RESUMEN
Different kinase-dependent cell signaling pathways are known to play important roles in glia-mediated neuroprotection and reprogramming of Müller glia (MG) into Müller glia-derived progenitor cells (MGPCs) in the retina. However, very little is known about the phosphatases that regulate kinase-dependent signaling in MG. Using single-cell RNA-sequencing (scRNA-seq) databases, we investigated patterns of expression of Dual Specificity Phosphatases (DUSP1/6) and other protein phosphatases in normal and damaged chick retinas. We found that DUSP1, DUSP6, PPP3CB, PPP3R1 and PPPM1A/B/D/E/G are widely expressed by many types of retinal neurons and are dynamically expressed by MG and MGPCs in retinas during the process of reprogramming. We find that inhibition of DUSP1/6 and PP2C phosphatases enhances the formation of proliferating MGPCs in damaged retinas and in retinas treated with insulin and FGF2 in the absence of damage. By contrast, inhibition of PP2B phosphatases suppressed the formation of proliferating MGPCs, but increased numbers of proliferating MGPCs in undamaged retinas treated with insulin and FGF2. In damaged retinas, inhibition of DUSP1/6 increased levels of pERK1/2 and cFos in MG whereas inhibition of PP2B's decreased levels of pStat3 and pS6 in MG. Analyses of scRNA-seq libraries identified numerous differentially activated gene modules in MG in damaged retinas versus MG in retinas treated with insulin+FGF2 suggesting significant differences in kinase-dependent signaling pathways that converge on the formation of MGPCs. Inhibition of phosphatases had no significant effects upon numbers of dying cells in damaged retinas. We conclude that the activity of different protein phosphatases acting through retinal neurons and MG "fine-tune" the cell signaling responses of MG in damaged retinas and during the reprogramming of MG into MGPCs.
Asunto(s)
Pollos , Células Ependimogliales , Retina , Animales , Células Ependimogliales/metabolismo , Retina/metabolismo , Retina/citología , Células Madre/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/genética , Proliferación Celular/fisiología , Neuroglía/metabolismoRESUMEN
Chromatin access and epigenetic control over gene expression play important roles in regulating developmental processes. However, little is known about how chromatin access and epigenetic gene silencing influence mature glial cells and retinal regeneration. Herein, we investigate the expression and functions of S-adenosylhomocysteine hydrolase (SAHH; AHCY) and histone methyltransferases (HMTs) during the formation of Müller glia (MG)-derived progenitor cells (MGPCs) in the chick and mouse retinas. In chick, AHCY, AHCYL1 and AHCYL2, and many different HMTs are dynamically expressed by MG and MGPCs in damaged retinas. Inhibition of SAHH reduced levels of H3K27me3 and potently blocks the formation of proliferating MGPCs. By using a combination of single cell RNA-seq and single cell ATAC-seq, we find significant changes in gene expression and chromatin access in MG with SAHH inhibition and NMDA-treatment; many of these genes are associated with glial and neuronal differentiation. A strong correlation across gene expression, chromatin access, and transcription factor motif access in MG was observed for transcription factors known to convey glial identity and promote retinal development. By comparison, in the mouse retina, inhibition of SAHH has no influence on the differentiation of neuron-like cells from Ascl1-overexpressing MG. We conclude that in the chick the activity of SAHH and HMTs are required for the reprogramming of MG into MGPCs by regulating chromatin access to transcription factors associated with glial differentiation and retinal development.
Asunto(s)
Cromatina , Transducción de Señal , Animales , Ratones , Transducción de Señal/fisiología , Cromatina/metabolismo , Células Madre/metabolismo , Células Ependimogliales/metabolismo , Retina , Neuroglía/metabolismo , Pollos/genética , Factores de Transcripción/metabolismo , Proliferación Celular/fisiologíaRESUMEN
Thermal burn injuries are an important environmental stressor that can result in considerable morbidity and mortality. The exact mechanism by which an environmental stimulus to skin results in local and systemic effects is an area of active research. One potential mechanism to allow skin keratinocytes to disperse bioactive substances is via microvesicle particles, which are subcellular bodies released directly from cellular membranes. Our previous studies have indicated that thermal burn injury of the skin keratinocyte in vitro results in the production of the lipid mediator platelet-activating factor (PAF). The present studies demonstrate that thermal burn injury to keratinocytes in vitro and human skin explants ex vivo, and mice in vivo generate microvesicle particles. Use of pharmacologic and genetic tools indicates that the optimal release of microvesicles is dependent upon the PAF receptor. Of note, burn injury-stimulated microvesicle particles do not carry appreciable protein cytokines yet contain high levels of PAF. These studies describe a novel mechanism involving microvesicle particles by which a metabolically labile bioactive lipid can travel from cells in response to environmental stimuli.
