RESUMEN
Wnt/ß-catenin signaling has an essential role in eye development. Faulty regulation of this pathway results in ocular malformations, owing to defects in cell-fate determination and differentiation. Herein, we show that disruption of Maz, the gene encoding Myc-associated zinc-finger transcription factor, produces developmental eye defects in mice and humans. Expression of key genes involved in the Wnt cascade, Sfrp2, Wnt2b and Fzd4, was significantly increased in mice with targeted inactivation of Maz, resulting in abnormal peripheral eye formation with reduced proliferation of the progenitor cells in the region. Paradoxically, the Wnt reporter TCF-Lef1 displayed a significant downregulation in Maz-deficient eyes. Molecular analysis indicates that Maz is necessary for the activation of the Wnt/ß-catenin pathway and participates in the network controlling ciliary margin patterning. Copy-number variations and single-nucleotide variants of MAZ were identified in humans that result in abnormal ocular development. The data support MAZ as a key contributor to the eye comorbidities associated with chromosome 16p11.2 copy-number variants and as a transcriptional regulator of ocular development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Anomalías del Ojo/metabolismo , Ojo/metabolismo , Factores de Transcripción/metabolismo , Adolescente , Adulto , Animales , Apoptosis , Proliferación Celular , Preescolar , Variaciones en el Número de Copia de ADN , Proteínas de Unión al ADN/genética , Ojo/patología , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Femenino , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Lactante , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis , Fenotipo , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genética , Vía de Señalización Wnt , Adulto JovenRESUMEN
The ascending thoracic aorta is designed to withstand biomechanical forces from pulsatile blood. Thoracic aortic aneurysms and acute aortic dissections (TAADs) occur as a result of genetically triggered defects in aortic structure and a dysfunctional response to these forces. Here, we describe mutations in the forkhead transcription factor FOXE3 that predispose mutation-bearing individuals to TAAD. We performed exome sequencing of a large family with multiple members with TAADs and identified a rare variant in FOXE3 with an altered amino acid in the DNA-binding domain (p.Asp153His) that segregated with disease in this family. Additional pathogenic FOXE3 variants were identified in unrelated TAAD families. In mice, Foxe3 deficiency reduced smooth muscle cell (SMC) density and impaired SMC differentiation in the ascending aorta. Foxe3 expression was induced in aortic SMCs after transverse aortic constriction, and Foxe3 deficiency increased SMC apoptosis and ascending aortic rupture with increased aortic pressure. These phenotypes were rescued by inhibiting p53 activity, either by administration of a p53 inhibitor (pifithrin-α), or by crossing Foxe3-/- mice with p53-/- mice. Our data demonstrate that FOXE3 mutations lead to a reduced number of aortic SMCs during development and increased SMC apoptosis in the ascending aorta in response to increased biomechanical forces, thus defining an additional molecular pathway that leads to familial thoracic aortic disease.
Asunto(s)
Aneurisma de la Aorta Torácica/genética , Disección Aórtica/genética , Factores de Transcripción Forkhead/genética , Adulto , Disección Aórtica/metabolismo , Disección Aórtica/patología , Animales , Aorta/metabolismo , Aorta/patología , Aneurisma de la Aorta Torácica/metabolismo , Aneurisma de la Aorta Torácica/patología , Apoptosis , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Expresión Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Músculo Liso Vascular/patología , Mutación Missense , Miocitos del Músculo Liso/fisiología , Linaje , Proteína p53 Supresora de Tumor/genética , Remodelación Vascular , Pez CebraRESUMEN
Wnt glycoproteins control key processes during development and disease by activating various downstream pathways. Wnt secretion requires post-translational modification mediated by the O-acyltransferase encoded by the Drosophila porcupine homolog gene (PORCN). In humans, PORCN mutations cause focal dermal hypoplasia (FDH, or Goltz syndrome), an X-linked dominant multisystem birth defect that is frequently accompanied by ocular abnormalities such as coloboma, microphthalmia, or even anophthalmia. Although genetic ablation of Porcn in mouse has provided insight into the etiology of defects caused by ectomesodermal dysplasia in FDH, the requirement for Porcn and the actual Wnt ligands during eye development have been unknown. In this study, Porcn hemizygosity occasionally caused ocular defects reminiscent of FDH. Conditional inactivation of Porcn in periocular mesenchyme led to defects in mid- and hindbrain and in craniofacial development, but was insufficient to cause ocular abnormalities. However, a combination of conditional Porcn depletion in optic vesicle neuroectoderm, lens, and neural crest-derived periocular mesenchyme induced severe eye abnormalities with high penetrance. In particular, we observed coloboma, transdifferentiation of the dorsal and ventral retinal pigment epithelium, defective optic cup periphery, and closure defects of the eyelid, as well as defective corneal morphogenesis. Thus, Porcn is required in both extraocular and neuroectodermal tissues to regulate distinct Wnt-dependent processes during morphogenesis of the posterior and anterior segments of the eye.
