RESUMEN
The transcription factor activating protein two gamma (AP2γ) is an important regulator of neurogenesis both during embryonic development as well as in the postnatal brain, but its role for neurophysiology and behavior at distinct postnatal periods is still unclear. In this work, we explored the neurogenic, behavioral, and functional impact of a constitutive and heterozygous AP2γ deletion in mice from early postnatal development until adulthood. AP2γ deficiency promotes downregulation of hippocampal glutamatergic neurogenesis, altering the ontogeny of emotional and memory behaviors associated with hippocampus formation. The impairments induced by AP2γ constitutive deletion since early development leads to an anxious-like phenotype and memory impairments as early as the juvenile phase. These behavioral impairments either persist from the juvenile phase to adulthood or emerge in adult mice with deficits in behavioral flexibility and object location recognition. Collectively, we observed a progressive and cumulative impact of constitutive AP2γ deficiency on the hippocampal glutamatergic neurogenic process, as well as alterations on limbic-cortical connectivity, together with functional behavioral impairments. The results herein presented demonstrate the modulatory role exerted by the AP2γ transcription factor and the relevance of hippocampal neurogenesis in the development of emotional states and memory processes.
Asunto(s)
Ansiedad/genética , Trastornos de la Memoria/genética , Factor de Transcripción AP-2/deficiencia , Factores de Edad , Animales , Masculino , RatonesRESUMEN
Mammary gland ductal morphogenesis depends on the differentiation of mammary stem cells (MaSCs) into basal and luminal lineages. The AP-2γ transcription factor, encoded by Tfap2c, has a central role in mammary gland development but its effect in mammary lineages and specifically MaSCs is largely unknown. Here, we utilized an inducible, conditional knockout of Tfap2c to elucidate the role of AP-2γ in maintenance and differentiation of MaSCs. Loss of AP-2γ in the basal epithelium profoundly altered the transcriptomes and decreased the number of cells within several clusters of mammary epithelial cells, including adult MaSCs and luminal progenitors. AP-2γ regulated the expression of genes known to be required for mammary development, including Cebpb, Nfkbia, and Rspo1. As a result, AP-2γ-deficient mice exhibited repressed mammary gland ductal outgrowth and inhibition of regenerative capacity. The findings demonstrate that AP-2γ can regulate development of mammary gland structures potentially regulating maintenance and differentiation of multipotent MaSCs.
Asunto(s)
Células Madre Multipotentes/metabolismo , Factor de Transcripción AP-2/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Noqueados , Células Madre Multipotentes/citología , Inhibidor NF-kappaB alfa/metabolismo , Regeneración , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Trombospondinas/metabolismo , Factor de Transcripción AP-2/deficienciaRESUMEN
BACKGROUND: AP-2δ is the most divergent member of the Activating Protein-2 (TFAP2) family of transcription factors. AP-2δ is restricted to specific regions of the CNS, including a subset of ganglion cells in the retina. Retinal ganglion cells (RGCs), the only output neurons of the retina, are responsible for transmitting the visual signal to the brain. RESULTS: AP-2δ knockout results in loss of Brn3c (Pou4f3) expression in AP-2δ -positive RGCs. While AP-2δ-/- mice have morphologically normal retinas at birth, there is a significant reduction in retinal ganglion cell numbers by P21, after eye opening. Chromatin immunoprecipitation indicates that Brn3c is a target of AP-2δ in the retina. Using fluorochrome-conjugated cholera toxin subunit B to trace ganglion cell axons from the eye to the major visual pathways in the brain, we found 87 % and 32 % decreases in ipsilateral and contralateral projections, respectively, to the superior colliculus in AP-2δ-/- mice. In agreement with anatomical data, visually evoked responses recorded from the brain confirmed that retinal outputs to the brain are compromised. CONCLUSIONS: AP-2δ is important for the maintenance of ganglion cell numbers in the retina. Loss of AP-2δ alters retinal axonal projections to visual centers of the brain, with ipsilaterial projections to the superior colliculus being the most dramatically affected. Our results have important implications for integration of the visual signal at the superior colliculus.
