analyzed. Swimming distance was recorded at the same age. Results: The obscurin distribution pattern was similar in human and zebrafish EOMs. The proportion of slow and fast myofibers was reduced in obscna-/-;obscnb-/- zebrafish EOMs but not in trunk muscle, whereas the number of myofibers containing cardiac myosin myh7 was significantly increased in EOMs of obscurin double mutants. Loss of obscurin resulted in less OKRs in zebrafish larvae but not in adult zebrafish. Conclusions: Obscurin expression is conserved in normal human and zebrafish EOMs. Loss of obscurin induces a myofiber type shift in the EOMs, with upregulation of cardiac myosin heavy chain, myh7, showing an adaptation strategy in EOMs. Our model will facilitate further studies in conditions related to obscurin.
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Músculos Oculomotores , Proteínas Serina-Treonina Quinasas , Factores de Intercambio de Guanina Nucleótido Rho , Pez Cebra , Animales , Humanos , Inmunohistoquímica , Músculo Esquelético/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Músculos Oculomotores/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT.
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G-Cuádruplex , Neoplasias , Fotoquimioterapia , Animales , ADN/metabolismo , Daño del ADN , Replicación del ADN , Inestabilidad Genómica , Neoplasias/genética , Neoplasias/terapia , Estrés Oxidativo , Fármacos Fotosensibilizantes/farmacología , Pez Cebra/genética , Pez Cebra/metabolismo , Fotoquimioterapia/métodosRESUMEN
BACKGROUND: Migrating muscle progenitors delaminate from the somite and subsequently form muscle tissue in distant anatomical regions such as the paired appendages, or limbs. In amniotes, this process requires a signaling cascade including the transcription factor paired box 3 (Pax3). RESULTS: In this study, we found that, unlike in mammals, pax3a/3b double mutant zebrafish develop near to normal appendicular muscle. By analyzing numerous mutant combinations of pax3a, pax3b and pax7a, and pax7b, we determined that there is a feedback system and a compensatory mechanism between Pax3 and Pax7 in this developmental process, even though Pax7 alone is not required for appendicular myogenesis. pax3a/3b/7a/7b quadruple mutant developed muscle-less pectoral fins. CONCLUSIONS: We found that Pax3 and Pax7 are redundantly required during appendicular myogenesis in zebrafish, where Pax7 is able to activate the same developmental programs as Pax3 in the premigratory progenitor cells.
Asunto(s)
Factores de Transcripción Paired Box , Pez Cebra , Animales , Mamíferos , Desarrollo de Músculos/genética , Músculo Esquelético , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX7/genética , Factores de Transcripción Paired Box/genética , Pez Cebra/genéticaRESUMEN
Purpose: To study the medial rectus (MR) muscle of zebrafish (Daniorerio) with respect to the pattern of distribution of desmin and its correlation to distinct types of myofibers and motor endplates. Methods: The MRs of zebrafish were examined using confocal microscopy in whole-mount longitudinal specimens and in cross sections processed for immunohistochemistry with antibodies against desmin, myosin heavy chain isoforms, and innervation markers. Desmin patterns were correlated to major myofiber type and type of innervation. A total of 1382 myofibers in nine MR muscles were analyzed. Results: Four distinct desmin immunolabeling patterns were found in the zebrafish MRs. Approximately a third of all slow myofibers lacked desmin, representing 8.5% of the total myofiber population. The adult zebrafish MR muscle displayed en grappe, en plaque, and multiterminal en plaque neuromuscular junctions (NMJs) with intricate patterns of desmin immunolabeling. Conclusions: The MRs of zebrafish showed important similarities with the human extraocular muscles with regard to the pattern of desmin distribution and presence of the major types of NMJs and can be regarded as an adequate model to further study the role of desmin and the implications of heterogeneity in cytoskeletal protein composition. Translational Relevance: The establishment of a zebrafish model to study the cytoskeleton in muscles that are particularly resistant to muscle disease opens new avenues to understand human myopathies and muscle dystrophies and may provide clues to new therapies.
