RESUMEN
The tRNA-histidine guanylyltransferase 1-like (THG1L), also known as induced in high glucose-1 (IHG-1), encodes for an essential mitochondria-associated protein highly conserved throughout evolution, that catalyses the 3'-5' addition of a guanine to the 5'-end of tRNA-histidine (tRNAHis). Previous data indicated that THG1L plays a crucial role in the regulation of mitochondrial biogenesis and dynamics, in ATP production, and is critically involved in the modulation of apoptosis, cell-cycle progression and survival, as well as in cellular stress responses and redox homeostasis. Dysregulations of THG1L expression play a central role in various pathologies, including nephropathies, and neurodevelopmental disorders often characterized by developmental delay and cerebellar ataxia. Despite the essential role of THG1L, little is known about its expression during vertebrate development. Herein, we examined the detailed spatio-temporal expression of this gene in the developing Xenopus laevis. Our results show that thg1l is maternally inherited and its temporal expression suggests a role during the earliest stages of embryogenesis. Spatially, thg1l mRNA localizes in the ectoderm and marginal zone mesoderm during early stages of development. Then, at tadpole stages, thg1l transcripts mostly localise in neural crests and their derivatives, somites, developing kidney and central nervous system, therefore largely coinciding with territories displaying intense energy metabolism during organogenesis in Xenopus.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Nucleotidiltransferasas , Proteínas de Xenopus , Xenopus laevis , Animales , Embrión no Mamífero/metabolismo , Embrión no Mamífero/embriología , Desarrollo Embrionario/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Xenopus laevis/metabolismo , Xenopus laevis/embriología , Xenopus laevis/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
The 4th Italian Zebrafish Meeting took place in Palermo from February 7 to 9, 2024. The primary aim of this meeting was to bring together a diverse group of principal investigators, young researchers, facility managers, commercial vendors, and others to provide an important forum for presentation and discussion of the most innovative and exciting scientific research currently ongoing in Italy using the zebrafish model. Nonetheless, the meeting program has been conceived to allow the dissemination of cutting-edge scientific research across a wide range of topics and to shed light on its future directions, without geographical boundaries. Indeed, people from various parts of the world joined the meeting, and 210 participants presented their latest work in talks and posters. Importantly, the meeting had designated time to foster open scientific exchange and informal networking opportunities among participants of all career stages, thus allowing initiation of new collaborations and strengthening of existing partnerships. The meeting was a tremendous success as testified by the highest participation ever since the first meeting of the series in 2017, coupled with the highly positive satisfaction rating expressed by the attendants. The full program and detailed information about the meeting can be found on the dedicated website at https://itazebrafishmeeting.wixsite.com/izm2024.
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Pez Cebra , Animales , Italia , Modelos Animales , SiciliaRESUMEN
Vision starts in retinal photoreceptors when specialized proteins (opsins) sense photons via their covalently bonded vitamin A derivative 11cis retinaldehyde (11cis-RAL). The reaction of non-enzymatic aldehydes with amino groups lacks specificity, and the reaction products may trigger cell damage. However, the reduced synthesis of 11cis-RAL results in photoreceptor demise and suggests the need for careful control over 11cis-RAL handling by retinal cells. This perspective focuses on retinoid(s) synthesis, their control in the adult retina, and their role during retina development. It also explores the potential importance of 9cis vitamin A derivatives in regulating retinoid synthesis and their impact on photoreceptor development and survival. Additionally, recent advancements suggesting the pivotal nature of retinoid synthesis regulation for cone cell viability are discussed.