Asunto(s)
Quemaduras/inmunología , Micropartículas Derivadas de Células/inmunología , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Piel/patología , Animales , Biopsia , Quemaduras/patología , Línea Celular , Micropartículas Derivadas de Células/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Queratinocitos/inmunología , Queratinocitos/metabolismo , Metabolismo de los Lípidos/inmunología , Ratones , Ratones Noqueados , Glicoproteínas de Membrana Plaquetaria/genética , Cultivo Primario de Células , Receptores Acoplados a Proteínas G/genética , Piel/inmunologíaRESUMEN
To investigate microRNA (miR) functions in early eye development, we asked whether eye field transcription factors (EFTFs) are targets of miR-dependent regulation in Xenopus embryos. Argonaute (AGO) ribonucleoprotein complexes, including miRs and targeted mRNAs, were coimmunoprecipitated from transgenic embryos expressing myc-tagged AGO under the control of the rax1 promoter; mRNAs for all EFTFs coimmunoprecipitated with Ago in late neurulae. Computational predictions of miR binding sites within EFTF 3'UTRs identified miR-199a-3p ("miR-199") as a candidate regulator of EFTFs, and miR-199 was shown to regulate rax1 in vivo. Targeted overexpression of miR-199 led to small eyes, a reduction in EFTF expression, and reduced cell proliferation. Inhibition of interactions between mir-199 and the rax1 3'UTR reversed the small eye phenotype. Although targeted knockdown of miR-199 left the eye field intact, it reduced optic cup outgrowth and disrupted eye formation. Computational identification of candidate miR-199 targets within the Xenopus transcriptome led to the identification of ptk7 as a candidate regulator. Targeted overexpression of ptk7 resulted in abnormal optic cup formation and a reduction or loss of eye development, recapitulating the range of eye phenotypes seen following miR-199 knockdown. Our results indicate that miR-199 plays both positive and negative regulatory roles in eye development.
Asunto(s)
Ojo/embriología , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Interferencia de ARN , Xenopus laevis/embriología , Xenopus laevis/genética , Animales , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Estudios de Asociación Genética , Mutación con Pérdida de Función , Organogénesis/genética , Fenotipo , Unión Proteica , Proteínas Tirosina Quinasas Receptoras/genética , Reproducibilidad de los Resultados , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
BACKGROUND: The retinal homeobox (rx/rax) gene is a transcription factor expressed in the developing eye field that is necessary for normal eye development. rax is necessary for retinal specification and stem cell development. The genetic program of early retinal development, including rax expression, can be induced in naïve ectoderm by activation of insulin-like growth factor (IGF) signaling. We have undertaken a microarray-based approach to identify rax-dependent IGF-induced genes. RESULTS: We identified 21 IGF-induced genes that exhibit at least a two-fold decrease in expression when rax expression is knocked down. Ten of these genes were expressed in the developing eye, eight were expressed in the ciliary marginal zone of the mature tadpole retina, and four could significantly rescue the rax knockdown phenotype. One of these, the nei endonuclease VIII-like 3 (neil3) gene, rescued the rax knockdown phenotype to a remarkable degree. We found that neil3 is necessary for normal retinal lamination and retinal neuron differentiation. CONCLUSIONS: We have identified neil3 as a component of the rax genetic pathway necessary for normal retinal progenitor cell development. neil3 is involved in the base excision DNA repair pathway, suggesting that this pathway is essential for normal rax-dependent progenitor cell development in the mature retina. Developmental Dynamics 247:1199-1210, 2018. © 2018 Wiley Periodicals, Inc.