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Músculos Oculomotores , Pez Cebra , Animales , Desmina , Humanos , Placa Motora , Cadenas Pesadas de MiosinaRESUMEN
The building blocks of DNA, dNTPs, can be produced de novo or can be salvaged from deoxyribonucleosides. However, to what extent the absence of de novo dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated de novo dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the de novo production of dNTPs, at embryonic day 13. All other tissues had normal de novo dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in WT postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for de novo dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in WT hearts.
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Desoxirribonucleótidos/biosíntesis , Corazón/crecimiento & desarrollo , Miocitos Cardíacos/metabolismo , Animales , Replicación del ADN , Corazón/fisiología , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismoRESUMEN
BACKGROUND: Tumour necrosis factor receptor associated factor 6 (TRAF6) promotes inflammation in response to various cytokines. Aberrant Wnt3a signals promotes cancer progression through accumulation of ß-Catenin. Here we investigated a potential role for TRAF6 in Wnt signaling. METHODS: TRAF6 expression was silenced by siRNA in human prostate cancer (PC3U) and human colorectal SW480 cells and by CRISPR/Cas9 in zebrafish. Several biochemical methods and analyses of mutant phenotype in zebrafish were used to analyse the function of TRAF6 in Wnt signaling. FINDINGS: Wnt3a-treatment promoted binding of TRAF6 to the Wnt co-receptors LRP5/LRP6 in PC3U and LNCaP cells in vitro. TRAF6 positively regulated mRNA expression of ß-Catenin and subsequent activation of Wnt target genes in PC3U cells. Wnt3a-induced invasion of PC3U and SW480 cells were significantly reduced when TRAF6 was silenced by siRNA. Database analysis revealed a correlation between TRAF6 mRNA and Wnt target genes in patients with prostate cancer, and high expression of LRP5, TRAF6 and c-Myc correlated with poor prognosis. By using CRISPR/Cas9 to silence TRAF6 in zebrafish, we confirm TRAF6 as a key molecule in Wnt3a signaling for expression of Wnt target genes. INTERPRETATION: We identify TRAF6 as an important component in Wnt3a signaling to promote activation of Wnt target genes, a finding important for understanding mechanisms driving prostate cancer progression. FUND: KAW 2012.0090, CAN 2017/544, Swedish Medical Research Council (2016-02513), Prostatacancerförbundet, Konung Gustaf V:s Frimurarestiftelse and Cancerforskningsfonden Norrland. The funders did not play a role in manuscript design, data collection, data analysis, interpretation nor writing of the manuscript.
Asunto(s)
Inflamación/genética , Neoplasias de la Próstata/genética , Factor 6 Asociado a Receptor de TNF/genética , Proteína Wnt3A/genética , Proteínas de Pez Cebra/genética , Animales , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación/patología , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Neoplasias de la Próstata/patología , Vía de Señalización Wnt/genética , Pez Cebra , beta Catenina/genéticaRESUMEN
The hepatocyte growth factor receptor C-met plays an important role in cellular migration, which is crucial for many developmental processes as well as for cancer cell metastasis. C-met has been linked to the development of mammalian appendicular muscle, which are derived from migrating muscle progenitor cells (MMPs) from within the somite. Mammalian limbs are homologous to the teleost pectoral and pelvic fins. In this study we used Crispr/Cas9 to mutate the zebrafish met gene and found that the MMP derived musculature of the paired appendages was severely affected. The mutation resulted in a reduced muscle fibre number, in particular in the pectoral abductor, and in a disturbed pectoral fin function. Other MMP derived muscles, such as the sternohyoid muscle and posterior hypaxial muscle were also affected in met mutants. This indicates that the role of met in MMP function and appendicular myogenesis is conserved within vertebrates.