Asunto(s)
Axones/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Colículos Superiores/metabolismo , Factor de Transcripción AP-2/deficiencia , Animales , Apoptosis , Recuento de Células , Inmunoprecipitación de Cromatina , Visión de Colores , Adaptación a la Oscuridad , Potenciales Evocados Visuales/fisiología , Cuerpos Geniculados/citología , Cuerpos Geniculados/metabolismo , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Ratones Endogámicos C57BL , Ratones Noqueados , Colículos Superiores/citología , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/metabolismo , Factor de Transcripción AP-2/metabolismo , Factor de Transcripción Brn-3C/metabolismoRESUMEN
INTRODUCTION: The transcription factor activating enhancer binding protein 2 epsilon (AP-2ε) was recently shown to be expressed during chondrogenesis as well as in articular chondrocytes of humans and mice. Furthermore, expression of AP-2ε was found to be upregulated in affected cartilage of patients with osteoarthritis (OA). Despite these findings, adult mice deficient for AP-2ε (Tfap2e(-/-)) do not exhibit an obviously abnormal cartilaginous phenotype. We therefore analyzed embryogenesis of Tfap2e(-/-) mice to elucidate potential transient abnormalities that provide information on the influence of AP-2ε on skeletal development. In a second part, we aimed to define potential influences of AP-2ε on articular cartilage function and gene expression, as well as on OA progression, in adult mice. METHODS: Murine embryonic development was accessed via in situ hybridization, measurement of skeletal parameters and micromass differentiation of mesenchymal cells. To reveal discrepancies in articular cartilage of adult wild-type (WT) and Tfap2e(-/-) mice, light and electron microscopy, in vitro culture of cartilage explants, and quantification of gene expression via real-time PCR were performed. OA was induced via surgical destabilization of the medial meniscus in both genotypes, and disease progression was monitored on histological and molecular levels. RESULTS: Only minor differences between WT and embryos deficient for AP-2ε were observed, suggesting that redundancy mechanisms effectively compensate for the loss of AP-2ε during skeletal development. Surprisingly, though, we found matrix metalloproteinase 13 (Mmp13), a major mediator of cartilage destruction, to be significantly upregulated in articular cartilage of adult Tfap2e(-/-) mice. This finding was further confirmed by increased Mmp13 activity and extracellular matrix degradation in Tfap2e(-/-) cartilage explants. OA progression was significantly enhanced in the Tfap2e(-/-) mice, which provided evidence for in vivo relevance. This finding is most likely attributable to the increased basal Mmp13 expression level in Tfap2e(-/-) articular chondrocytes that results in a significantly higher total Mmp13 expression rate during OA as compared with the WT. CONCLUSIONS: We reveal a novel role of AP-2ε in the regulation of gene expression in articular chondrocytes, as well as in OA development, through modulation of Mmp13 expression and activity.
Asunto(s)
Regulación de la Expresión Génica/genética , Metaloproteinasa 13 de la Matriz/biosíntesis , Osteoartritis/genética , Osteogénesis/genética , Factor de Transcripción AP-2/deficiencia , Animales , Cartílago Articular/embriología , Cartílago Articular/metabolismo , Cartílago Articular/patología , Modelos Animales de Enfermedad , Femenino , Inmunohistoquímica , Hibridación in Situ , Masculino , Metaloproteinasa 13 de la Matriz/genética , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Osteoartritis/metabolismo , Osteoartritis/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción AP-2/genéticaRESUMEN
Maintenance and maturation of primordial germ cells is controlled by complex genetic and epigenetic cascades, and disturbances in this network lead to either infertility or malignant aberration. Transcription factor TFAP2C has been described to be essential for primordial germ cell maintenance and to be upregulated in several human germ cell cancers. Using global gene expression profiling, we identified genes deregulated upon loss of Tfap2c in embryonic stem cells and primordial germ cell-like cells. We show that loss of Tfap2c affects many aspects of the genetic network regulating germ cell biology, such as downregulation of maturation markers and induction of markers indicative for somatic differentiation, cell cycle, epigenetic remodeling and pluripotency. Chromatin-immunoprecipitation analyses demonstrated binding of TFAP2C to regulatory regions of deregulated genes (Sfrp1, Dmrt1, Nanos3, c-Kit, Cdk6, Cdkn1a, Fgf4, Klf4, Dnmt3b and Dnmt3l) suggesting that these genes are direct transcriptional targets of TFAP2C in primordial germ cells. Since Tfap2c deficient primordial germ cell-like cells display cancer related deregulations in epigenetic remodeling, cell cycle and pluripotency control, the Tfap2c-knockout allele was bred onto 129S2/Sv genetic background. There, mice heterozygous for Tfap2c develop with high incidence germ cell cancer resembling human pediatric germ cell tumors. Precursor lesions can be observed as early as E16.5 in developing testes displaying persisting expression of pluripotency markers. We further demonstrate that mice with a heterozygous deletion of the TFAP2C target gene Nanos3 are also prone to develop teratomas. These data highlight TFAP2C as a critical and dose-sensitive regulator of germ cell fate.
