El más complejo de los viajes : el embrión en la sociedad contemporánea

La palabra embrión evoca en nosotros, de forma inmediata, extrañas y misteriosas imágenes de probetas y laboratorios. Sin duda no deja de ser una paradoja sorprendente que aquello que todos nosotros hemos sido alguna vez, nos sea tan ajen

Detecting hydroxyl radical with a new two-photon fluorescent probe in living cells

Reactive oxygen species (ROS) are known to be involved in the onset and development of multiple diseases, including cancer, cardiovascular diseases, neurodegenerative diseases and diabetes among others. Generation of ROS is a phenomenon that results from normal cell metabolism as well as from the response to certain pathologic stimuli like certain cytokines, xenobiotics and bacterial infection. These ROS are highly reactive, short-lived molecules that play critical roles in the living cell and it is well accepted that cellular oxidative stress results from the imbalance between generation and elimination of ROS in cells. In this work, we describe the design and synthesis of a two photon (TP) organelle-targeting activatable fluorescent probe. This probe is a naphthalene-indoline compound that targets specifically lysosomes, reaching these organelles in the fluorescence “off” state and minimizing background reactions. Upon arrival at the lysosomes, the probe is triggered and a fluorescence “on” signal is observed that can be combined with TP microscopy to image the lysosomal •OH in living cells.Real Sociedad Española de Química y Grupo Especializado de Química Orgánica de la RSEQ•Universidade de Santiago de Compostela • CIQUS • Lilly • Mestrelab Research •Thieme •Scharlab • Acros • ABCR • AMSLab • GalChimia • Elsevier • The Journal of the American Chemical Society • ACS Omega • Organic Letters • The Journal of Organic Chemistry • Accounts of Chemical Research. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Three embryonic cell lineages related with the epicardium show distinct developmental fates

The embryonic epicardium generates a population of mesenchymal cells that contribute to the coronary vessels and the connective tissue of the adult heart. We have used murine cell-tracing models to compare the developmental fate of three different lineages related with the epicardium. Mice bearing R26R-EYFP reporters were crossed with mice expressing Cre-recombinase under control of the promoters of the cardiac troponin gene (cTnT), the Wilms tumor suppressor gene (Wt1), and the G2 enhancer of the GATA4 gene. Thus, we could trace, using confocal microscopy and flow cytometry, the lineage of the cardiac cells expressing Wt1, cTnT and GATA4 under control of the G2 enhancer, from midgestation to adults. Additionally we have studied a knockin Wt1-GFP reporter model to detect cells with Wt1 expression in the developing and adult heart. During development, Wt1 is expressed in a major part of the proepicardium and epicardium, and also in some epicardial-derived mesenchymal cells, in part of the mesenchymal and coronary endothelial cells and also in a fraction of the cardiomyocytes. GATA4 expression is activated by the enhancer G2 in lateral mesoderm and pro/epicardium, but not in epicardial-derived cells, although most of these cells as well as some cardiomyocytes originate from a G2-GATA4 expressing lineage. cTnT is expressed in all the cardiomyocytes, but a part of the epicardial cells also derives from a cTnTCre-EYFP positive cell lineage. The developmental fate of these linages reveals interesting differences, according to our preliminary results. For example, the G2-GATA4 cell linage contributes more than the Wt1 cell lineage to the coronary endothelium during development. However, both lineages are highly represented in the adult cardiac endothelium, suggesting postnatal expression of Wt1 in the coronary endothelium and incorporation of endothelial progenitor cells from bone marrow (where the G2-GATA4 reporter is active in 20% of hematopoietic stem cells).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A developmental model for the pathogenenesis of cardiac arterio-ventricular fistulae