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Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Sistemas CRISPR-Cas , Movimiento Celular/fisiología , Extremidades/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Músculos/metabolismo , Células Madre/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Using Caenorhabditis elegans as an infection host model for Vibrio cholerae predator interactions, we discovered a bacterial cytotoxin, MakA, whose function as a virulence factor relies on secretion via the flagellum channel in a proton motive force-dependent manner. The MakA protein is expressed from the polycistronic makDCBA (motility-associated killing factor) operon. Bacteria expressing makDCBA induced dramatic changes in intestinal morphology leading to a defecation defect, starvation and death in C. elegans. The Mak proteins also promoted V. cholerae colonization of the zebrafish gut causing lethal infection. A structural model of purified MakA at 1.9 Å resolution indicated similarities to members of a superfamily of bacterial toxins with unknown biological roles. Our findings reveal an unrecognized role for V. cholerae flagella in cytotoxin export that may contribute both to environmental spread of the bacteria by promoting survival and proliferation in encounters with predators, and to pathophysiological effects during infections.
RESUMEN
The pigment pattern of many animal species is a result of the arrangement of different types of pigment-producing chromatophores. The zebrafish has three different types of chromatophores: black melanophores, yellow xanthophores, and shimmering iridophores arranged in a characteristic pattern of golden and blue horizontal stripes. In the zebrafish embryo, chromatophores derive from the neural crest cells. Using pax7a and pax7b zebrafish mutants, we identified a previously unknown requirement for Pax7 in xanthophore lineage formation. The absence of Pax7 results in a severe reduction of xanthophore precursor cells and a complete depletion of differentiated xanthophores in embryos as well as in adult zebrafish. In contrast, the melanophore lineage is increased in pax7a/pax7b double-mutant embryos and larvae, whereas juvenile and adult pax7a/pax7b double-mutant zebrafish display a severe decrease in melanophores and a pigment pattern disorganization indicative of a xanthophore- deficient phenotype. In summary, we propose a novel role for Pax7 in the early specification of chromatophore precursor cells.
Asunto(s)
Factor de Transcripción PAX2/genética , Factor de Transcripción PAX2/metabolismo , Pigmentación/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Diferenciación Celular/genética , Cromatóforos , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Melanóforos/metabolismo , Cresta Neural/metabolismo , Pigmentación/fisiología , Pez Cebra/genéticaRESUMEN
Numerous muscle lineages are formed during myogenesis within both slow- and fast-specific cell groups. In this study, we show that six fast muscle-specific myosin heavy chain genes have unique expression patterns in the zebrafish embryo. The expression of tail-specific myosin heavy chain (fmyhc2.1) requires wnt signaling and is essential for fast muscle organization within the tail. Retinoic acid treatment results in reduced wnt signaling, which leads to loss of the fmyhc2.1 domain. Retinoic acid treatment also results in a shift of muscle identity within two trunk domains defined by expression of fmyhc1.2 and fmyhc1.3 in favor of the anteriormost myosin isoform, fmyhc1.2. In summary, we identify new muscle domains along the anteroposterior axis in the zebrafish that are defined by individual nonoverlapping, differentially regulated expression of myosin heavy chain isoforms.