Asunto(s)
Retinoides , Animales , Humanos , Retina/metabolismo , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Retinaldehído/metabolismo , Retinoides/metabolismo , Vitamina A/metabolismoRESUMEN
Haploinsufficiency of the PRR12 gene is implicated in a human neuro-ocular syndrome. Although identified as a nuclear protein highly expressed in the embryonic mouse brain, PRR12 molecular function remains elusive. This study explores the spatio-temporal expression of zebrafish PRR12 co-orthologs, prr12a and prr12b, as a first step to elucidate their function. In silico analysis reveals high evolutionary conservation in the DNA-interacting domains for both orthologs, with significant syntenic conservation observed for the prr12b locus. In situ hybridization and RT-qPCR analyses on zebrafish embryos and larvae reveal distinct expression patterns: prr12a is expressed early in zygotic development, mainly in the central nervous system, while prr12b expression initiates during gastrulation, localizing later to dopaminergic telencephalic and diencephalic cell clusters. Both transcripts are enriched in the ganglion cell and inner neural layers of the 72 hpf retina, with prr12b widely distributed in the ciliary marginal zone. In the adult brain, prr12a and prr12b are found in the cerebellum, amygdala and ventral telencephalon, which represent the main areas affected in autistic patients. Overall, this study suggests PRR12's potential involvement in eye and brain development, laying the groundwork for further investigations into PRR12-related neurobehavioral disorders.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana , Proteínas del Tejido Nervioso , Proteínas de Pez Cebra , Animales , Encéfalo/metabolismo , Encéfalo/crecimiento & desarrollo , Retina/metabolismo , Retina/crecimiento & desarrollo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas de la Membrana/genéticaRESUMEN
BACKGROUND: Post-translational histone modifications are among the most common epigenetic modifications that orchestrate gene expression, playing a pivotal role during embryonic development and in various pathological conditions. Among histone lysine demethylases, KDM7A, also known as KIAA1718 or JHDM1D, catalyzes the demethylation of H3K9me1/2 and H3K27me1/2, leading to transcriptional regulation. Previous data suggest that KDM7A plays a central role in several biological processes, including cell proliferation, commitment, differentiation, apoptosis, and maintenance. However, information on the expression pattern of KDM7A in whole organisms is limited, and its functional role is still unclear. RESULTS: In Xenopus development, kdm7a is expressed early, undergoing spatiotemporal regulation in various organs and tissues, including the central nervous system and the eye. Focusing on retinal development, we found that kdm7a overexpression does not affect the expression of genes critically involved in early neural development and eye-field specification, whereas unbalances the distribution of neural cell subtypes in the mature retina by disfavoring the development of ganglion cells while promoting that of horizontal cells. CONCLUSIONS: Kdm7a is dynamically expressed during embryonic development, and its overexpression influences late retinal development, suggesting a potential involvement in the molecular machinery regulating the spatiotemporally ordered generation of retinal neuronal subtypes.
RESUMEN
In proliferating multipotent retinal progenitors, transcription factors dynamics set the fate of postmitotic daughter cells, but postmitotic cell fate plasticity driven by extrinsic factors remains controversial. Transcriptome analysis reveals the concurrent expression by postmitotic rod precursors of genes critical for the Müller glia cell fate, which are rarely generated from terminally-dividing progenitors as a pair with rod precursors. By combining gene expression and functional characterisation in single cultured rod precursors, we identified a time-restricted window where increasing cell culture density switches off the expression of genes critical for Müller glial cells. Intriguingly, rod precursors in low cell culture density maintain the expression of genes of rod and glial cell fate and develop a mixed rod/Muller glial cells electrophysiological fingerprint, revealing rods derailment toward a hybrid rod-glial phenotype. The notion of cell culture density as an extrinsic factor critical for preventing rod-fated cells diversion toward a hybrid cell state may explain the occurrence of hybrid rod/MG cells in the adult retina and provide a strategy to improve engraftment yield in regenerative approaches to retinal degenerative disease by stabilising the fate of grafted rod precursors.