Asunto(s)
Ojo/embriología , Hipoplasia Dérmica Focal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/metabolismo , Aciltransferasas , Alelos , Animales , Modelos Animales de Enfermedad , Ojo/metabolismo , Femenino , Genotipo , Glicoproteínas/metabolismo , Hemicigoto , Hibridación in Situ , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Recombinación Genética , Epitelio Pigmentado de la Retina/embriología , Epitelio Pigmentado de la Retina/metabolismo , Proteínas Wnt/metabolismoRESUMEN
The present study examined the consequences of eliminating horizontal cells from the outer retina during embryogenesis upon the organization and assembly of the outer plexiform layer (OPL). Retinal horizontal cells exhibit a migration defect in Lim1-conditional knock-out (Lim1-CKO) mice and become mispositioned in the inner retina before birth, redirecting their dendrites into the inner plexiform layer. The resultant (mature) OPL, developing in the absence of horizontal cells, shows a retraction of rod spherules into the outer nuclear layer and a sprouting of rod bipolar cell dendrites to reach ectopic ribbon-protein puncta. Cone pedicles and the dendrites of type 7 cone bipolar cells retain their characteristic stratification and colocalization within the collapsed OPL, although both are atrophic and the spatial distribution of the pedicles is disrupted. Developmental analysis of Lim1-CKO retina reveals that components of the rod and cone pathways initially co-assemble within their normal strata in the OPL, indicating that horizontal cells are not required for the correct targeting of photoreceptor terminals or bipolar cell dendrites. As the rod spherules begin to retract during the second postnatal week, rod bipolar cells initially show no signs of ectopic growth, sprouting only subsequently and continuing to do so well after the eighth postnatal week. These results demonstrate the critical yet distinctive roles for horizontal cells on the rod and cone pathways and highlight a unique and as-yet-unrecognized maintenance function of an inhibitory interneuron that is not required for the initial targeting and co-stratification of other components in the circuit.
Asunto(s)
Plasticidad Neuronal/fisiología , Células Horizontales de la Retina/fisiología , Neuronas Retinianas/fisiología , Vías Visuales/crecimiento & desarrollo , Animales , Femenino , Ratones , Ratones Transgénicos , Sinapsis/metabolismo , Vías Visuales/fisiologíaRESUMEN
Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.