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Cadenas Pesadas de Miosina/biosíntesis , Tretinoina/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Animales , Expresión Génica , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Morfolinos , Desarrollo de Músculos/genética , Fibras Musculares de Contracción Rápida/citología , Cadenas Pesadas de Miosina/genética , Isoformas de Proteínas/biosíntesis , Estructura Terciaria de Proteína , Cola (estructura animal)/embriología , Pez CebraRESUMEN
BACKGROUND: Previously we have identified a distal region of the rainbow trout (Oncorhynchus mykiss) metallothionein-A (rtMT-A) enhancer region, being essential for free radical activation of the rtMT-A gene. The distal promoter region included four activator protein 1 (AP1) cis-acting elements and a single nuclear factor interleukin-6 (NF-IL6) element. In the present study we used the rainbow trout hepatoma (RTH-149) cell line to further examine the involvement of NF-IL6 and AP1 in rtMT-A gene expression following exposure to oxidative stress and tumour promotion. RESULTS: Using enhancer deletion studies we observed strong paraquat (PQ)-induced rtMT-A activation via NF-IL6 while the AP1 cis-elements showed a weak but significant activation. In contrast to mammals the metal responsive elements were not activated by oxidative stress. Electrophoretic mobility shift assay (EMSA) mutation analysis revealed that the two most proximal AP1 elements, AP11,2, exhibited strong binding to the AP1 consensus sequence, while the more distal AP1 elements, AP13,4 were ineffective. Phorbol-12-myristate-13-acetate (PMA), a known tumor promoter, resulted in a robust induction of rtMT-A via the AP1 elements alone. To determine the conservation of regulatory functions we transfected human Hep G2 cells with the rtMT-A enhancer constructs and were able to demonstrate that the cis-elements were functionally conserved. The importance of NF-IL6 in regulation of teleost MT is supported by the conservation of these elements in MT genes from different teleosts. In addition, PMA and PQ injection of rainbow trout resulted in increased hepatic rtMT-A mRNA levels. CONCLUSIONS: These studies suggest that AP1 primarily is involved in PMA regulation of the rtMT-A gene while NF-IL6 is involved in free radical regulation. Taken together this study demonstrates the functionality of the NF-IL6 and AP-1 elements and suggests an involvement of MT in protection during pathological processes such as inflammation and cancer.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Carcinoma Hepatocelular/metabolismo , Metalotioneína/genética , Oncorhynchus mykiss/genética , Factor de Transcripción AP-1/metabolismo , Animales , Sitios de Unión , Proteína beta Potenciadora de Unión a CCAAT/genética , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Elementos de Facilitación Genéticos , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Metalotioneína/metabolismo , Mutación , Oncorhynchus mykiss/metabolismo , Estrés Oxidativo , Paraquat/farmacología , Ésteres del Forbol/farmacología , Regiones Promotoras Genéticas , Factor de Transcripción AP-1/genética , TransfecciónRESUMEN
In the embryonic zebrafish, skeletal muscle fibres are formed from muscle progenitors in the paraxial mesoderm. The embryonic myotome is mostly constituted of fast-twitch-specific fibres, which are formed from a fast-specific progenitor cell pool. The most lateral fraction of the fast domain in the myotome of zebrafish embryos derives from the Pax7-positive dermomyotome-like cells. In this study, we show that two genes, belonging to the sine oculus class 1 (six1) genes (six1a and six1b), are both essential for the regulation of Pax7(+) cell proliferation and, consequently, in their differentiation during the establishment of the zebrafish dermomyotome. In both six1a and six1b morphant embryos, Pax7(+) cells are initially formed but fail to proliferate, as detected by reduced levels of the proliferation marker phosphohistone3 and reduced brdU incorporation. In congruence, overexpression of six1a or six1b leads to increased Pax7(+) cell number and reduced or alternatively delayed fibre cell differentiation. Bone morphogenetic protein signalling has previously been suggested to inhibit differentiation of Pax7(+) cells in the dermomyotome. Here we show that the remaining Pax7(+) cells in six1a and six1b morphant embryos also have significantly reduced pSmad1/5/8 levels and propose that this leads to a reduced proliferative activity, which may result in a premature differentiation of Pax7(+) cells in the zebrafish dermomyotome. In summary, we show a mechanism for Six1a and Six1b in establishing the Pax7(+) cell derived part of the fast muscle and suggest new important roles for Six1 in the regulation of the Pax7(+) muscle cell population through pSmad1/5/8 signalling.