Asunto(s)
Neuroglía , Retina , Retina/metabolismo , Neuroglía/metabolismo , Diferenciación Celular/genética , Factores de Transcripción/metabolismo , Técnicas de Cultivo de CélulaRESUMEN
Haploinsufficiency of the SETD5 gene, encoding a SET domain-containing histone methyltransferase, has been identified as a cause of intellectual disability and Autism Spectrum Disorder (ASD). Recently, the zebrafish has emerged as a valuable model to study neurodevelopmental disorders because of its genetic tractability, robust behavioral traits and amenability to high-throughput drug screening. To model human SETD5 haploinsufficiency, we generated zebrafish setd5 mutants using the CRISPR/Cas9 technology and characterized their morphological, behavioral and molecular phenotypes. According to our observation that setd5 is expressed in adult zebrafish brain, including those areas controlling social behavior, we found that setd5 heterozygous mutants exhibit defective aggregation and coordination abilities required for shoaling interactions, as well as indifference to social stimuli. Interestingly, impairment in social interest is rescued by risperidone, an antipsychotic drug used to treat behavioral traits in ASD individuals. The molecular analysis underscored the downregulation of genes encoding proteins involved in the synaptic structure and function in the adult brain, thus suggesting that brain hypo-connectivity could be responsible for the social impairments of setd5 mutant fishes. The zebrafish setd5 mutants display ASD-like features and are a promising setd5 haploinsufficiency model for drug screening aimed at reversing the behavioral phenotypes.
Asunto(s)
Trastorno del Espectro Autista , Metiltransferasas , Animales , Humanos , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Metiltransferasas/genética , Metiltransferasas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Conducta SocialRESUMEN
Microgravity and space radiation (SR) are two highly influential factors affecting humans in space flight (SF). Many health problems reported by astronauts derive from endothelial dysfunction and impaired homeostasis. Here, we describe the adaptive response of human, capillary endothelial cells to SF. Reference samples on the ground and at 1g onboard permitted discrimination between the contribution of microgravity and SR within the combined responses to SF. Cell softening and reduced motility occurred in SF cells, with a loss of actin stress fibers and a broader distribution of microtubules and intermediate filaments within the cytoplasm than in control cells. Furthermore, in space the number of primary cilia per cell increased and DNA repair mechanisms were found to be activated. Transcriptomics revealed the opposing effects of microgravity from SR for specific molecular pathways: SR, unlike microgravity, stimulated pathways for endothelial activation, such as hypoxia and inflammation, DNA repair and apoptosis, inhibiting autophagic flux and promoting an aged-like phenotype. Conversely, microgravity, unlike SR, activated pathways for metabolism and a pro-proliferative phenotype. Therefore, we suggest microgravity and SR should be considered separately to tailor effective countermeasures to protect astronauts' health.
Asunto(s)
Autofagia , Capilares/citología , Radiación Cósmica , Células Endoteliales/efectos de la radiación , Transducción de Señal , Ingravidez , Apoptosis , Biomarcadores/metabolismo , Línea Celular , Supervivencia Celular , Cromosomas Humanos/metabolismo , Citoesqueleto/metabolismo , Daño del ADN , Fluorescencia , Regulación de la Expresión Génica , Genoma Humano , Humanos , Masculino , Mecanotransducción Celular , Modelos Biológicos , Transducción de Señal/efectos de la radiación , Vuelo Espacial , Estrés Fisiológico , Homeostasis del Telómero , Transcriptoma/genéticaRESUMEN
Inherited retinal degenerations (IRD) affecting either photoreceptors or pigment epithelial cells cause progressive visual loss and severe disability, up to complete blindness. Retinal organoids (ROs) technologies opened up the development of human inducible pluripotent stem cells (hiPSC) for disease modeling and replacement therapies. However, hiPSC-derived ROs applications to IRD presently display limited maturation and functionality, with most photoreceptors lacking well-developed outer segments (OS) and light responsiveness comparable to their adult retinal counterparts. In this review, we address for the first time the microenvironment where OS mature, i.e., the subretinal space (SRS), and discuss SRS role in photoreceptors metabolic reprogramming required for OS generation. We also address bioengineering issues to improve culture systems proficiency to promote OS maturation in hiPSC-derived ROs. This issue is crucial, as satisfying the demanding metabolic needs of photoreceptors may unleash hiPSC-derived ROs full potential for disease modeling, drug development, and replacement therapies.