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Permeabilidad de la Membrana Celular/fisiología , ADN (Citosina-5-)-Metiltransferasas/genética , Morfogénesis/genética , Segmento Externo de las Células Fotorreceptoras Retinianas/fisiología , Epitelio Pigmentado de la Retina/fisiología , Animales , Permeabilidad de la Membrana Celular/genética , Polaridad Celular/genética , ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN/genética , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Morfogénesis/fisiología , Especificidad de Órganos/genética , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Epitelio Pigmentado de la Retina/embriología , Epitelio Pigmentado de la Retina/crecimiento & desarrollo , Epitelio Pigmentado de la Retina/metabolismo , TranscriptomaRESUMEN
The brain plays a central role in controlling energy, glucose, and lipid homeostasis, with specialized neurons within nuclei of the mediobasal hypothalamus, namely the arcuate (ARC) and ventromedial (VMH), tasked with proper signal integration. Exactly how the exquisite cytoarchitecture and underlying circuitry becomes established within these nuclei remains largely unknown, in part because hypothalamic developmental programs are just beginning to be elucidated. Here, we demonstrate that the Retina and anterior neural fold homeobox (Rax) gene plays a key role in establishing ARC and VMH nuclei in mice. First, we show that Rax is expressed in ARC and VMH progenitors throughout development, consistent with genetic fate mapping studies demonstrating that Rax+ lineages give rise to VMH neurons. Second, the conditional ablation of Rax in a subset of VMH progenitors using a Shh::Cre driver leads to a fate switch from a VMH neuronal phenotype to a hypothalamic but non-VMH identity, suggesting that Rax is a selector gene for VMH cellular fates. Finally, the broader elimination of Rax throughout ARC/VMH progenitors using Six3::Cre leads to a severe loss of both VMH and ARC cellular phenotypes, demonstrating a role for Rax in both VMH and ARC fate specification. Combined, our study illustrates that Rax is required in ARC/VMH progenitors to specify neuronal phenotypes within this hypothalamic brain region. Rax thus provides a molecular entry point for further study of the ontology and establishment of hypothalamic feeding circuits.
Asunto(s)
Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Hipotálamo Medio/metabolismo , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Animales , Linaje de la Célula , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Hipotálamo Medio/embriología , Hipotálamo Medio/crecimiento & desarrollo , Ratones , Ratones Transgénicos , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Mutation of the polarity gene Crumbs homolog 1 (CRB1) is responsible for >10% of Leber congenital amaurosis (LCA) cases worldwide; LCA is characterized by early-onset degenerative retinal dystrophy. The role of CRB1 in LCA8 pathogenesis remains elusive since Crb1 mouse mutants, including a null allele, have failed to mimic the early-onset of LCA, most likely due to functional compensation by closely related genes encoding Crb2 and Crb3. Crb proteins form an evolutionarily conserved, apical polarity complex with the scaffolding protein associated with lin-seven 1 (Pals1), also known as MAGUK p55 subfamily member 5 (MPP5). Pals1 and Crbs are functionally inter-dependent in establishing and maintaining epithelial polarity. Pals1 is a single gene in the mouse and human genomes; therefore, we ablated Pals1 to establish a mouse genetic model mimicking human LCA. In our study, the deletion of Pals1 leads to the disruption of the apical localization of Crb proteins in retinal progenitors and the adult retina, validating their mutual interaction. Remarkably, the Pals1 mutant mouse exhibits the critical features of LCA such as early visual impairment as assessed by electroretinogram, disorganization of lamination and apical junctions and retinal degeneration. Our data uncover the indispensible role of Pals1 in retinal development, likely involving the maintenance of retinal polarity and survival of retinal neurons, thus providing the basis for the pathologic mechanisms of LCA8.