Asunto(s)
Proteínas del Ojo/genética , N-Glicosil Hidrolasas/genética , Proteínas de Xenopus/genética , Animales , Proteínas del Ojo/análisis , Proteínas de Homeodominio/genética , Larva/crecimiento & desarrollo , Análisis por Matrices de Proteínas , Retina/química , Retina/citología , Células Madre , Proteínas de Xenopus/análisis , Xenopus laevis/embriologíaRESUMEN
Studies, including ours, have shown that pro-oxidative stressors, such as chemotherapeutic agents, generate oxidized lipids with agonistic platelet-activating factor (PAF) activity. Importantly, recent reports have implicated that these PAF-agonists are transported extracellularly via microvesicle particles (MVPs). While the role of PAF-receptor (PAF-R) has been implicated in mediating chemotherapy effects, its significance in chemotherapy-mediated MVP release in pancreatic cancer has not been studied. The current studies determined the functional significance of PAF-R in gemcitabine chemotherapy-mediated MVP release in human pancreatic cancer cells. Using PAF-R-expressing (PANC-1) and PAF-R-deficient (Hs766T) cells, we demonstrate that gemcitabine induces MVP release in a PAF-R-dependent manner. Blocking of PAF-R via PAF-R antagonist or inhibition of MVP generation via inhibitor of acid sphingomyelinase (aSMase) enzyme, significantly attenuated gemcitabine-mediated MVP release from PANC-1 cells, however, exerted no effects in Hs766T cells. Notably, MVPs from gemcitabine-treated PANC-1 cells, contained a measurable amount of PAF-agonists. Mechanistically, pretreatment with ERK1/2 or p38 inhibitors significantly abrogated gemcitabine-mediated MVP release, indicating the involvement of mitogen-activated protein kinase (MAPK) pathway in PAF-R-dependent gemcitabine-mediated MVP release. These findings demonstrate the significance of PAF-R in gemcitabine-mediated MVP release, as well as the rationale of evaluating PAF-R targeting agents with gemcitabine against pancreatic cancer.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Micropartículas Derivadas de Células/metabolismo , Desoxicitidina/análogos & derivados , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias Pancreáticas/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Línea Celular Tumoral , Desoxicitidina/farmacología , Humanos , Glicoproteínas de Membrana Plaquetaria/agonistas , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/metabolismo , GemcitabinaRESUMEN
The Retinal homeobox (rax) gene is expressed in vertebrate retinal progenitor and stem cells and is essential for retinal development. In frogs, rax is expressed in the ciliary marginal zone (CMZ), a region containing retinal progenitor and stem cells at the anterior of the eye. Little is known regarding regulation of rax transcription and regulation of transcription of rax targets. We found that three ultra-conserved genomic elements (UCEs) flanking the rax coding region regulate expression of a rax promoter-GFP transgene in Xenopus tadpoles. One of these elements, UCE1, regulates expression of the transgene in the dorsal CMZ. UCE1 contains a Rax binding site, PCE-1. We demonstrate that rax regulates expression of the transgene through the PCE-1 site found in UCE1. Therefore, rax transcription in the CMZ is controlled, in part, by autoregulatory mechanisms.