Asunto(s)
Bioingeniería/métodos , Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Organoides/citología , Degeneración Retiniana/terapia , Epitelio Pigmentado de la Retina/citología , Animales , Humanos , Degeneración Retiniana/patologíaRESUMEN
The plant-derived natural alkaloid berberine displays therapeutic potential to treat several pathological conditions, including dyslipidemias, diabetes and cardiovascular disorders. However, data on berberine effects during embryonic development are scarce and in part controversial. In this study, using zebrafish embryos as vertebrate experimental model, we address the effects of berberine treatment on cardiovascular system development and functionality. Starting from the observation that berberine induces developmental toxicity and pericardial edema in a time- and concentration-dependent manner, we found that treated embryos display cardiac looping defects and, at later stages, present an abnormal heart characterized by a stretched morphology and atrial endocardial/myocardial detachment. Furthermore, berberine affected cardiac functionality of the embryos, promoting bradycardia and reducing the cardiac output, the atrial shortening fraction percentage and the atrial stroke volume. We also found that, during development, berberine interferes with the angiogenic process, without altering vascular permeability. These alterations are associated with increased levels of vascular endothelial growth factor aa (vegfaa) mRNA, suggesting an important role for Vegfaa as mediator of berberine-induced cardiovascular defects. Altogether, these data indicate that berberine treatment during vertebrate development leads to an impairment of cardiovascular system morphogenesis and functionality, suggesting a note of caution in its use during pregnancy and lactation.
Asunto(s)
Berberina/toxicidad , Sistema Cardiovascular/embriología , Morfogénesis/efectos de los fármacos , Pez Cebra/embriología , Animales , Teratógenos/toxicidadRESUMEN
BACKGROUND: Understanding the signalling pathways involved in angiogenesis, and developing anti-angiogenic drugs are one of the major focuses on cancer research. Herein, we assessed the effect of CPTH6, a lysine acetyltransferase inhibitor and anti-tumoral compound, on angiogenesis-related properties of both endothelial and cancer cells. METHODS: The in vitro effect of CPTH6 on protein acetylation and anti-angiogenic properties on endothelial and lung cancer cells was evaluated via wound healing, trans-well invasion and migration, tube formation, immunoblotting and immunofluorescence. Matrigel plug assay, zebrafish embryo and mouse xenograft models were used to evaluate in vivo anti-angiogenic effect of CPTH6. RESULTS: CPTH6 impaired in vitro endothelial angiogenesis-related functions, and decreased the in vivo vascularization both in mice xenografts and zebrafish embryos. Mechanistically, CPTH6 reduced α-tubulin acetylation and induced accumulation of acetylated microtubules in the perinuclear region of endothelial cells. Interestingly, CPTH6 also affected the angiogenesis-related properties of lung cancer cells, and conditioned media derived from CPTH6-treated lung cancer cells impaired endothelial cells morphogenesis. CPTH6 also modulated the VEGF/VEGFR2 pathway, and reshaped cytoskeletal organization of lung cancer cells. Finally, anti-migratory effect of CPTH6, dependent on α-tubulin acetylation, was also demonstrated by genetic approaches in lung cancer cells. CONCLUSION: Overall, this study indicates that α-tubulin acetylation could play a role in the anti-angiogenic effect of CPTH6 and, more in general, it adds information to the role of histone acetyltransferases in tumor angiogenesis, and proposes the inhibition of these enzymes as an antiangiogenic therapy of cancer.
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Inhibidores de la Angiogénesis/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Neoplasias Pulmonares/irrigación sanguínea , Lisina Acetiltransferasas/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Tiazoles/farmacología , Animales , Apoptosis , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Transducción de Señal , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mutations in one SETD5 allele are genetic causes of intellectual disability and autistic spectrum disorders. However, the mechanisms by which SETD5 regulates brain development and function remain largely elusive. Herein, we found that Setd5 haploinsufficiency impairs the proliferative dynamics of neural progenitors and synaptic wiring of neurons, ultimately resulting in behavioral deficits in mice. Mechanistically, Setd5 inactivation in neural stem cells, zebrafish, and mice equally affects genome-wide levels of H3K36me3 on active gene bodies. Notably, we demonstrated that SETD5 directly deposits H3K36me3, which is essential to allow on-time RNA elongation dynamics. Hence, Setd5 gene loss leads to abnormal transcription, with impaired RNA maturation causing detrimental effects on gene integrity and splicing. These findings identify SETD5 as a fundamental epigenetic enzyme controlling the transcriptional landscape in neural progenitors and their derivatives and illuminate the molecular events that connect epigenetic defects with neuronal dysfunctions at the basis of related human diseases.