Asunto(s)
Amaurosis Congénita de Leber/metabolismo , Proteínas de la Membrana/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Retina/metabolismo , Células Madre/metabolismo , Animales , Animales Recién Nacidos , Proliferación Celular , Electrorretinografía , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/patología , Masculino , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Microscopía Electrónica , Proteínas del Tejido Nervioso/metabolismo , Nucleósido-Fosfato Quinasa/genética , Retina/embriología , Retina/crecimiento & desarrollo , Células Madre/patología , Células Madre/ultraestructura , Agudeza VisualRESUMEN
The development of topographic maps of the sensory periphery is sensitive to the disruption of adenylate cyclase 1 (AC1) signaling. AC1 catalyzes the production of cAMP in a Ca2+/calmodulin-dependent manner, and AC1 mutant mice (AC1−/−) have disordered visual and somatotopic maps. However, the broad expression of AC1 in the brain and the promiscuous nature of cAMP signaling have frustrated attempts to determine the underlying mechanism of AC1-dependent map development. In the mammalian visual system, the initial coarse targeting of retinal ganglion cell (RGC) projections to the superior colliculus (SC) and lateral geniculate nucleus (LGN) is guided by molecular cues, and the subsequent refinement of these crude projections occurs via an activity-dependent process that depends on spontaneous retinal waves. Here, we show that AC1−/− mice have normal retinal waves but disrupted map refinement. We demonstrate that AC1 is required for the emergence of dense and focused termination zones and elimination of inaccurately targeted collaterals at the level of individual retinofugal arbors. Conditional deletion of AC1 in the retina recapitulates map defects, indicating that the locus of map disruptions in the SC and dorsal LGN of AC1−/− mice is presynaptic. Finally, map defects in mice without AC1 and disrupted retinal waves (AC1−/−;ß2−/− double KO mice) are no worse than those in mice lacking only ß2−/−, but loss of AC1 occludes map recovery in ß2−/− mice during the second postnatal week. These results suggest that AC1 in RGC axons mediates the development of retinotopy and eye-specific segregation in the SC and dorsal LGN.
Asunto(s)
Adenilil Ciclasas/metabolismo , Mapeo Encefálico/métodos , Células Ganglionares de la Retina/metabolismo , Vías Visuales/metabolismo , Animales , Electrofisiología , Electroporación , Cuerpos Geniculados/citología , Cuerpos Geniculados/crecimiento & desarrollo , Cuerpos Geniculados/metabolismo , Imagenología Tridimensional/métodos , Ratones , Ratones Noqueados , Ratones Mutantes , Células Ganglionares de la Retina/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Colículos Superiores/citología , Colículos Superiores/crecimiento & desarrollo , Colículos Superiores/metabolismo , Vías Visuales/citologíaRESUMEN
The membrane-associated palmitoylated protein 5 (MPP5 or PALS1) is thought to organize intracellular PALS1-CRB-MUPP1 protein scaffolds in the retina that are involved in maintenance of photoreceptor-Müller glia cell adhesion. In humans, the Crumbs homolog 1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, especially PALS1, we generated and analyzed conditional knockdown mice for Pals1. Small irregularly shaped spots were detected throughout the PALS1 deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. The electroretinography a- and b-wave was severely attenuated in the aged mutant retinas, suggesting progressive degeneration of photoreceptors. The histological analysis showed abnormal retinal pigment epithelium structure, ectopic photoreceptor nuclei in the subretinal space, an irregular outer limiting membrane, half rosettes of photoreceptors in the outer plexiform layer, and a thinner photoreceptor synaptic layer suggesting improper photoreceptor cell layering during retinal development. The PALS1 deficient retinas showed reduced levels of Crumbs complex proteins adjacent to adherens junctions, upregulation of glial fibrillary acidic protein indicative of gliosis, and persisting programmed cell death after retinal maturation. The phenotype suggests important functions of PALS1 in the retinal pigment epithelium in addition to the neural retina.
Asunto(s)
Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Nucleósido-Fosfato Quinasa/deficiencia , Nucleósido-Fosfato Quinasa/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Animales , Femenino , Masculino , Marmota , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/ultraestructura , Oftalmoscopía , Retina/metabolismo , Retina/ultraestructura , Tomografía de Coherencia ÓpticaRESUMEN
Retinal degeneration causes vision impairment and blindness in humans. If one day we are to harness the potential of stem cell-based cell replacement therapies to treat these conditions, it is imperative that we better understand normal retina development. Currently, the genes and mechanisms that regulate the specification of the neuroretina during vertebrate eye development remain unknown. Here, we identify sine oculis-related homeobox 3 (Six3) as a crucial player in this process in mice. In Six3 conditional-mutant mouse embryos, specification of the neuroretina was abrogated, but that of the retinal pigmented epithelium was normal. Conditional deletion of Six3 did not affect the initial development of the optic vesicle but did arrest subsequent neuroretina specification. Ectopic rostral expansion of Wnt8b expression was the major response to Six3 deletion and the leading cause for the specific lack of neuroretina, as ectopic Wnt8b expression in transgenic embryos was sufficient to suppress neuroretina specification. Using chromatin immunoprecipitation assays, we identified Six3-responsive elements in the Wnt8b locus and demonstrated that Six3 directly repressed Wnt8b expression in vivo. Our findings provide a molecular framework to the program leading to neuroretina differentiation and may be relevant for the development of novel strategies aimed at characterizing and eventually treating different abnormalities in eye formation.