Coronary Artery Fistulae (CAF) are congenital coronary artery (CA) anomalies consisting of an abnormal communication of a coronary artery with either a cardiac chamber or a large cardiac vessel. Although their incidence in the Western population is low, CAF can lead to complications such as myocardial hypertrophy, endocarditis, heart dilatation and cardiac failure. CAFs can appear as an isolated anomaly or linked to some other forms of congenital heart disease like Left Ventricular Non-Compaction (LVNC) and intrinsic CA anatomy anomalies, but their etiology remains unknown. In this work we have used two different experimental models (transgenic mice and avian embryos) to investigate on the developmental mechanics of CAF formation. In order to tackle this goal, we have manipulated epicardial development and ventricular wall compaction, two inextricably related developmental events during coronary embryogenesis. Conditional integrin α4 gene deletion in the septum transversum/proepicardial (ST/PE) region (G2-Gata4+) disrupts early epicardium development and reduces cardiomyocyte proliferation, leading to the thinning of the ventricular compact myocardial layer. Reduction in compact myocardium thickness associates to the presence of multiple ventricular myocardial discontinuities and focal endocardial extrusion. This same phenotype can be experimentally reproduced in chick embryos using a cryocauterization method (Palmquist-Gomes et al., 2016). Our results suggest that the partial absence of epicardium in α4integrin;G2-Gata4Cre mouse embryos and the cryoinjury in avian embryos generate myocardial discontinuities in the embryonic ventricular wall, which promote endocardial extrusion towards the pericardial cavity and the early contact of the endocardium with coronary progenitors at the epicardial surface of the heart. In the case of avian embryos, this phenomenon leads to precocious smooth muscle differentiation from epicardial mesenchymal cells, and the formation of pouch-like structures that closely resemble CAF. We conclude that anomalous compact myocardial embryonic growth can originate CAF.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A developmental model for the pathogenenesis of cardiac arterio-ventricular fistulae

Coronary Artery Fistulae (CAF) are congenital coronary artery (CA) anomalies consisting of an abnormal communication of a coronary artery with either a cardiac chamber or a large cardiac vessel. Although their incidence in the Western population is low, CAF can lead to complications such as myocardial hypertrophy, endocarditis, heart dilatation and cardiac failure. CAFs can appear as an isolated anomaly or linked to some other forms of congenital heart disease like Left Ventricular Non-Compaction (LVNC) and intrinsic CA anatomy anomalies, but their etiology remains unknown. In this work we have used two different experimental models (transgenic mice and avian embryos) to investigate on the developmental mechanics of CAF formation. In order to tackle this goal, we have manipulated epicardial development and ventricular wall compaction, two inextricably related developmental events during coronary embryogenesis. Conditional integrin α4 gene deletion in the septum transversum/proepicardial (ST/PE) region (G2-Gata4+) disrupts early epicardium development and reduces cardiomyocyte proliferation, leading to the thinning of the ventricular compact myocardial layer. Reduction in compact myocardium thickness associates to the presence of multiple ventricular myocardial discontinuities and focal endocardial extrusion. This same phenotype can be experimentally reproduced in chick embryos using a cryocauterization method (Palmquist-Gomes et al., 2016). Our results suggest that the partial absence of epicardium in α4integrin;G2-Gata4Cre mouse embryos and the cryoinjury in avian embryos generate myocardial discontinuities in the embryonic ventricular wall, which promote endocardial extrusion towards the pericardial cavity and the early contact of the endocardium with coronary progenitors at the epicardial surface of the heart. In the case of avian embryos, this phenomenon leads to precocious smooth muscle differentiation from epicardial mesenchymal cells, and the formation of pouch-like structures that closely resemble CAF. We conclude that anomalous compact myocardial embryonic growth can originate CAF.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Coup-TFII expression defines two different septum transversum cell compartments crucial to cardiac septation and compact ventricular wall growth