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Encéfalo/embriología , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Código de Histonas/genética , Metiltransferasas/genética , Proteínas de Pez Cebra/fisiología , Animales , Conducta Animal , Encéfalo/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Cognición , Epigénesis Genética , Histona Metiltransferasas/genética , Metiltransferasas/fisiología , Ratones , Mutación , Células-Madre Neurales/metabolismo , Empalme del ARN/genética , RNA-Seq , Conducta Social , Elongación de la Transcripción Genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Collagen prolyl 4-hydroxylases (c-P4Hs) are evolutionary conserved enzymes whose activity is essential for the correct folding of stable triple helical molecules of collagen and collagen-like proteins. They play crucial roles in embryo development, connective tissue functional organization, tumor growth and metastasis. Despite the important function of these enzymes, little is known about their expression during vertebrate development. In this study, we determine and compare the previously undescribed spatio-temporal expression patterns of the p4ha1 and p4ha2 genes, which encode the main subunits containing the enzyme active site, during Xenopus development. The two genes are maternally inherited and share expression in dorsal mesoderm, branchial arches and their derivatives, as well as in the central nervous system, although with distinct spatio-temporal patterns. A major co-expression domain for p4ha1 and p4ha2 is represented by the developing notochord, where these genes are transcribed from early neurula stage to stage 42 tadpole, thus paralleling the profile of collagen II production and suggesting a coordination between collagen synthesis and its post-translational modifications.
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Regulación del Desarrollo de la Expresión Génica , Procolágeno-Prolina Dioxigenasa/clasificación , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Procolágeno-Prolina Dioxigenasa/genética , Análisis Espacio-Temporal , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
Glaucoma and other optic neuropathies are characterized by a loss of retinal ganglion cells (RGCs), a cell layer located in the posterior eye segment. Several preclinical studies demonstrate that neurotrophins (NTs) prevent RGC loss. However, NTs are rarely investigated in the clinic due to various issues, such as difficulties in reaching the retina, the very short half-life of NTs, and the need for multiple injections. We demonstrate that NTs can be conjugated to magnetic nanoparticles (MNPs), which act as smart drug carriers. This combines the advantages of the self-localization of the drug in the retina and drug protection from fast degradation. We tested the nerve growth factor and brain-derived neurotrophic factor by comparing the neuroprotection of free versus conjugated proteins in a model of RGC loss induced by oxidative stress. Histological data demonstrated that the conjugated proteins totally prevented RGC loss, in sharp contrast to the equivalent dose of free proteins, which had no effect. The overall data suggest that the nanoscale MNP-protein hybrid is an excellent tool in implementing ocular drug delivery strategies for neuroprotection and therapy.
Asunto(s)
Nanopartículas/química , Factores de Crecimiento Nervioso/farmacología , Neuroprotección/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Retina/efectos de los fármacos , Animales , Factor Neurotrófico Derivado del Encéfalo/administración & dosificación , Factor Neurotrófico Derivado del Encéfalo/química , Factor Neurotrófico Derivado del Encéfalo/farmacología , Sistemas de Liberación de Medicamentos , Glaucoma/metabolismo , Glaucoma/patología , Humanos , Factor de Crecimiento Nervioso/administración & dosificación , Factor de Crecimiento Nervioso/química , Factor de Crecimiento Nervioso/farmacología , Factores de Crecimiento Nervioso/administración & dosificación , Factores de Crecimiento Nervioso/química , Células PC12 , Ratas , Retina/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Células Tumorales CultivadasRESUMEN
P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro. We, then, translated this approach in vivo by delivering the CRISPR/Cas9 components in murine Rho+/P23H mutant retinae. Targeted retinae presented a high rate of cleavage in the P23H but not WT Rho allele. This gene manipulation was sufficient to slow photoreceptor degeneration and improve retinal functions. To improve the translational potential of our approach, we tested intravitreal delivery of this system by means of adeno-associated viruses (AAVs). To this purpose, the employment of the AAV9-PHP.B resulted the most effective in disrupting the P23H Rho mutant. Finally, this approach was translated successfully in human cells engineered with the homozygous P23H RHO gene mutation. Overall, this is a significant proof-of-concept that gene allele specific targeting by CRISPR/Cas9 technology is specific and efficient and represents an unprecedented tool for treating RP and more broadly dominant genetic human disorders affecting the eye, as well as other tissues.
