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Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.
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Regulación de la Expresión Génica , Genómica , Anfioxos/genética , Vertebrados/genética , Animales , Tipificación del Cuerpo/genética , Metilación de ADN , Humanos , Anfioxos/embriología , Anotación de Secuencia Molecular , Regiones Promotoras Genéticas , Transcriptoma/genéticaRESUMEN
The path from a fertilised egg to an embryo involves the coordinated formation of cell types, tissues and organs. Developmental modules comprise discrete units specified by self-sufficient genetic programs that can interact with each other during embryogenesis. Here, we have taken advantage of the different span of embryonic development between two distantly related teleosts, zebrafish (Danio rerio) and medaka (Oryzias latipes) (3 and 9â days, respectively), to explore modularity principles. We report that inter-species blastula transplantations result in the ectopic formation of a retina formed by donor cells - a module. We show that the time taken for the retina to develop follows a genetic program: an ectopic zebrafish retina in medaka develops with zebrafish dynamics. Heterologous transplantation results in a temporal decoupling between the donor retina and host organism, illustrated by two paradigms that require retina-host interactions: lens recruitment and retino-tectal projections. Our results uncover a new experimental system for addressing temporal decoupling along embryonic development, and highlight the presence of largely autonomous but interconnected developmental modules that orchestrate organogenesis.
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Blástula , Oryzias/embriología , Retina/embriología , Quimera por Trasplante/embriología , Pez Cebra/embriología , Animales , Blástula/embriología , Blástula/trasplante , Xenoinjertos , Retina/citologíaRESUMEN
The pathogenesis of complex diseases such as pulmonary arterial hypertension (PAH) is entirely rooted in changes in the expression of some vasoactive factors. These play a significant role in the onset and progression of the disease. Indeed, PAH has been associated with pathophysiologic alterations in vascular function. These are often dictated by increased oxidative stress and impaired modulation of the nitric oxide (NO) pathway. NO reduces the uncontrolled proliferation of vascular smooth muscle cells that leads to occlusion of vessels and an increase in pulmonary vascular resistances, which is the mainstay of PAH development. To date, two classes of NO-pathway modulating drugs are approved for the treatment of PAH: the phosphodiesterase-5 inhibitors (PD5i), sildenafil and tadalafil, and the soluble guanylate cyclase activator (sGC), riociguat. Both drugs provide considerable improvement in exercise capacity and pulmonary hemodynamics. PD5i are the recommended drugs for first-line PAH treatment, whereas sGCs are also the only drug approved for the treatment of resistant or inoperable chronic thromboembolic pulmonary hypertension. In this review, we will focus on the current information regarding the nitric oxide pathway and its modulation in PAH.
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Hipertensión Arterial Pulmonar , Humanos , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Óxido Nítrico , Hipertensión Pulmonar Primaria Familiar , Inhibidores de Fosfodiesterasa 5/farmacología , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Citrato de Sildenafil/farmacología , Citrato de Sildenafil/uso terapéutico , Guanilil Ciclasa SolubleRESUMEN
Yap1/Taz are well-known Hippo effectors triggering complex transcriptional programs controlling growth, survival and cancer progression. Here, we describe yap1b, a new Yap1/Taz family member with a unique transcriptional activation domain that cannot be phosphorylated by Src/Yes kinases. We show that yap1b evolved specifically in euteleosts (i.e. including medaka but not zebrafish) by duplication and adaptation of yap1. Using DamID-seq, we generated maps of chromatin occupancy for Yap1, Taz (Wwtr1) and Yap1b in gastrulating zebrafish and medaka embryos. Our comparative analyses uncover the genetic programs controlled by Yap family proteins during early embryogenesis, and show largely overlapping targets for Yap1 and Yap1b. CRISPR/Cas9-induced mutation of yap1b in medaka does not result in an overt phenotype during embryogenesis or adulthood. However, yap1b mutation strongly enhances the embryonic malformations observed in yap1 mutants. Thus yap1-/-; yap1b-/- double mutants display more severe body flattening, eye misshaping and increased apoptosis than yap1-/- single mutants, thus revealing overlapping gene functions. Our results indicate that, despite its divergent transactivation domain, Yap1b cooperates with Yap1 to regulate cell survival and tissue morphogenesis during early development.