Asunto(s)
Proteínas del Ojo/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Retina/crecimiento & desarrollo , Proteínas de Xenopus/genética , Xenopus/genética , Animales , Sitios de Unión , Secuencia Conservada/genética , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Larva/genética , Larva/crecimiento & desarrollo , Regiones Promotoras Genéticas , Retina/metabolismo , Xenopus/crecimiento & desarrollo , Proteínas de Xenopus/biosíntesis , Proteínas de Xenopus/metabolismoRESUMEN
Nkx5 family members are homeobox transcription factors important for sensory organ development. Several members of the Nkx5 family are expressed in the eye, brain, developing ear, and lateral line. Members of this family have been previously identified in medaka, chick, and mouse. Here, we characterize two members of the Nkx5 family, Nkx5.3 and SOHo, in Xenopus laevis. We verify the identity of X. laevis Nkx5.3 and SOHo by phylogenetic comparison to chicken, medaka, and zebrafish orthologs. Both Nkx5.3 and SOHo are expressed in the developing eye, ear, lateral line system, and cranial neurons as determined by in situ hybridization.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Órganos de los Sentidos/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Ganglios/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Hibridación in Situ , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Filogenia , Órganos de los Sentidos/metabolismo , Alineación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/anatomía & histología , Xenopus laevis/metabolismoRESUMEN
PURPOSE: The photoreceptor conserved element-1 (PCE-1) sequence is found in the transcriptional regulatory regions of many genes expressed in photoreceptors. The retinal homeobox (Rx or Rax) gene product functions by binding to PCE-1 sites. However, other transcriptional regulators have also been reported to bind to PCE-1. One of these, vsx2, is expressed in retinal progenitor and bipolar cells. The purpose of this study is to identify Xenopus laevis vsx gene products and characterize vsx gene product expression and function with respect to the PCE-1 site. METHODS: X. laevis vsx gene products were amplified with PCR. Expression patterns were determined with in situ hybridization using whole or sectioned X. laevis embryos and digoxigenin- or fluorescein-labeled antisense riboprobes. DNA binding characteristics of the vsx gene products were analyzed with electrophoretic mobility shift assays (EMSAs) using in vitro translated proteins and radiolabeled oligonucleotide probes. Gene transactivation assays were performed using luciferase-based reporters and in vitro transcribed effector gene products, injected into X. laevis embryos. RESULTS: We identified one vsx1 and two vsx2 gene products. The two vsx2 gene products are generated by alternate mRNA splicing. We verified that these gene products are expressed in the developing retina and that expression resolves into distinct cell types in the mature retina. Finally, we found that vsx gene products can bind the PCE-1 site in vitro and that the two vsx2 isoforms have different gene transactivation activities. CONCLUSIONS: vsx gene products are expressed in the developing and mature neural retina. vsx gene products can bind the PCE-1 site in vitro and influence the expression of a rhodopsin promoter-luciferase reporter gene. The two isoforms of vsx have different gene transactivation activities in this reporter gene system.
Asunto(s)
Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Células Fotorreceptoras/metabolismo , Elementos Reguladores de la Transcripción/genética , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Animales , Ensayo de Cambio de Movilidad Electroforética , Amplificación de Genes , Genes Homeobox , Hibridación in Situ , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Transcripción Genética , Xenopus laevisRESUMEN
Different kinase-dependent cell signaling pathways are known to play important roles in glia-mediated neuroprotection and reprogramming of Müller glia (MG) into Müller glia-derived progenitor cells (MGPCs) in the retina. However, very little is known about the phosphatases that regulate kinase-dependent signaling in MG. Using single-cell RNA-sequencing (scRNA-seq) databases, we investigated patterns of expression of Dual Specificity Phosphatases (DUSP1/6) and other protein phosphatases in normal and damaged chick retinas. We found that DUSP1, DUSP6, PPP3CB, PPP3R1 and PPPM1A/B/D/E/G are dynamically expressed by MG and MGPCs in retinas during the process of reprogramming. We find that inhibition of DUSP1/6 and PP2C phosphatases enhances the formation of proliferating MGPCs in damaged retinas and in retinas treated with insulin in FGF2 in the absence of damage. By contrast, inhibition of PP2B phosphatases suppressed the formation of proliferating MGPCs, but increased numbers of proliferating MGPCs in undamaged retinas treated with insulin and FGF2. In damaged retinas, inhibition of DUSP1/6 increased levels of pERK1/2 and cFos in MG whereas inhibition of PP2B's decreased levels of pStat3 and pS6 in MG. Analyses of scRNA-seq libraries identified numerous differentially activated gene modules in MG in damaged retinas versus MG in retinas treated with insulin+FGF2 suggesting significant differences in kinase-dependent signaling pathways that converge on the formation of MGPCs. Inhibition of phosphatases had no significant effects upon numbers of dying cells in damaged retinas. We conclude that the activity of different protein phosphatases "fine-tune" the cell signaling responses of MG in damaged retinas and during the reprogramming of MG into MGPCs.