Asunto(s)
Predisposición Genética a la Enfermedad , Células Germinativas/metabolismo , Haploinsuficiencia , Teratoma/genética , Teratoma/metabolismo , Factor de Transcripción AP-2/metabolismo , Animales , Diferenciación Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Células Madre Embrionarias , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/citología , Células Germinativas/patología , Heterocigoto , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones Noqueados , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Reproducibilidad de los Resultados , Teratoma/patología , Factor de Transcripción AP-2/deficiencia , Factor de Transcripción AP-2/genética , Activación TranscripcionalRESUMEN
Ap-2 transcription factors comprise a family of 5 closely related sequence-specific DNA binding proteins that play pivotal and non-redundant roles in embryonic organogenesis. To investigate the function of Ap-2δ, wδe analyzed its expression during embryogenesis and generated Ap-2δ-deficient mice. In line with the specific expression pattern of Ap-2δ in the mesencephalic tectum and the dorsal midbrain, Ap-2δ-deficient mice failed to maintain the colliculus inferior, a derivative of the dorsal midbrain, as a consequence of increased apoptotic cell death. To identify specific Ap-2δ target genes in cells of the developing dorsal midbrain, we performed whole genome analysis of cDNA expression levels. This approach identified a set of 12 putative target genes being expressed in the developing midbrain, including the transcription factors Pitx2, Mef2c, Bhlhb4 and Pou4f3. Using chromatin immunoprecipitation (CHIP) we showed that some of these genes are direct targets of Ap-2δ. Consistently, we demonstrate that Ap-2δ occupies and activates the Pou4f3 and Bhlhb4 promoters. In addition, known Pou4f3 target genes were downregulated in the posterior midbrain of Ap-2δ-deficient mice. Despite the absence of a central part of the auditory pathway, the presence of neuronal responses to sounds in the neocortex of Ap-2δ-deficient mice indicates that auditory information from the brainstem still reaches the neocortex. In summary, our data define Ap-2δ as an important transcription factor, specifying gene expression patterns required for the development of the posterior midbrain.