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Pérdida del Embrión/genética , Regulación del Desarrollo de la Expresión Génica , Morfogénesis/genética , Transactivadores/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Pérdida del Embrión/veterinaria , Embrión no Mamífero , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Mutación , Oryzias/embriología , Oryzias/genética , Dominios Proteicos/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAP , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND: Long non-coding RNAs are RNAs longer than 200 bps that do not encode any proteins and are able to alter gene expression by acting on different steps of regulation, including DNA methylation and chromatin structure. They represent a class of biomarkers of crescent interest in the hematologic and oncologic fields. Recent studies showed that the expression levels of specific lncRNAs correlate with the prognosis of paediatric patients with Acute Lymphoblastic Leukaemia. METHODS: We used NGS approaches to analyse the transcriptome of 9 childhood B-ALL patients and 6 childhood T-ALL patients, in comparison with B and T healthy lymphocytes from cord blood. We validate our findings both ex vivo, in a different cohort of 10 B-ALL and 10 T-ALL patients, and in silico using public datasets. RESULTS: We characterised the lncRNA landscape for B-ALL, T-ALL, healthy B, and T cell progenitors. From the characterised signature, we selected candidate lncRNAs able to discriminate not only B-ALL and T-ALL from healthy subjects but also between the two types of leukaemia, and subsequently validated their potential as a diagnostic tool in an additional cohort of paediatric patients. We confirmed our finding with open access transcriptomic data, comparing ALL lncRNAs with AML lncRNA landscape as well. Finally, expression correlation analyses of T-ALL selected lncRNA biomarkers suggested a possible role in lymphocyte activation and the ß-catenin signalling pathway for AC247036.1 and involvement in hedgehog signalling for HHIP-AS1. CONCLUSIONS: Our work identified a lncRNA signature discriminating paediatric B-ALL and T-ALL from healthy subjects, between them and from AML. This study provides the keystone to future clinical studies determining the theragnostic value of the characterised long non coding transcriptome panorama in a clinical setting for childhood patient management.
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The ontogeny of the vertebrate retina has been a topic of interest to developmental biologists and human geneticists for many decades. Understanding the unfolding of the genetic program that transforms a field of progenitors cells into a functionally complex and multi-layered sensory organ is a formidable challenge. Although classical genetic studies succeeded in identifying the key regulators of retina specification, understanding the architecture of their gene network and predicting their behavior are still a distant hope. The emergence of next-generation sequencing platforms revolutionized the field unlocking the access to genome-wide datasets. Emerging techniques such as RNA-seq, ChIP-seq, ATAC-seq, or single cell RNA-seq are used to characterize eye developmental programs. These studies provide valuable information on the transcriptional and cis-regulatory profiles of precursors and differentiated cells, outlining the trajectories that connect each intermediate state. Here, recent systems biology efforts are reviewed to understand the genetic programs shaping the vertebrate retina.
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Secuenciación de Inmunoprecipitación de Cromatina/métodos , Redes Reguladoras de Genes , Organogénesis/genética , RNA-Seq/métodos , Retina/crecimiento & desarrollo , Biología de Sistemas/métodos , Vertebrados/crecimiento & desarrollo , Vertebrados/genética , Animales , Genoma , Código de Histonas/genética , Histonas/genética , Humanos , Elementos Reguladores de la Transcripción , Análisis de la Célula Individual/métodos , TranscriptomaRESUMEN