RESUMEN
The Retinal Homeobox (Rx) gene is essential for vertebrate eye development. Rx function is required for the specification and maintenance of retinal progenitor cells (RPCs). Loss of Rx function leads to a lack of eye development in a variety of species. Here we show that Rx function is also necessary during retinal regeneration. We performed a thorough characterization of retinal regeneration after partial retinal resection in pre-metamorphic Xenopus laevis. We show that after injury the wound is repopulated with retinal progenitor cells (RPCs) that express Rx and other RPC marker genes. We used an shRNA-based approach to specifically silence Rx expression in vivo in tadpoles. We found that loss of Rx function results in impaired retinal regeneration, including defects in the cells that repopulate the wound and the RPE at the wound site. We show that the regeneration defects can be rescued by provision of exogenous Rx. These results demonstrate for the first time that Rx, in addition to being essential during retinal development, also functions during retinal regeneration.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Homeodominio/metabolismo , Regeneración , Retina/fisiología , Factores de Transcripción/metabolismo , Xenopus laevis/embriología , Animales , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Larva/fisiología , Retina/citología , Células Madre/fisiología , Factores de Transcripción/genética , TransgenesRESUMEN
The regenerative potential of Müller glia (MG) is extraordinary in fish, poor in chick and terrible in mammals. In the chick model, MG readily reprogram into proliferating Müller glia-derived progenitor cells (MGPCs), but neuronal differentiation is very limited. The factors that suppress the neurogenic potential of MGPCs in the chick are slowly being revealed. Isoforms of Nuclear Factor I (NFI) are cell-intrinsic factors that limit neurogenic potential; these factors are required for the formation of MG in the developing mouse retina and deletion of these factors reprograms MG into neuron-like cells in mature mouse retina. Accordingly, we sought to characterize the patterns of expression of NFIs in the developing, mature and damaged chick retina. In addition, we characterized patterns of expression of NFIs in the retinas of large mammals, pigs and monkeys. Using a combination of single-cell RNA-sequencing (scRNA-seq) and immunolabeling, we probed for patterns of expression. In embryonic chick, levels of NFIs are very low in early E5 (embryonic day 5) retinal progenitor cells (RPCs), upregulated in E8 RPCs, further upregulated in differentiating MG at E12 and E15. NFIs are maintained in mature resting MG, microglia and neurons. Levels of NFIs are reduced in activated MG in retinas treated with NMDA and/or insulin+FGF2, and further downregulated in proliferating MGPCs. However, levels of NFIs in MGPCs were significantly higher than those seen in RPCs. Immunolabeling for NFIA and NFIB closely matched patterns of expression revealed in different types of retinal neurons and glia, consistent with findings from scRNA-seq. In addition, we find expression of NFIA and NFIB through progenitors in the circumferential marginal zone at the far periphery of the retina. We find similar patterns of expression for NFIs in scRNA-seq databases for pig and monkey retinas. Patterns of expression of NFIA and NFIB were validated with immunofluorescence in pig and monkey retinas wherein these factors were predominantly detected in MG and a few types of inner retinal neurons. In summary, NFIA and NFIB are prominently expressed in developing chick retina and by mature neurons and glia in the retinas of chicks, pigs and monkeys. Although levels of NFIs are decreased in chick, in MGPCs these levels remain higher than those seen in neurogenic RPCs. We propose that the neurogenic potential of MGPCs in the chick retina is suppressed by NFIs.