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Marcación de Gen/métodos , Vectores Genéticos , Retina/fisiología , Degeneración Retiniana/terapia , Rodopsina/genética , Alelos , Animales , Sistemas CRISPR-Cas , Electroporación/métodos , Fibroblastos , Terapia Genética/métodos , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Mutación , ARN Guía de Kinetoplastida , Retina/patología , Degeneración Retiniana/genéticaRESUMEN
Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endothelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system. Little is known about its expression during kidney development, although genetic polymorphisms and alterations around the human pdzrn3 chromosomal region have been found to be associated with renal cell carcinomas and other kidney diseases. We investigated the pdzrn3 spatio-temporal expression pattern in Xenopus laevis embryos by in situ hybridization. We focused our study on the development of the pronephros, which is the embryonic amphibian kidney, functionally similar to the most primitive nephric structures of human kidney. To explore the role of pdzrn3 during renal morphogenesis, we performed loss-of-function experiments, through antisense morpholino injections and analysed the morphants using specific pronephric markers. Dynamic pdzrn3 expression was observed in embryonic tissues, such as somites, brain, eye, blood islands, heart, liver and pronephros. Loss of function experiments resulted in specific alterations of pronephros development. In particular, at early stages, pdzrn3 depletion was associated with a reduction of the pronephros anlagen and later, with perturbations of the tubulogenesis, including deformation of the proximal tubules. Rescue experiments, in which mRNA of the zebrafish pdzrn3 orthologue was injected together with the morpholino, allowed recovery of the kidney phenotypes. These results underline the importance of pdzrn3 expression for correct nephrogenesis.
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Proteínas Portadoras/genética , Regulación del Desarrollo de la Expresión Génica , Morfogénesis/genética , Pronefro/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animales , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Prueba de Complementación Genética , Humanos , Hibridación in Situ , Mutación , Pronefro/embriología , Dominios RING Finger/genética , ARN Mensajero/genética , Xenopus laevis/embriología , Proteínas de Pez Cebra/genéticaRESUMEN
The MICAL (Molecules Interacting with CasL) proteins catalyze actin oxidation-reduction reactions destabilizing F-actin in cytoskeletal dynamics. Here we show for the first time that MICAL2 mRNA is significantly over-expressed in aggressive, poorly differentiated/undifferentiated, primary human epithelial cancers (gastric and renal). Immunohistochemistry showed MICAL2-positive cells on the cancer invasive front and in metastasizing cancer cells inside emboli, but not at sites of metastasis, suggesting MICAL2 expression was 'on' in a subpopulation of primary cancer cells seemingly detaching from the tissue of origin, enter emboli and travel to distant sites, and was turned 'off' upon homing at metastatic sites. In vitro, MICAL2 knock-down resulted in mesenchymal to epithelial transition, reduction of viability, and loss of motility and invasion properties of human cancer cells. Moreover, expression of MICAL2 cDNA in MICAL2-depleted cells induced epithelial to mesenchymal transition. Altogether our data indicate that MICAL2 over-expression is associated with cancer progression and metastatic disease. MICAL2 might be an important regulator of epithelial to mesenchymal transition and therefore a promising target for anti-metastatic therapy.