A new petroleum-degrading bacterium, designated strain GC2T, was isolated from Bozkus 1 petroleum station in Diyarbakir, located in the southeast of Turkey. Cells were Gram-negative staining, aerobic, coccoid-rods, non-motile, non-spore-forming. The bacterium was found to degrade 100% of n-alkanes ranging from C11 to C34 presented in the 1% crude oil after incubation of 7 days. The membrane phospholipids were 1,2 diacylglycero-3-phosphorylethanolamine (PEA), phosphatidylglycerol (PG), dipalmitoyl-sn-glycerol 1- phosphocholine (PC1), 1,2 dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (PC3), cardiolipin also called diphosphatidylglycerol (CL) and l-α- phosphatidic acid, dipalmitoyl (AP); predominant respiratory ubiquinone was Q-8 and C16:0, C18:1ω9c and C16:1 were the major cellular fatty acids. The 16S rRNA sequence analysis revealed that the strain GC2T was a member of genus Acinetobacter and was most closely related to Acinetobacter lwoffii DSM 2403 T (99.79%), Acinetobacter pseudolwoffii ANC 5318 T (98.83%) and Acinetobacter harbinensis HITLi 7 T (98.14%). The rpoB and gyrB gene sequence analysis confirmed that the strain GC2T was a member of genus Acinetobacter and that the closest relative was Acinetobacter lwoffii DSM 2403 T (99.08% and 100% similarity, respectively). DNA-DNA hybridization values between GC2T and its closest relatives ranged from 65.6% (with A. lwoffii) to 5.1% (with A. venetianus). The whole genome sequence of strain GC2T was obtained. The DNA G + C content of this strain was determined to be 42.9 mol %. ANI indexes, in silico estimations of DDH values and wet lab DDH values demonstrated that strain GC2T represents an independent genomospecies. On the basis of phenotypic characteristics, chemotaxonomic, phylogenetic data and DNA-DNA hybridization and whole genome analysis, we propose to assign strain GC2T as a new species of the genus Acinetobacter, for which the name Acinetobacter mesopotamicus sp. nov. is proposed. The type strain of this species is GC2T (DSM 26953 T = JCM 31073 T). The whole genome of strain GC2T has been deposited at DDBJ/ENA/GenBank under the accession JAALFF010000000.
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Acinetobacter , Petróleo , Acinetobacter/genética , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Ácidos Grasos/análisis , Hibridación de Ácido Nucleico , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Suelo , Microbiología del Suelo , TurquíaRESUMEN
In the hypothalamic arcuate nucleus (ARC), proopiomelanocortin (POMC) neurons and the POMC-derived peptide α-melanocyte-stimulating hormone (α-MSH) promote satiety. POMC neurons receive orexin-A (OX-A)-expressing inputs and express both OX-A receptor type 1 (OX-1R) and cannabinoid receptor type 1 (CB1R) on the plasma membrane. OX-A is crucial for the control of wakefulness and energy homeostasis and promotes, in OX-1R-expressing cells, the biosynthesis of the endogenous counterpart of marijuana's psychotropic and appetite-inducing component Δ(9)-tetrahydrocannabinol, i.e., the endocannabinoid 2-arachidonoylglycerol (2-AG), which acts at CB1R. We report that OX-A/OX-1R signaling at POMC neurons promotes 2-AG biosynthesis, hyperphagia, and weight gain by blunting α-MSH production via CB1R-induced and extracellular-signal-regulated kinase 1/2 activation- and STAT3 inhibition-mediated suppression of Pomc gene transcription. Because the systemic pharmacological blockade of OX-1R by SB334867 caused anorectic effects by reducing food intake and body weight, our results unravel a previously unsuspected role for OX-A in endocannabinoid-mediated promotion of appetite by combining OX-induced alertness with food seeking. Notably, increased OX-A trafficking was found in the fibers projecting to the ARC of obese mice (ob/ob and high-fat diet fed) concurrently with elevation of OX-A release in the cerebrospinal fluid and blood of mice. Furthermore, a negative correlation between OX-A and α-MSH serum levels was found in obese mice as well as in human obese subjects (body mass index > 40), in combination with elevation of alanine aminotransferase and γ-glutamyl transferase, two markers of fatty liver disease. These alterations were counteracted by antagonism of OX-1R, thus providing the basis for a therapeutic treatment of these diseases.