Asunto(s)
Factores de Transcripción NFI , Transducción de Señal , Animales , Proliferación Celular/fisiología , Mamíferos , Ratones , Factores de Transcripción NFI/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Primates/metabolismo , Retina , Transducción de Señal/fisiología , Células Madre , PorcinosRESUMEN
The retinal homeobox (Rx) gene product is essential for eye development. However little is known about its molecular function. It has been demonstrated that Rx binds to photoreceptor conserved element (PCE-1), a highly conserved element found in the promoter region of photoreceptor-specific genes such as rhodopsin and red cone opsin. We verify that Rx is co-expressed with rhodopsin and red cone opsin in maturing photoreceptors and demonstrate that Rx binds to the rhodopsin and red cone opsin promoters in vivo. We also find that Rx can cooperate with the Xenopus analogs of Crx and Nrl, otx5b and XLMaf (respectively), to activate a Xenopus opsin promoter-dependent reporter. Finally, we demonstrate that reduction of Rx expression in tadpoles results in decreases in expression of several PCE-1 containing photoreceptor genes, abnormal photoreceptor morphology, and impaired vision. Our data suggests that Rx, in combination with other transcription factors, is necessary for normal photoreceptor gene expression, maintenance, and function. This establishes a direct role for Rx in regulation of genes expressed in a differentiated cell type.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Células Fotorreceptoras/metabolismo , Rodopsina/genética , Opsinas de Bastones/genética , Proteínas de Xenopus/genética , Animales , Embrión no Mamífero/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/genética , Retinaldehído , Rodopsina/metabolismo , Opsinas de Bastones/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
A complete carcinogen, ultraviolet B (UVB) radiation (290-320 nm), is the major cause of skin cancer. UVB-induced systemic immunosuppression that contributes to photocarcinogenesis is due to the glycerophosphocholine-derived lipid mediator platelet-activating factor (PAF). A major question in photobiology is how UVB radiation, which only absorbs appreciably in the epidermal layers of skin, can generate systemic effects. UVB exposure and PAF receptor (PAFR) activation in keratinocytes induce the release of large numbers of microvesicle particles (MVPs; extracellular vesicles ranging from 100 to 1000 nm in size). MVPs released from skin keratinocytes in vitro in response to UVB (UVB-MVPs) are dependent on the keratinocyte PAFR. Here, we used both pharmacologic and genetic approaches in cells and mice to show that both the PAFR and enzyme acid sphingomyelinase (aSMase) were necessary for UVB-MVP generation. Our discovery that the calcium-sensing receptor is a keratinocyte-selective MVP marker allowed us to determine that UVB-MVPs leaving the keratinocyte can be found systemically in mice and humans following UVB exposure. Moreover, we found that UVB-MVPs contained bioactive contents including PAFR agonists that allowed them to serve as effectors for UVB downstream effects, in particular UVB-mediated systemic immunosuppression.
Asunto(s)
Micropartículas Derivadas de Células/inmunología , Tolerancia Inmunológica/efectos de la radiación , Queratinocitos/inmunología , Rayos Ultravioleta , Animales , Línea Celular , Micropartículas Derivadas de Células/genética , Femenino , Humanos , Ratones , Ratones Noqueados , Factor de Activación Plaquetaria/genética , Factor de Activación Plaquetaria/inmunología , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/inmunología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/inmunologíaRESUMEN
The aristaless-related gene, Arx, plays a fundamental role in patterning the brain in humans and mice. Arx mutants exhibit lissencephaly among other anomalies. We have cloned a Xenopus aristaless homolog that appears to define specific regions of the developing forebrain. xArx2 is transcribed in blastula through neurula stages, and comes to be restricted to the ventral and lateral telencephalon, lateral diencephalon, neural floor plate of the anterior spinal cord, and somites. In this respect, Arx2 expresses in regions similar to Arx with the exception of the somites. Overexpression enlarges the telencephalon, and interference by means of antisense morpholino-mediated translation knockdown reduces growth of this area. Overexpression and inhibition studies demonstrate that misregulation of xArx2 imposes dire consequences upon patterns of differentiation not only in the forebrain where the gene normally expresses, but also in more caudal brain territories and derivatives as well. This suggests that evolutionary changes that expanded Arx-expression from ventral to dorsal prosencephalon might be one of the determinants that marked development and expansion of the telencephalon.
Asunto(s)
Encéfalo/embriología , Encéfalo/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/crecimiento & desarrollo , Secuencia Conservada , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Filogenia , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Alineación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/clasificación , Factores de Transcripción/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/clasificación , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
In situ hybridization performed using whole fixed embryos provides accurate and detailed visualization of gene expression patterns. These patterns are useful for investigating spatial patterns of gene expression in normally developing embryos but can also be useful in investigating the effects of genetic or environmental changes on expression of genetic markers characteristic of particular tissues, organs, or genetic pathways. Our lab's protocol for whole-mount in situ hybridization is presented.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Transcriptoma , Xenopus laevis/embriología , Xenopus laevis/genética , Animales , Embrión no Mamífero , Perfilación de la Expresión Génica/métodos , Hibridación in Situ/métodos , FenotipoRESUMEN
Thermal burn injuries in patients who are alcohol-intoxicated result in greater morbidity and mortality. Murine models combining ethanol and localized thermal burn injury reproduce the systemic toxicity seen in human subjects, which consists of both acute systemic cytokine production with multiple organ dysfunction, as well as a delayed systemic immunosuppression. However, the exact mechanisms for these acute and delayed effects are unclear. These studies sought to define the role of the lipid mediator platelet-activating factor in the acute and delayed effects of intoxicated burn injury. Combining ethanol and thermal burn injury resulted in increased enzymatic platelet-activating factor generation in a keratinocyte cell line in vitro, human skin explants ex vivo, as well as in murine skin in vivo. Further, the acute increase in inflammatory cytokines, such as IL-6, and the systemic immunosuppressive effects of intoxicated thermal burn injury were suppressed in mice lacking platelet-activating factor receptors. Together, these studies provide a potential mechanism and treatment strategies for the augmented toxicity and immunosuppressive effects of thermal burn injury in the setting of acute ethanol exposure, which involves the pleotropic lipid mediator platelet-activating factor.