Asunto(s)
Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Mesencéfalo/metabolismo , Factor de Transcripción AP-2/genética , Animales , Apoptosis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sitios de Unión/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Potenciales Evocados , Proteínas de Homeodominio/genética , Hibridación in Situ , Colículos Inferiores/embriología , Colículos Inferiores/metabolismo , Mesencéfalo/embriología , Ratones , Ratones Noqueados , Neocórtex/citología , Neocórtex/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción AP-2/deficiencia , Factor de Transcripción AP-2/metabolismo , Factor de Transcripción Brn-3C/genéticaRESUMEN
Development of the secondary palate in mammals is a complex process that can be easily perturbed, leading to the common and distressing birth defect cleft palate. Animal models are particularly useful tools for dissecting underlying genetic components of cleft palate. We describe a new cleft palate model resulting from a transgene insertion mutation. Transgene insertional mutagenesis disrupts the genomic organization and expression of the Ap2ß1 gene located on chromosome 11. This gene encodes the ß2-adaptin subunit of the heterotetrameric adaptor protein 2 complex involved in clathrin-dependent endocytosis. Homozygous cleft palate mutant mice express no Ap2ß1 messenger RNA or ß2-adaptin protein and die during the perinatal period. Heterozygous mice are phenotypically normal despite expressing diminished ß2-adaptin messenger RNA and protein compared with wildtype. Remarkably, the paralogous ß1-adaptin subunit of the adaptor protein 1 complex partially substitutes for the missing ß2-adaptin in embryonic fibroblasts from homozygous mutant mice, resulting in assembly of reduced levels of an adaptor protein 2 complex bearing ß1-adaptin. This variant adaptor protein 2 complex is, therefore, apparently capable of maintaining viability of the homozygous mutant embryos until birth but insufficient to support palatogenesis. Nonsyndromic cleft palate in an animal model is associated with disruption of the Ap2ß1 gene.
Asunto(s)
Fisura del Paladar/genética , Factor de Transcripción AP-2/deficiencia , Factor de Transcripción AP-2/genética , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Mutantes , Ratones Transgénicos , Mutagénesis Insercional , SíndromeRESUMEN
Tcfap2a, the gene encoding the mouse AP-2alpha transcription factor, is required for normal development of multiple structures during embryogenesis, including the face and limbs. Using comparative sequence analysis and transgenic-mouse experiments we have identified an intronic enhancer within this gene that directs expression to the face and limb mesenchyme. There are two conserved sequence blocks within this intron, and the larger of these directs tissue-specific activity and is found in all vertebrate Tcfap2a genes analyzed. To assess the role of the enhancer in regulating endogenous mouse Tcfap2a expression, we have deleted this cis-regulatory sequence from the genome. Loss of this element severely impairs Tcfap2a expression in the limb bud mesenchyme but generates only a modest reduction in the facial mesenchyme. The reduction in Tcfap2a transcription is accompanied by altered patterning of the forelimb, resulting in postaxial polydactyly. These results indicate that the major role for this enhancer resides within the limb bud, and it serves to maintain a level of Tcfap2a expression that limits the size of the hand plate and the associated number of digit primordia. The potential role of this cis-acting sequence in modeling the size and shape of the face and limbs during evolution is discussed.
Asunto(s)
Cara , Regulación del Desarrollo de la Expresión Génica , Intrones/genética , Esbozos de los Miembros/metabolismo , Mesodermo/metabolismo , Factor de Transcripción AP-2/metabolismo , Alelos , Animales , Secuencia de Bases , Secuencia Conservada , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Elementos de Facilitación Genéticos , Cara/embriología , Humanos , Esbozos de los Miembros/embriología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Especificidad de Órganos , Alineación de Secuencia , Factor de Transcripción AP-2/deficiencia , Factor de Transcripción AP-2/genéticaRESUMEN
Activating protein 2alpha (AP-2alpha) is known to be expressed in the retina, and AP-2alpha-null mice exhibit defects in the developing optic cup, including patterning of the neural retina (NR) and a replacement of the dorsal retinal pigmented epithelium (RPE) with NR. In this study, we analyzed the temporal and spatial retinal expression patterns of AP-2alpha and created a conditional deletion of AP-2alpha in the developing retina. AP-2alpha exhibited a distinct expression pattern in the developing inner nuclear layer of the retina, and colocalization studies indicated that AP-2alpha was exclusively expressed in postmitotic amacrine cell populations. Targeted deletion of AP-2alpha in the developing retina did not result in observable retinal defects. Further examination of AP-2alpha-null mutants revealed that the severity of the RPE defect was variable and, although defects in retinal lamination occur at later embryonic stages, earlier stages showed normal lamination and expression of markers for amacrine and ganglion cells. Together, these data demonstrate that, whereas AP-2alpha alone does not play an intrinsic role in retinogenesis, it has non-cell-autonomous effects on optic cup development. Additional expression analyses showed that multiple AP-2 proteins are present in the developing retina, which will be important to future studies.