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Endocannabinoides/metabolismo , Neuronas/metabolismo , Obesidad/metabolismo , Orexinas/metabolismo , Proopiomelanocortina/metabolismo , Respuesta de Saciedad , alfa-MSH/metabolismo , Adulto , Animales , Núcleo Hipotalámico Anterior/metabolismo , Núcleo Hipotalámico Anterior/patología , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Inhibición Neural , Transducción de Señal , Regulación hacia ArribaRESUMEN
Strain Corallo1T was isolated from mucus of red coral (Corallium rubrum) at Punta Pizzaco (Procida island, Naples, Italy). It was characterised as a Gram-stain negative, motile, rod-shaped bacterium. Strain Corallo1T was found to show positive responses for cytochrome-c oxidase, catalase, reduction of nitrate and nitrite, ß-galactosidase activity and hydrolysis of starch, xylan, peptone, Tween 40, Tween 80 and casein. Strain Corallo1T was found to be mesophilic, neutrophilic to alkalophilic and slightly halophilic. According to analysis of the almost-complete 16S rRNA gene, strain Corallo1T is closely related to Vibrio celticus (100% sequence similarity), Vibrio gigantis (100%), Vibrio crassostreae (99.7%), Vibrio artabrorum (99.7%) and Vibrio pomeroyi (99.6%). MLSA of five housekeeping genes (atpA, pyrH, recA, rpoA and rpoD) was performed to refine the phylogenetic relationships of strain Corallo1T. A draft genome sequence of strain Corallo1T was obtained. The DNA G+C content of this strain was determined to be 44.5 mol %. The major cellular fatty acids of strain Corallo1T are C16:1, n-C16:0 and C18:1, and the major isoprenoid ubiquinone is Q8. ANI indexes, in silico estimations of DDH values and wet lab DDH values demonstrated that strain Corallo1T represents an independent genomospecies. Based on a polyphasic taxonomic characterisation, strain Corallo1T is concluded to represent a novel species of the genus Vibrio, for which the name Vibrio coralliirubri sp. nov. is proposed. The type strain is Corallo1T (= DSM 27495T = CIP 110630T).
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Antozoos/microbiología , Vibrio/aislamiento & purificación , Animales , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , ADN Ribosómico/genética , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Italia , Moco/microbiología , Filogenia , ARN Ribosómico 16S/genética , Vibrio/clasificación , Vibrio/genética , Vibrio/metabolismoRESUMEN
Colorectal cancer (CRC) is a major health problem in Western countries. The endocannabinoid 2-arachidonoyl-glycerol (2-AG) exerts antiproliferative actions in a number of tumoral cell lines, including CRC cells. Monoacylglycerol lipase (MAGL), a serine hydrolase that inactivates 2-AG, is highly expressed in aggressive human cancer cells. Here, we investigated the role of MAGL in experimental colon carcinogenesis. The role of MAGL was assessed in vivo by using the xenograft and the azoxymethane models of colon carcinogenesis; MAGL expression was evaluated by RT-PCR and immunohistochemistry; 2-AG levels were measured by liquid chromatography mass spectrometry; angiogenesis was evaluated in tumor tissues [by microvessel counting and by investigating the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) proteins] as well as in human umbilical vein endothelial cells (HUVEC); cyclin D1 was evaluated by RT-PCR. MAGL and 2-AG were strongly expressed in tumor tissues. The MAGL inhibitor URB602 reduced xenograft tumor volume, this effect being associated to down-regulation of VEGF and FGF-2, reduction in the number of vessels and down-regulation of cyclin D1. In HUVEC, URB602 exerted a direct antiangiogenic effect by inhibiting FGF-2 induced proliferation and migration, and by modulating pro/anti-angiogenic agents. In experiments aiming at investigating the role of MAGL in chemoprevention, URB602 attenuated azoxymethane-induced preneoplastic lesions, polyps and tumors. MAGL, possibly through modulation of angiogenesis, plays a pivotal role in experimental colon carcinogenesis. Pharmacological inhibition of MAGL could represent an innovative therapeutic approach to reduce colorectal tumor progression.