Asunto(s)
Quemaduras/inmunología , Etanol/metabolismo , Queratinocitos/fisiología , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Receptores Acoplados a Proteínas G/genética , Enfermedad Aguda , Intoxicación Alcohólica , Animales , Línea Celular , Citocinas/metabolismo , Femenino , Calor , Humanos , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regulación hacia ArribaRESUMEN
Forkhead proteins are involved in gene regulation in a large variety of developmental situations. Several forkhead gene products are expressed in the developing eye and brain. Here we characterize the expression of FoxN4 during Xenopus development. We report that FoxN4 is expressed in the eye from the earliest stages of specification through retinal maturation. FoxN4 is also expressed in the pallium, optic tectum, isthmus, reticular formation, and in cells lining the ventricle of the tadpole brain.
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Encéfalo/embriología , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Retina/embriología , Células Madre/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Secuencia de Aminoácidos , Animales , Encéfalo/citología , Embrión no Mamífero/metabolismo , Factores de Transcripción Forkhead/química , Datos de Secuencia Molecular , Retina/citología , Alineación de Secuencia , Proteínas de Xenopus/química , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
PURPOSE: The retinal homeobox (Rx) gene plays an essential role in retinal development. An Rx-like (Rx-L) gene from Xenopus laevis has been identified. The purpose of this study was to analyze the function of Rx-L in the developing retina. METHODS: DNA-binding properties of Rx-L were analyzed by electrophoretic mobility shift assay (EMSA), with in vitro-translated proteins and radiolabeled oligonucleotide probe. The Rx-L expression pattern was analyzed by in situ hybridization using whole or sectioned embryos and digoxigenin-labeled antisense riboprobes. Rx-L loss of function was studied by using antisense morpholino oligonucleotides targeted to the Rx-L translation initiation site. Embryos injected with control or Rx-L morpholinos were analyzed at stage 41 or 45. RESULTS: Rx-L shares homology with Rx at the homeo-, OAR, and Rx domains, but lacks an octapeptide motif. Rx-L is expressed in the developing retina beginning in the early tailbud stage. In the maturing retina, Rx-L expression is restricted primarily to the developing photoreceptor layer and the ciliary marginal zone. Rx-L can bind a photoreceptor conserved element-1 (PCE-1) oligonucleotide, an element conserved among all known photoreceptor gene promoters. In a promoter activity assay, Rx-L functions as a stronger transcriptional activator than Rx. Antisense morpholino-mediated knockdown of Rx-L expression resulted in a decrease in rhodopsin and red cone opsin expression levels in Xenopus retinas. Injection of the Rx-L antisense morpholino oligonucleotide also resulted in a decrease in the length of both rod and cone outer segments. CONCLUSIONS: The results suggest that Rx-L functions to regulate rod and cone development by activating photoreceptor-specific gene expression.
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Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Células Fotorreceptoras de Vertebrados/fisiología , Retina/embriología , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Ensayo de Cambio de Movilidad Electroforética , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/química , Células Fotorreceptoras de Vertebrados/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
The members of the FoxE subfamily of Fox (forkhead) genes are expressed in the developing pituitary, thyroid and lens. Mammalian Foxe1 is expressed primarily in the developing pituitary and thyroid gland, Foxe3 is expressed in the developing lens, while Xenopus FoxE4 is expressed in the developing lens and thyroid. Here we report the identification of Xenopus FoxE1, a gene that is primarily expressed in the developing pituitary and thyroid.