Asunto(s)
Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias Renales/genética , Proteínas de Microfilamentos/genética , Oxidorreductasas/genética , Neoplasias Gástricas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Proteínas de Microfilamentos/metabolismo , Microscopía Fluorescente , Invasividad Neoplásica , Oncogenes/genética , Oxidorreductasas/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
We recently identified pfdn6a and tcp1α (also known as cct-α) as genes coregulated by the transcription factor Rx1. The proteins encoded by these genes belong to two interacting complexes (Prefoldin and "chaperonin containing t-complex polypeptide 1"), which promote the folding of actin and tubulin and have more recently been reported to be involved in a variety of additional functions including cell cycle control and transcription regulation. However, little is known about the expression and function of these two genes during vertebrate development. To assess whether pfdn6a and tcp1α display a general coordinated expression during Xenopus development, we determined, by RT-PCR and in situ hybridization, the spatio-temporal expression pattern of pfnd6a, which was not previously described, and compared it to that of tcp1α, extending the analysis to stages not previously investigated for this gene. We detected maternal transcripts of pfnd6a in the animal hemisphere at early blastula stage. During gastrulation, pfdn6a was expressed in the involuting mesoderm and subsequently in the anterior and dorsal neural plate. At tailbud and tadpole stages, pfdn6a RNA was mainly detected in the forebrain, midbrain, eye vesicle, otic vesicle, branchial arches, and developing pronephros. The pfnd6a expression pattern largely overlaps with that of tcp1α indicating a spatio-temporal transcriptional coregulation of these genes in the majority of their expression sites, which is suggestive of a possible involvement in the same developmental events.
Asunto(s)
Chaperoninas/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animales , Tipificación del Cuerpo/genética , Embrión no Mamífero/embriología , Hibridación in Situ , Larva/genética , Larva/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Xenopus laevis/embriología , Xenopus laevis/crecimiento & desarrolloRESUMEN
It has long been known that the depletion of bone morphogenetic protein (BMP) is one of the key factors necessary for the development of anterior neuroectodermal structures. However, the precise molecular mechanisms that underlie forebrain regionalization are still not completely understood. Here, we show that Noggin1 is involved in the regionalization of anterior neural structures in a dose-dependent manner. Low doses of Noggin1 expand prosencephalic territories, while higher doses specify diencephalic and retinal regions at the expense of telencephalic areas. A similar dose-dependent mechanism determines the ability of Noggin1 to convert pluripotent cells in prosencephalic or diencephalic/retinal precursors, as shown by transplant experiments and molecular analyses. At a molecular level, the strong inhibition of BMP signaling exerted by high doses of Noggin1 reinforces the Nodal/transforming growth factor (TGF)ß signaling pathway, leading to activation of Gli1 and Gli2 and subsequent activation of Sonic Hedgehog (SHH) signaling. We propose a new role for Noggin1 in determining specific anterior neural structures by the modulation of TGFß and SHH signaling.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras/metabolismo , Células Madre Pluripotentes/metabolismo , Retina/embriología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Portadoras/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Retina/citología , Telencéfalo/citología , Telencéfalo/embriología , Factor de Crecimiento Transformador beta/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
BACKGROUND: The transcription factor Rx1, also known as Rax, controls key properties of retinal precursors including migration behavior, proliferation, and maintenance of multipotency. However, Rx1 effector genes are largely unknown. RESULTS: To identify genes controlled by Rx1 in early retinal precursors, we compared the transcriptome of Xenopus embryos overexpressing Rx1 to that of embryos in which Rx1 was knocked-down. In particular, we selected 52 genes coherently regulated, i.e., actived in Rx1 gain of function and repressed in Rx1 loss of function experiments, or vice versa. RT-qPCR and in situ hybridization confirmed the trend of regulation predicted by microarray data for the selected genes. Most of the genes upregulated by Rx1 are coexpressed with this transcription factor, while downregulated genes are either not expressed or expressed at very low levels in the early developing retina. Putative direct Rx1 target genes, activated by GR-Rx1 in the absence of protein synthesis, include Ephrin B1 and Sh2d3c, an interactor of ephrinB1 receptor, which represent candidate novel effectors for the migration promoting activity of Rx1. CONCLUSIONS: This study identifies previously undescribed Rx1 regulated genes mainly involved in transcription regulation, cell migration/adhesion, and cell proliferation that contribute to delineate the molecular mechanisms underlying Rx1 activities.