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Proteínas de Homeodominio/metabolismo , Músculos/citología , Factor de Transcripción PAX7/metabolismo , Células Madre/citología , Células Madre/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Proliferación Celular , Proteínas de Homeodominio/genética , Desarrollo de Músculos/genética , Desarrollo de Músculos/fisiología , Factor de Transcripción PAX7/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaAsunto(s)
Acuicultura , Peces/crecimiento & desarrollo , Medicina , Desarrollo de Músculos/fisiología , Músculos/patología , Cicatrización de Heridas , Animales , Animales Modificados Genéticamente , Modelos Biológicos , Proteína MioD/metabolismo , Regeneración , Transducción de Señal , Temperatura , Pez Cebra/crecimiento & desarrolloRESUMEN
Although the function of the adult olfactory system has been thoroughly studied, the molecular mechanisms regulating the initial formation of the olfactory nerve, the first cranial nerve, remain poorly defined. Here, we provide evidence that both modulated Notch and bone morphogenetic protein (BMP) signaling affect the generation of neurons in the olfactory epithelium and reduce the number of migratory neurons, so called epithelioid cells. We show that this reduction of epithelial and migratory neurons is followed by a subsequent failure or complete absence of olfactory nerve formation. These data provide new insights into the early generation of neurons in the olfactory epithelium and the initial formation of the olfactory nerve tract. Our results present a novel mechanism in which BMP signals negatively affect Notch activity in a dominant manner in the olfactory epithelium, thereby regulating neurogenesis and explain why a balance of BMP and Notch activity is critical for the generation of neurons and proper development of the olfactory nerve.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Neurogénesis/genética , Nervio Olfatorio/fisiología , Receptores Notch/fisiología , Animales , Animales Modificados Genéticamente , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/genética , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , Mucosa Olfatoria/citología , Mucosa Olfatoria/inervación , Mucosa Olfatoria/fisiología , Nervio Olfatorio/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
The olfactory sensory epithelium and the respiratory epithelium are derived from the olfactory placode. However, the molecular mechanisms regulating the differential specification of the sensory and the respiratory epithelium have remained undefined. To address this issue, we first identified Msx1/2 and Id3 as markers for respiratory epithelial cells by performing quail chick transplantation studies. Next, we established chick explant and intact chick embryo assays of sensory/respiratory epithelial cell differentiation and analyzed two mice mutants deleted of Bmpr1a;Bmpr1b or Fgfr1;Fgfr2 in the olfactory placode. In this study, we provide evidence that in both chick and mouse, Bmp signals promote respiratory epithelial character, whereas Fgf signals are required for the generation of sensory epithelial cells. Moreover, olfactory placodal cells can switch between sensory and respiratory epithelial cell fates in response to Fgf and Bmp activity, respectively. Our results provide evidence that Fgf activity suppresses and restricts the ability of Bmp signals to induce respiratory cell fate in the nasal epithelium. In addition, we show that in both chick and mouse the lack of Bmp or Fgf activity results in disturbed placodal invagination; however, the fate of cells in the remaining olfactory epithelium is independent of morphological movements related to invagination. In summary, we present a conserved mechanism in amniotes in which Bmp and Fgf signals act in an opposing manner to regulate the respiratory versus sensory epithelial cell fate decision.
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Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Mucosa Olfatoria/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , Células Receptoras Sensoriales/efectos de los fármacos , Animales , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/fisiología , Células CHO , Diferenciación Celular/genética , Células Cultivadas , Embrión de Pollo , Cricetinae , Cricetulus , Antagonismo de Drogas , Embrión de Mamíferos , Embrión no Mamífero , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/fisiología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Ratones , Modelos Biológicos , Mucosa Olfatoria/metabolismo , Mucosa Olfatoria/fisiología , Vías Olfatorias/efectos de los fármacos , Vías Olfatorias/metabolismo , Vías Olfatorias/fisiología , Codorniz/embriología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/fisiología , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The zebrafish u-boot (ubo) gene encodes the transcription factor Prdm1, which is essential for the specification of the primary slow-twitch muscle fibres that derive from adaxial cells. Here, we show that Prdm1 functions by acting as a transcriptional repressor and that slow-twitch-specific muscle gene expression is activated by Prdm1-mediated repression of the transcriptional repressor Sox6. Genes encoding fast-specific isoforms of sarcomeric proteins are ectopically expressed in the adaxial cells of ubo(tp39) mutant embryos. By using chromatin immunoprecipitation, we show that these are direct targets of Prdm1. Thus, Prdm1 promotes slow-twitch fibre differentiation by acting as a global repressor of fast-fibre-specific genes, as well as by abrogating the repression of slow-fibre-specific genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Sitios de Unión , Miosinas Cardíacas/genética , Diferenciación Celular , Inmunoprecipitación de Cromatina , Regulación del Desarrollo de la Expresión Génica , Fibras Musculares de Contracción Rápida/citología , Fibras Musculares Esqueléticas/citología , Fibras Musculares de Contracción Lenta/citología , Mutación/genética , Cadenas Ligeras de Miosina/genética , Especificidad de Órganos , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Regiones Promotoras Genéticas/genética , Regulación hacia Arriba/genética , Pez Cebra/genéticaRESUMEN
Recent studies have shown that only breast cancer epithelial cells with intact p53 can induce metallothionein (MT) synthesis after exposure to metals. In this study, the potential role of p53 on regulation of MT was investigated. Results demonstrate that zinc and copper increased metal response elements (MREs) activity and MTF-1 expression in p53 positive MN1 and parental MCF7 cells. However, inactivation of p53 by treatment with pifithrin-alpha or the presence of inactive p53 inhibited MRE-dependent reporter gene expression in response to metals. MTF-1 levels remained unchanged after treatment with zinc in cells with nonfunctional p53. The introduction of wild-type p53 in MDD2 cells, containing nonfunctional p53, enhanced the ability of zinc to increase MRE-dependent reporter gene expression. The cellular level of p21Cip1/WAF1 was increased in MDD2 cells after p53 transfection, confirming the presence of active p53. The treatment of MN1 and parental MCF7 with trichostatin A led to a sixfold increase in the MRE activity in response to zinc. On the contrary, MRE activity remained unaltered in MDD2 cells with inactive p53. The above results demonstrate that activation of p53 is an important factor in metal regulation of MT.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Metalotioneína/genética , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Bencimidazoles/metabolismo , Neoplasias de la Mama/patología , Carbocianinas/metabolismo , Cationes Bivalentes/farmacología , Línea Celular Tumoral , Cobre/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Colorantes Fluorescentes/metabolismo , Genes Reporteros , Humanos , Luciferasas/metabolismo , Potenciales de la Membrana/fisiología , Metales Pesados/farmacología , Mitocondrias/fisiología , Plásmidos , Elementos de Respuesta/genética , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Zinc/farmacología , Factor de Transcripción MTF-1RESUMEN
The developing oocyte is surrounded by an acellular envelope that is composed of 2-4 isoforms of zona pellucida (ZP) proteins. The ZP proteins comprise the ZP1, ZP2, ZP3, and ZPX isoforms. While ZP1 (ZPB) and ZP3 (ZPC) are present in all species, ZP2 (ZPA) is not found in teleost fish and ZPX is not found in mammals. In the present study, we identify and characterize the ZP1, ZP3 and ZPX isoforms of gilthead seabream. Furthermore, by analyzing the conserved domains, which include the external hydrophobic patch and the internal hydrophobic patch, we show that ZP2 and ZPX are closely related isoforms. ZP proteins are synthesized in either the liver or ovary of most teleosts. Only in rainbow trout has it been shown that zp3 has dual transcription sites. In gilthead seabream, all four mRNA isoforms are transcribed in both the liver and ovary, with zp1a, zp1b, and zp3 being highly expressed in the liver, and zpx being primarily expressed in the ovary. However, determination of the ZP proteins in plasma showed high levels of ZP1b, ZP3, and ZPX, with low or non-detectable levels of ZP1a. In similarity to other teleost ZPs, the hepatic transcription of all four ZP isoforms is under estrogenic control. Previously, we have shown that cortisol can potentiate estrogen-induced ZP synthesis in salmonids, and now we show that this is not the case in the gilthead seabream. The present study shows for the first time the endocrine regulation of a teleost ZPX isoform, and demonstrates the dual-organ transcriptional activities of all the ZP proteins in one species.