Asunto(s)
Proteínas del Ojo/fisiología , Proteínas de Homeodominio/fisiología , Proteínas del Tejido Nervioso/fisiología , Placa Neural/embriología , Retina/embriología , Proteínas Wnt/antagonistas & inhibidores , Animales , Ratones , Ratones Transgénicos , Factores de Transcripción SOXB1/fisiología , Proteínas Wnt/fisiología , beta Catenina/fisiología , Proteína Homeobox SIX3RESUMEN
The majority of human skeletal dysplasias are caused by dysregulation of growth plate homeostasis. As TGF-beta signaling is a critical determinant of growth plate homeostasis, skeletal dysplasias are often associated with dysregulation of this pathway. The context-dependent action of TFG-beta signaling is tightly controlled by numerous mechanisms at the extracellular level and downstream of ligand-receptor interactions. However, TGF-beta is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus, and the regulation of this posttranslational intracellular processing and trafficking is much less defined. Here, we report that a cysteine-rich protein, E-selectin ligand-1 (ESL-1), acts as a negative regulator of TGF-beta production by binding TGF-beta precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-beta by a furin-like protease, leading to reduced secretion of mature TGF-beta by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-beta/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation. Gain-of-function and rescue studies of the Xenopus ESL-1 ortholog in the context of early embryogenesis showed that this regulation of TGF-beta/Nodal signaling was evolutionarily conserved. This study identifies what we believe to be a novel intracellular mechanism for regulating TGF-beta during skeletal development and homeostasis.
Asunto(s)
Condrocitos/metabolismo , Placa de Crecimiento/metabolismo , Homeostasis , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Animales , Diferenciación Celular/fisiología , Condrocitos/citología , Citoplasma/metabolismo , Selectina E/metabolismo , Furina/metabolismo , Placa de Crecimiento/citología , Ligandos , Ratones , Ratones Noqueados , Ratones Transgénicos , Receptores de Factores de Crecimiento de Fibroblastos , Selectinas/metabolismo , Sialoglicoproteínas , Transducción de Señal/fisiología , Xenopus laevisRESUMEN
FoxF genes are essential for visceral mesoderm development from Drosophila to human. However, part of the difficulty of studying the visceral mesoderm is its relative inaccessibility during early development. Owing to its external development Xenopus laevis presents considerable advantages for the study of visceral mesoderm formation, yet FoxF2 has not been identified in this system. Here, we describe the cloning and expression pattern of XFoxF2 during embryonic development, metamorphosis and adulthood, and compare and contrast it to the expression of FoxF1 in Xenopus laevis and FoxF2 in mouse.
Asunto(s)
Factores de Transcripción Forkhead/genética , Tracto Gastrointestinal/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas de Xenopus/genética , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Tracto Gastrointestinal/embriología , Tracto Gastrointestinal/crecimiento & desarrollo , Hibridación in Situ , Metamorfosis Biológica/genética , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Factores de Tiempo , Xenopus laevis/embriología , Xenopus laevis/crecimiento & desarrolloRESUMEN
Transcriptional networks, which are initiated by secreted proteins, cooperate with each other to orchestrate eye development. The establishment of dorsal/ventral polarity, especially dorsal specification in the optic vesicle, is poorly understood at a molecular and cellular level. Here, we show that COUP-TFI (Nr2f1) and COUP-TFII (Nr2f2) are highly expressed in the progenitor cells in the developing murine eye. Phenotype analysis of COUP-TFI and COUP-TFII single-gene conditional knockout mouse models suggests that COUP-TFs compensate for each other to maintain morphogenesis of the eye. However, in eye-specific COUP-TFI/TFII double-knockout mice, progenitor cells at the dorso-distal optic vesicle fail to differentiate appropriately, causing the retinal pigmented epithelium cells to adopt a neural retina fate and abnormal differentiation of the dorsal optic stalk; the development of proximo-ventral identities, neural retina and ventral optic stalk is also compromised. These cellular defects in turn lead to congenital ocular colobomata and microphthalmia. Immunohistochemical and in situ hybridization assays reveal that the expression of several regulatory genes essential for early optic vesicle development, including Pax6, Otx2, Mitf, Pax2 and Vax1/2, is altered in the corresponding compartments of the mutant eye. Using ChIP assay, siRNA treatment and transient transfection in ARPE-19 cells in vitro, we demonstrate that Pax6 and Otx2 are directly regulated by COUP-TFs. Taken together, our findings reveal novel and distinct cell-intrinsic mechanisms mediated by COUP-TF genes to direct the specification and differentiation of progenitor cells, and that COUP-TFs are crucial for dorsalization of the eye.
Asunto(s)
Factores de Transcripción COUP/fisiología , Proteínas del Ojo/genética , Ojo/embriología , Regulación del Desarrollo de la Expresión Génica , Morfogénesis/genética , Animales , Factores de Transcripción COUP/genética , Células Cultivadas , Coloboma/embriología , Coloboma/genética , Embrión de Mamíferos/metabolismo , Ojo/metabolismo , Anomalías del Ojo/embriología , Anomalías del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Transgénicos , Microftalmía/embriología , Microftalmía/genética , Modelos Biológicos , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Retina/embriología , Retina/metabolismo , Vías Visuales/embriologíaRESUMEN
The transcription factor Pitx3 is critical for lens formation. Deletions in the promoter of this gene cause abnormal lens development in the aphakia (ak) mouse mutant, which has only rudimentary lenses. In this study, we investigated the role of Pitx3 in lens development and differentiation. We found that reduced expression of Pitx3 leads to changes in the proliferation, differentiation and survival of lens cells. The genetic interactions between Pitx3 and Foxe3 were investigated, as these two transcription factors are expressed at the same time in lens development and their absence has similar consequences for lens development. We found no evidence that these two genes genetically interact. In general, our study shows that the abnormal phenotype of the ak lenses is not due to just one molecular pathway, rather in the absence of Pitx3 expression multiple aspects of lens development are disrupted.
Asunto(s)
Proteínas de Homeodominio/fisiología , Cristalino/embriología , Cristalino/metabolismo , Factores de Transcripción/fisiología , Animales , Afaquia/embriología , Afaquia/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Mutantes , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The gastrointestinal (GI) tract forms from the endoderm (which gives rise to the epithelium) and the mesoderm (which develops into the smooth muscle layer, the mesenchyme, and numerous other cell types). Much of what is known of GI development has been learned from studies of the endoderm and its derivatives, because of the importance of epithelial biology in understanding and treating human diseases. Although the necessity of epithelial-mesenchymal cross talk for GI development is uncontested, the role of the mesoderm remains comparatively less well understood. The transformation of the visceral mesoderm during development is remarkable; it differentiates from a very thin layer of cells into a complex tissue comprising smooth muscle cells, myofibroblasts, neurons, immune cells, endothelial cells, lymphatics, and extracellular matrix molecules, all contributing to the form and function of the digestive system. Understanding the molecular processes that govern the development of these cell types and elucidating their respective contribution to GI patterning could offer insight into the mechanisms that regulate cell fate decisions in the intestine, which has the unique property of rapid cell renewal for the maintenance of epithelial integrity. In reviewing evidence from both mammalian and nonmammalian models, we reveal the important role of the visceral mesoderm in the ontogeny of the GI tract.