COUP-TFII encodes for an orphan nuclear receptor expressed by multiple embryonic tissues. COUP-TFII functions include the regulation of mesodermal progenitor differentiation and cell fate specification, and is required for completion of cardiovascular development as shown by the early death (E9.5) of COUP-TFII-null mice. In this study, we show that COUP-TFII, which is strongly expressed in the atrial myocardium, is also expressed in two different compartments of the septum transversum (ST, E9.5), a mesodermal folding adjacent to cardiac inflow myocardium. The first ST compartment is COUP-TFII+/Isl1+; cells in this compartment concentrate in the posterior part of the ST, overlap with SHF, and are continuous with the dorsal mesenchymal protrusion (DMP, also known as spina vestibuli). The second compartment is characteristically COUP-TFII+/Isl1-, and comprises the majority of proepicardial cells. To dissect the role of COUP-TFII+ ST cells in cardiac development, we conditionally deleted COUP-TFII in the ST using two different Cre constructs (Wt1Cre; G2-Gata4Cre). We show that COUP-TFII deletion in the ST is most severe in G2-Gata4Cre;COUP-TFII-/- mice, containing various cardiovascular progenitor lineages. Mutant mice display atrial septation and atrioventricular septal defects as well as a severe disruption of compact ventricular myocardial growth and coronary vascularization. We conclude that COUP-TFII plays critical, pleiotropic, tissue-dependent roles during cardiac septation, growth and vascularization.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Centro Nacional de Investigaciones Cardiovasculares (CNIC-ISCIII

“Off-on” two-photon indolenine probes for pH bioimaging

Fluorescence-based biosensors have become an indispensable tool in modern biology. These molecules can be used to monitor processes inside living cells and in real-time. The control of the pH is crucial in multiple biological process such as proliferation, apoptosis and defense. Therefore, the use of such pH probes are widespread in fluorescence microscopy. Recently, we have described a biosensor with excellent photophysical and suitable two-photon absorption (TPA) properties. This sensor allows the detection of hydroxyl radicals inside lysosomes. Based on this scaffold, we have designed, synthesized and characterized a new TPA fluorescent probe with an “off-on” response to different pH environments. These naphthalene-indolenine derivatives have a high synthetic versatility through affordable and efficient synthesis. The preliminary results suggest that this sensor has promising properties for detecting pH changes inside living cells.Real Sociedad Española de Química. Universidad de Murcia. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Pt(II)-dendrimers as bio-imaging marker for bacteria in two-photon excitation microscopy

The use of luminescent markers based on metal complexes in two-photon excitation microscopy techniques are of great interest in the field of bioimaging. However, despite the excellent luminescent properties of Pt(II) complexes, their application in this field is still limited, due to their poor solubility and quenching problems in aqueous media [1]. The insertion of a Pt(II) complex into a dendritic structure, gives as a result an unique luminescent marker soluble in biological media. Dendrimers provides excellent properties to the metal complex such as solubility in aqueous media, protection against quenching processes and binding to bacterial surfaces. The new probe can be used as bacteria cells marker in luminescent microscopy, operating under one or two-photon excitation (OPE/TPE) conditions, as well as in electron microscopy, thus providing a powerful tool in the field of bioimaging.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Tailoring two-photon fluorescent probes for pH bioimaging in living cells

Fluorescence biosensors are indispensable basic tools in modern biology. These type of molecules allow real-time visualization of biological events inside living cells. Especially important in many of these processes (proliferation, apoptosis or defense tasks) is the control of the cellular pH. In consequence, a great variety of structural models have been developed for pH bioimaging in fluorescence microscopy. Nonetheless, these efforts have been mainly focused on the development of one-photon (OP) probes. Recently, we described a biosensor with excellent photophysical properties and appropriate two-photon absorption (TPA) behavior. This sensor allows selective and specific detection of hydroxyl radicals solely inside lysosomes.Based on this scaffold, we have synthesized and characterized new TPA fluorescent probes. These molecules have an “off-on” response to different pH environments with a strong selectivity and sensitivity toward H+. These naphthalene-indolenine derivatives have a high synthetic versatility through affordable and efficient synthesis. The synthetic modification of this model allows tuning subcellular targets through minor modifications and without affecting their emission properties. The effectiveness of these probes and their structural modifications for different pH-related applications has been probed in mouse embrionary fibroblast (MEF) cells.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech