Mecanismos regularodes asociados a la generación de diversidad muscular en Drosophila melanogaster: regulación del gen para paramiosina/miniparamiosina y efectos de la sobreexpresión de latroponina T

Tesis doctoral inédita leÍda en la Universidad Autónoma de Madrid. Facultad de Medicina. Departamento de Bioquímica. Fecha de lectura: 26 de Marzo 200

A trans-Regulatory code for the forebrain expression of Six3.2 in the Medaka fish

A well integrated and hierarchically organized gene regulatory network is responsible for the progressive specification of the forebrain. The transcription factor Six3 is one of the central components of this network. As such, Six3 regulates several components of the network, but its upstream regulators are still poorly characterized. Here we have systematically identified such regulators, taking advantage of the detailed functional characterization of the regulatory region of the medaka fish Six3.2 ortholog and of a time/cost-effective trans-regulatory screening, which complemented and overcame the limitations of in silico prediction approaches. The candidates resulting from this search were validated with dose-response luciferase assays and expression pattern criteria. Reconfirmed candidates with a matching expression pattern were also tested with chromatin immunoprecipitation and functional studies. Our results confirm the previously proposed direct regulation of Pax6 and further demonstrate that Msx2 and Pbx1 are bona fide direct regulators of early Six3.2 distribution in distinct domains of the medaka fish forebrain. They also point to other transcription factors, including Tcf3, as additional regulators of different spatial-temporal domains of Six3.2 expression. The activity of these regulators is discussed in the context of the gene regulatory network proposed for the specification of the forebrain.Spanish Ministerio de Economía y Competitividad (MINECO) Grants BFU2010-16031 and BFU2013-43213-P, cofounded by FEDER Funds; Comunidad Autónoma de Madrid (CAM) Grant CELL-DD S2010/BMD-2315; Fundaluce; Fundación ONCE; the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) del Instituto Carlos III (ISCIII); and an Institutional Grant from the Fundación Ramón Areces.Peer Reviewe

Accurate genome-wide predictions of spatio-temporal gene expression during embryonic development

Comprehensive information on the timing and location of gene expression is fundamental to our understanding of embryonic development and tissue formation. While high-throughput in situ hybridization projects provide invaluable information about developmental gene expression patterns for model organisms like Drosophila, the output of these experiments is primarily qualitative, and a high proportion of protein coding genes and most non-coding genes lack any annotation. Accurate data-centric predictions of spatio-temporal gene expression will therefore complement current in situ hybridization efforts. Here, we applied a machine learning approach by training models on all public gene expression and chromatin data, even from whole-organism experiments, to provide genome-wide, quantitative spatiotemporal predictions for all genes. We developed structured in silico nano-dissection, a computational approach that predicts gene expression in >200 tissue-developmental stages. The algorithm integrates expression signals from a compendium of 6,378 genome-wide expression and chromatin profiling experiments in a cell lineage-aware fashion. We systematically evaluated our performance via cross-validation and experimentally confirmed 22 new predictions for four different embryonic tissues. The model also predicts complex, multi-tissue expression and developmental regulation with high accuracy. We further show the potential of applying these genome-wide predictions to extract tissue specificity signals from non-tissue-dissected experiments, and to prioritize tissues and stages for disease modeling. This resource, together with the exploratory tools are freely available at our webserver http://find.princeton.edu, which provides a valuable tool for a range of applications, from predicting spatio-temporal expression patterns to recognizing tissue signatures from differential gene expression profiles.Fil: Zhou, Jian*. University of Princeton; Estados UnidosFil: Schor, Ignacio Esteban. European Molecular Biology Laboratory; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Yao, Victoria. University of Princeton; Estados UnidosFil: Theesfeld, Chandra L.. University of Princeton; Estados UnidosFil: Marco-Ferreres, Raquel. European Molecular Biology Laboratory; AlemaniaFil: Tadych, Alicja. University of Princeton; Estados UnidosFil: Furlong, Eileen E. M.. European Molecular Biology Laboratory; AlemaniaFil: Troyanskaya, Olga G.. University of Princeton; Estados Unido

The combination of transcriptomics and informatics identifies pathways targeted by miR-204 during neurogenesis and axon guidance

Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development. \uc2\ua9 The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

The logic of gene regulatory networks in early vertebrate forebrain patterning

The vertebrate forebrain or prosencephalon is patterned at the beginning of neurulation into four major domains: the telencephalic, hypothalamic, retinal and diencephalic anlagen. These domains will then give rise to the majority of the brain structures involved in sensory integration and the control of higher intellectual and homeostatic functions. Understanding how forebrain pattering arises has thus attracted the interest of developmental neurobiologists for decades. As a result, most of its regulators have been identified and their hierarchical relationship is now the object of active investigation. Here, we summarize the main morphogenetic pathways and transcription factors involved in forebrain specification and propose the backbone of a possible gene regulatory network (GRN) governing its specification, taking advantage of the GRN principles elaborated by pioneer studies in simpler organisms. We will also discuss this GRN and its operational logic in the context of the remarkable morphological and functional diversification that the forebrain has undergone during evolution. © 2012 Elsevier Ireland Ltd.the Spanish MICINN (BFU2010-16031); Comunidad Autonoma de Madrid (CAM, CELL-DD S2010/BMD-2315); Fundaluce; Fundación ONCE; CIBERER.Peer Reviewe

Retinal development: Embryology and early patterning

Peer Reviewe

Co-operation between enhancers modulates quantitative expression from the Drosophila Paramyosin/miniparamyosin gene in different muscle types

The distinct muscles of an organism accumulate different quantities of structural proteins, but always maintaining their stoichiometry. However, the mechanisms that control the levels of these proteins and that co-ordinate muscle gene expression remain to be defined. The paramyosin/miniparamyosin gene encodes two thick filament proteins transcribed from two different promoters. We have analysed the regulatory regions that control expression of this gene and that are situated in the two promoters, the 5′ and the internal promoters, both in vivo and in silico. A distal muscle enhancer containing three conserved MEF2 motifs is essential to drive high levels of paramyosin expression in all the major embryonic, larval and adult muscles. This enhancer shares sequence motifs, as well as its structure and organisation, with at least four co-regulated muscle enhancers that direct similar patterns of expression. However, other elements located downstream of the enhancer are also required for correct gene expression. Other muscle genes with different patterns of expression, such as miniparamyosin, are regulated by other basic mechanisms. The expression of miniparamyosin is controlled by two enhancers, AB and TX, but a BF modulator is required to ensure the correct levels of expression in each particular muscle. We propose a mechanism of transcriptional regulation in which similar enhancers are responsible for the spatio-temporal expression of co-regulated genes. However, it is the interaction between enhancers which ensures that the correct amounts of protein are expressed at any particular time in a cell, adapting these levels to their specific needs. These mechanisms may not be exclusive to neural or muscle tissue and might represent a general mechanism for genes that are spatially and temporally co-regulated. © 2005 Elsevier Ireland Ltd. All rights reserved.This research was supported by a grant BMC2001-1454 from the DGICYT (Spanish Ministry of Education, Culture and Sports). R. Marco Ferreres was a pre-doctoral fellow of the Spanish Ministry of Education, Culture and Sports and J. Vivar is a pre-doctoral fellow at the Universidad Autónoma de Madrid.Peer Reviewe

Secondary enhancers synergise with primary enhancers to guarantee fine-tuned muscle gene expression

Although tight quantitative control of gene expression is required to ensure that organs and tissues function correctly, the transcriptional mechanisms underlying this process still remain poorly understood. Here, we describe novel and evolutionary conserved secondary enhancers that are needed for the regulation of the expression of Troponin I genes. Secondary enhancers are silent when tested individually in electroporated muscles but interact with the primary enhancers and are required to precisely control the appropriate timing, the tissue and fibre specificity, and the quantitative expression of these genes during muscle differentiation. Synergism is completely dependent of the fully conserved MEF2 site present on the primary enhancers core of skeletal muscle Troponin I genes. Thus, while each of these paired enhancers has a different function, the concerted action of both is crucial to recapitulate endogenous gene expression. Through comparative genomics, we predict that this mechanism has also arisen in other mammalian muscle genes. Our results reveal the existence of a novel mechanism, conserved from flies to mammals, to fine-tune gene expression in each muscle and probably other tissuesThis research was supported by grants BFU2004-05384, BFU2007-61711BMC from the DGICYT (Spanish Ministry of Education, Culture and Sports), FIS-PS09/01267, Marato-TV3 and by Fundación Mutua Madrileñ

Dual functionality of cis -regulatory elements as developmental enhancers and Polycomb response elements

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