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Desarrollo Embrionario/fisiología , Tracto Gastrointestinal/embriología , Mesodermo/embriología , Animales , Embrión de Pollo , Desarrollo Fetal , Tracto Gastrointestinal/fisiología , Intestino Grueso/embriología , Intestino Delgado/embriología , Mesodermo/fisiología , Ratones , Modelos Animales , Especificidad de la Especie , Estómago/embriología , XenopusRESUMEN
Rx is a paired-like homeobox gene that is required for vertebrate eye formation. Mice lacking Rx function do not develop eyes or the posterior pituitary. To determine whether Rx is required cell autonomously in these tissues, we generated embryonic chimeras consisting of wild type and Rx-/- cells. We found that in the eye, Rx-deficient cells cannot participate in the formation of the neuroretina, retina pigment epithelium and the distal part of the optic stalk. In addition, in the ventral forebrain, Rx function is required cell autonomously for the formation of the posterior pituitary. Interestingly, Rx-/- and wild type cells segregate before the morphogenesis of these two tissues begins. Our observations suggest that Rx function is not only required for the morphogenesis of the retina and posterior pituitary, but also prior to morphogenesis, for the sorting out of cells to form distinct fields of retinal/pituitary cells.
Asunto(s)
Proteínas del Ojo/fisiología , Proteínas de Homeodominio/fisiología , Morfogénesis , Neurohipófisis/crecimiento & desarrollo , Retina/crecimiento & desarrollo , Animales , Movimiento Celular , Quimera , Embrión de Mamíferos , Ratones , Ratones Noqueados , Neurohipófisis/embriología , Retina/embriologíaRESUMEN
The complement system is the central component of innate immunity and an important player in the adaptive immunity of vertebrates. We analyzed the expression patterns of several key members of the complement cascade during Xenopus development. We found extensive expression of these molecules already during gastrula/early neurula stage. Remarkably, several genes also showed an organ-specific expression pattern during early organogenesis. Early expression is notable for two different expression patterns in the neuroectoderm. In one group, there is early strong neural plate and neural precursor expression. This is the case of properdin, C1qA, C3 and C9. The second pattern, seen with C1qR and C6, is noteworthy for its expression at the periphery of the neural plate, in the presumptive neural crest. Two genes stand out for their predominantly mesodermal expression. C3aR, the message for the cognate receptor for C3 in the complement cascade, is expressed at the same time as C3, but in a complementary, reciprocal pattern in the mesoderm. C1qA expression also predominates in somites, pronephros, visceral mesoderm and ventral blood islands. Finally, several genes are characterized by later expression in developing organs. C1qR displays a reticular pattern consistent with expression in the developing vasculature. The late expression of C1qA and C3bC4b is strongest in the pronephros. Finally, the expression of properdin in the hindbrain and in the developing lens are novel findings. The expression patterns of these molecules suggest that these components of the complement system may have in Xenopus a so far undefined developmental role.
Asunto(s)
Tipificación del Cuerpo/genética , Tipificación del Cuerpo/inmunología , Proteínas del Sistema Complemento/genética , Xenopus laevis/embriología , Xenopus laevis/inmunología , Animales , Vasos Sanguíneos/embriología , Vasos Sanguíneos/inmunología , Complemento C1q/genética , Complemento C3/genética , Complemento C9/genética , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Cristalino/embriología , Cristalino/inmunología , Mesodermo/embriología , Mesodermo/inmunología , Tubo Neural/embriología , Tubo Neural/inmunología , Organogénesis/genética , Organogénesis/inmunología , Properdina/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/inmunología , Xenopus laevis/genéticaRESUMEN
The dynamics of G protein-mediated signal transduction depend on the two-dimensional diffusion of membrane-bound G proteins and receptors, which has been suggested to be rate-limiting for vertebrate phototransduction, a highly amplified G protein-coupled signaling pathway. Using fluorescence recovery after photobleaching (FRAP), we measured the diffusion of the G protein transducin alpha-subunit (Galpha(t)) and the G protein-coupled receptor rhodopsin on disk membranes of living rod photoreceptors from transgenic Xenopus laevis. Treatment with either methyl-beta-cyclodextrin or filipin III to disrupt cholesterol-containing lipid microdomains dramatically accelerated diffusion of Galpha(t) in its GTP-bound state and of the rhodopsin-Galphabetagamma(t) complex but not of rhodopsin or inactive GDP-bound Galphabetagamma. These results imply an activity-dependent sequestration of G proteins into cholesterol-dependent lipid microdomains, which limits diffusion and exclude the majority of free rhodopsin and the free G protein heterotrimer. Our data offer a novel demonstration of lipid microdomains in the internal membranes of living sensory neurons.