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Antineoplásicos/uso terapéutico , Compuestos de Bifenilo/uso terapéutico , Colon/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Monoacilglicerol Lipasas/antagonistas & inhibidores , Recto/efectos de los fármacos , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Ácidos Araquidónicos/metabolismo , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Colon/irrigación sanguínea , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación hacia Abajo/efectos de los fármacos , Endocannabinoides/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glicéridos/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones Endogámicos ICR , Ratones Desnudos , Monoacilglicerol Lipasas/genética , Monoacilglicerol Lipasas/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Recto/irrigación sanguínea , Recto/metabolismo , Recto/patologíaRESUMEN
PURPOSE: PEA is an endogenous mediator released together with the endocannabinoid anandamide from membrane phospholipids. It is a plant derived compound with analgesic and anti-inflammatory properties. We verified whether the pathophysiology of experimental cystitis involves changes in the levels of PEA and of some of its targets, ie CB1 and CB2 receptors, and PPARα. We also determined whether exogenously administered PEA could be proposed as a preventive measure for cystitis. MATERIALS AND METHODS: Cystitis was induced by cyclophosphamide in female rats. Nociceptive responses, voiding episodes, gross damage, myeloperoxidase activity, bladder weight, bladder PEA and endocannabinoid levels (measured by liquid chromatography-mass spectrometry) and the expression of PEA targets (measured by quantitative reverse transcriptase-polymerase chain reaction) were recorded. RESULTS: Cyclophosphamide induced pain behavior, bladder inflammation and voiding dysfunction associated with increased bladder levels of PEA, up-regulation of CB1 receptor mRNA expression, down-regulation of PPARα mRNA and no change in CB2 receptor mRNA expression. Exogenously administered, ultramicronized PEA attenuated pain behavior, voids and bladder gross damage. The CB1 antagonist rimonabant and the PPARα antagonist GW6471 counteracted the beneficial effect of PEA on gross damage. Also, GW6471 further decreased voiding episodes in rats treated with PEA. CONCLUSIONS: The current study provides strong evidence for a protective role of PEA as well as an alteration in bladder levels of PEA and of some of its targets in cyclophosphamide induced cystitis.
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Antiinflamatorios no Esteroideos/uso terapéutico , Cistitis/prevención & control , Etanolaminas/uso terapéutico , Ácidos Palmíticos/uso terapéutico , Amidas , Animales , Ciclofosfamida , Cistitis/inducido químicamente , Modelos Animales de Enfermedad , Femenino , Ratas , Ratas WistarRESUMEN
Cannabigerol (CBG) is a safe non-psychotropic Cannabis-derived cannabinoid (CB), which interacts with specific targets involved in carcinogenesis. Specifically, CBG potently blocks transient receptor potential (TRP) M8 (TRPM8), activates TRPA1, TRPV1 and TRPV2 channels, blocks 5-hydroxytryptamine receptor 1A (5-HT1A) receptors and inhibits the reuptake of endocannabinoids. Here, we investigated whether CBG protects against colon tumourigenesis. Cell growth was evaluated in colorectal cancer (CRC) cells using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide and 3-amino-7-dimethylamino-2-methylphenazine hydrochloride assays; apoptosis was examined by histology and by assessing caspase 3/7 activity; reactive oxygen species (ROS) production by a fluorescent probe; CB receptors, TRP and CCAAT/enhancer-binding protein homologous protein (CHOP) messenger RNA (mRNA) expression were quantified by reverse transcription-polymerase chain reaction; small hairpin RNA-vector silencing of TRPM8 was performed by electroporation. The in vivo antineoplastic effect of CBG was assessed using mouse models of colon cancer. CRC cells expressed TRPM8, CB1, CB2, 5-HT1A receptors, TRPA1, TRPV1 and TRPV2 mRNA. CBG promoted apoptosis, stimulated ROS production, upregulated CHOP mRNA and reduced cell growth in CRC cells. CBG effect on cell growth was independent from TRPA1, TRPV1 and TRPV2 channels activation, was further increased by a CB2 receptor antagonist, and mimicked by other TRPM8 channel blockers but not by a 5-HT1A antagonist. Furthermore, the effect of CBG on cell growth and on CHOP mRNA expression was reduced in TRPM8 silenced cells. In vivo, CBG inhibited the growth of xenograft tumours as well as chemically induced colon carcinogenesis. CBG hampers colon cancer progression in vivo and selectively inhibits the growth of CRC cells, an effect shared by other TRPM8 antagonists. CBG should be considered translationally in CRC prevention and cure.
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Apoptosis/efectos de los fármacos , Cannabinoides/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Canales Catiónicos TRPM/antagonistas & inhibidores , Animales , Azoximetano/toxicidad , Western Blotting , Cannabis/química , Carcinógenos/toxicidad , Células Cultivadas , Colon/citología , Colon/efectos de los fármacos , Colon/metabolismo , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/patología , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Desnudos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Throughout embryonic development, the shaping of the functional and morphological characteristics of embryos is orchestrated by an intricate interaction between transcription factors and cis-regulatory elements. In this study, we conducted a comprehensive analysis of deuterostome cis-regulatory landscapes during gastrulation, focusing on four paradigmatic species: the echinoderm Strongylocentrotus purpuratus, the cephalochordate Branchiostoma lanceolatum, the urochordate Ciona intestinalis, and the vertebrate Danio rerio. Our approach involved comparative computational analysis of ATAC-seq datasets to explore the genome-wide blueprint of conserved transcription factor binding motifs underlying gastrulation. We identified a core set of conserved DNA binding motifs associated with 62 known transcription factors, indicating the remarkable conservation of the gastrulation regulatory landscape across deuterostomes. Our findings offer valuable insights into the evolutionary molecular dynamics of embryonic development, shedding light on conserved regulatory subprograms and providing a comprehensive perspective on the conservation and divergence of gene regulation underlying the gastrulation process.
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Ciona intestinalis , Gastrulación , Regulación del Desarrollo de la Expresión Génica , Animales , Gastrulación/genética , Ciona intestinalis/genética , Ciona intestinalis/embriología , Pez Cebra/genética , Pez Cebra/embriología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/embriología , Secuencia Conservada/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Anfioxos/genética , Anfioxos/embriología , Evolución MolecularRESUMEN
Background: Paediatric acute B-cell lymphoblastic leukaemia is the most common cancer of the paediatric age. Although the advancement of scientific and technological knowledge has ensured a huge step forward in the management of this disease, there are 15%-20% cases of recurrence leading to serious complications for the patient and sometimes even death. It is therefore necessary to identify new and increasingly personalised biomarkers capable of predicting the degree of risk of B-ALL in order to allow the correct management of paediatric leukaemia patients. Methods: Starting from our previously published results, we validate the expression level of LINC00958 in a cohort of 33 B-ALL and 9 T-ALL childhood patients, using in-silico public datasets as support. Expression levels of LINC00958 in B-ALL patients stratified by risk (high risk vs. standard/medium risk) and who relapsed 3 years after the first leukaemia diagnosis were also evaluated. Results: We identified the lncRNA LINC00958 as a biomarker of B-ALL, capable of discriminating B-ALL from T-ALL and healthy subjects. Furthermore, we associated LINC00958 expression levels with the disease risk classification (high risk and standard risk). Finally, we show that LINC00958 can be used as a predictor of relapses in patients who are usually stratified as standard risk and thus not always targeted for marrow transplantation. Conclusions: Our results open the way to new diagnostic perspectives that can be directly used in clinical practice for a better management of B-ALL paediatric patients.
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Genetic studies in human and mice have established a dual role for Vsx genes in retina development: an early function in progenitors' specification, and a later requirement for bipolar-cells fate determination. Despite their conserved expression patterns, it is currently unclear to which extent Vsx functions are also conserved across vertebrates, as mutant models are available only in mammals. To gain insight into vsx function in teleosts, we have generated vsx1 and vsx2 CRISPR/Cas9 double knockouts (vsxKO) in zebrafish. Our electrophysiological and histological analyses indicate severe visual impairment and bipolar cells depletion in vsxKO larvae, with retinal precursors being rerouted toward photoreceptor or Müller glia fates. Surprisingly, neural retina is properly specified and maintained in mutant embryos, which do not display microphthalmia. We show that although important cis-regulatory remodelling occurs in vsxKO retinas during early specification, this has little impact at a transcriptomic level. Our observations point to genetic redundancy as an important mechanism sustaining the integrity of the retinal specification network, and to Vsx genes regulatory weight varying substantially among vertebrate species.
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Proteínas de Homeodominio , Pez Cebra , Animales , Humanos , Ratones , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Homeodominio/metabolismo , Retina/metabolismo , Genes Homeobox , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Mutación , Mamíferos/genética , Factores de Transcripción/metabolismo , Proteínas del Ojo/metabolismoRESUMEN
Acute leukemia is the most common pediatric cancer. In most cases, this disease results from the malignant transformation of either the B-cell (B-ALL) or, less frequently, T-cell progenitors (T-ALL). Recently, a marked overexpression of KCTD15, a member of the emerging class of the potassium (K) channel tetramerization domain-containing proteins (KCTDs) has been detected in both patients and continuous cell lines as in vitro model systems. Because there is growing evidence of the key, yet diversified, roles played by KCTDs in cancers, we here report an exhaustive analysis of their expression profiles in both B-ALL and T-ALL patients. Although for most KCTDs, no significant alterations were found in these pathological states, for some members of the family, significant up- and down-regulations were detected in comparison with the values found in healthy subjects in the transcriptome analysis. Among these, particularly relevant is the upregulation of the closely related KCTD1 and KCTD15 in T-ALL patients. Interestingly, KCTD1 is barely expressed in both unaffected controls and B-ALL patients. Therefore, not only does this analysis represent the first study in which the dysregulation of all KCTDs is simultaneously evaluated in specific pathological contexts, but it also provides a promising T-ALL biomarker that could be suitable for clinical applications.
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The assembly of the embryo's primary axis is a fundamental landmark for the establishment of the vertebrate body plan. Although the morphogenetic movements directing cell convergence towards the midline have been described extensively, little is known on how gastrulating cells interpret mechanical cues. Yap proteins are well-known transcriptional mechanotransducers, yet their role in gastrulation remains elusive. Here we show that the double knockout of yap and its paralog yap1b in medaka results in an axis assembly failure, due to reduced displacement and migratory persistence in mutant cells. Accordingly, we identified genes involved in cytoskeletal organization and cell-ECM adhesion as potentially direct Yap targets. Dynamic analysis of live sensors and downstream targets reveal that Yap is acting in migratory cells, promoting cortical actin and focal adhesions recruitment. Our results indicate that Yap coordinates a mechanoregulatory program to sustain intracellular tension and maintain the directed cell migration for embryo axis development.
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Proteínas Adaptadoras Transductoras de Señales , Factores de Transcripción , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP , Adhesiones Focales/genética , Adhesiones Focales/metabolismo , Movimiento Celular/genéticaRESUMEN
Developmental and physiological processes depend on the transcriptional and translational activity of heterogeneous cell populations. A main challenge in gene expression studies is dealing with this intrinsic complexity while keeping sequencing efficiency. Translating ribosome affinity purification (TRAP) methods have allowed cell-specific recovery of polyribosome-associated RNAs by genetic tagging of ribosomes in selected cell populations. Here we combined the TRAP approach with adapted enhancer trap methods (trap-TRAP) to systematically generate zebrafish transgenic lines suitable for tissue-specific translatome interrogation. Through the random integration of a GFP-tagged version of the large subunit ribosomal protein L10a (EGFP-Rpl10a), we have generated stable lines driving expression in a variety of tissues, including the retina, skeletal muscle, lateral line primordia, rhombomeres, or jaws. To increase the range of applications, a UAS:TRAP transgenic line compatible with available Gal4 lines was also generated and tested. The resulting collection of lines and applications constitutes a resource for the zebrafish community in developmental genetics, organ physiology and disease modelling.
RESUMEN
Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the flattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE specification network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Morfogénesis/genética , Epitelio Pigmentado de la Retina/metabolismo , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Análisis por Conglomerados , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , RNA-Seq/métodos , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/embriología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/clasificación , Factores de Transcripción/genética , Pez Cebra/embriologíaRESUMEN
While lower vertebrates contain adult stem cells (aSCs) that maintain homeostasis and drive un-exhaustive organismal growth, mammalian aSCs display mainly the homeostatic function. Here, we use lineage analysis in the medaka fish gill to address aSCs and report separate stem cell populations for homeostasis and growth. These aSCs are fate-restricted during the entire post-embryonic life and even during re-generation paradigms. We use chimeric animals to demonstrate that p53 mediates growth coordination among fate-restricted aSCs, suggesting a hierarchical organisation among lineages in composite organs like the fish gill. Homeostatic and growth aSCs are clonal but differ in their topology; modifications in tissue architecture can convert the homeostatic zone into a growth zone, indicating a leading role for the physical niche defining stem cell output. We hypothesise that physical niches are main players to restrict aSCs to a homeostatic function in animals with fixed adult size.