Asunto(s)
Proteínas de Unión al GTP/química , Lípidos/química , Microdominios de Membrana/química , Animales , Colesterol/metabolismo , Densitometría , Proteínas de Unión al GTP/metabolismo , Guanosina Trifosfato/química , Insectos , Microscopía Fluorescente , Neuronas/metabolismo , Células Fotorreceptoras/metabolismo , Estructura Terciaria de Proteína , Rodopsina/química , Transducción de Señal , Xenopus laevisRESUMEN
Eye development is a complex process that involves the formation of the retina and the lens, collectively called the eyeball, as well as the formation of auxiliary eye structures such as the eyelid, lacrimal gland, cornea and conjunctiva. The developmental requirements for the formation of each individual structure are only partially understood. We have shown previously that the homeobox-containing gene Rx is a key component in eye formation, as retinal structures do not develop and retina-specific gene expression is not observed in Rx-deficient mice. In addition, Rx-/- embryos do not develop any lens structure, despite the fact that Rx is not expressed in the lens. This demonstrates that during normal mammalian development, retina-specific gene expression is necessary for lens formation. In this paper we show that lens formation can be restored in Rx-deficient embryos experimentally, by the elimination of beta-catenin expression in the head surface ectoderm. This suggests that beta-catenin is involved in lens specification either through Wnt signaling or through its function in cell adhesion. In contrast to lens formation, we demonstrate that the development of auxiliary eye structures does not depend on retina-specific gene expression or retinal morphogenesis. These results point to the existence of two separate developmental processes involved in the formation of the eye and its associated structures. One involved in the formation of the eyeball and the second involved in the formation of the auxiliary eye structures.
Asunto(s)
Proteínas del Ojo/fisiología , Ojo/embriología , Proteínas de Homeodominio/fisiología , Retina/embriología , beta Catenina/fisiología , Animales , Conjuntiva/citología , Conjuntiva/embriología , Ojo/citología , Proteínas del Ojo/genética , Párpados/citología , Párpados/embriología , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas de Homeodominio/genética , Hibridación in Situ , Aparato Lagrimal/citología , Aparato Lagrimal/embriología , Cristalino/citología , Cristalino/embriología , Ratones , Ratones Noqueados , Retina/citología , beta Catenina/genéticaRESUMEN
In this article, we investigate the expression, regulation, and function of the zebrafish forkhead gene foxe3. In wild type embryos, foxe3 is first expressed in a crescent-shaped area at the anterior end of the prechordal plate, corresponding to the polster. At later stages, the hatching gland, the lens, and the anterior pituitary express this gene. Using morpholinos against the zinc finger Kruppel-like factor 4 (KLF4) we show that foxe3 is regulated differently in the polster and in the lens. In the absence of KLF4, expression of foxe3 in the polster is not activated, whereas in the lens placode the expression of KLF4 is not required for the transcription of foxe3. The expression of foxe3 is also regulated by the hedgehog and nodal signaling pathways. foxe3 expression is altered in the hedgehog pathway mutants iguana and you-too and the nodal pathway mutant cyclops. foxe3 function is necessary for the execution of lens-specific gene expression and lens morphogenesis, as the knockdown of foxe3 results in a loss of platelet-derived growth factor receptor alpha (pdgfralpha) expression and in the vacuolization of the lens.