Browsing by Author "Torres, Miguel"
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Publication A Network of Macrophages Supports Mitochondrial Homeostasis in the Heart.(Cell Press, 2020-10-01) Nicolas-Avila, Jose A.; Lechuga-Vieco, Ana V.; Esteban-Martinez, Lorena; Sanchez-Diaz, Maria; Díaz-García, Elena; Santiago, Demetrio J; Rubio-Ponce, Andrea; Li, Jackson LiangYao; Balachander, Akhila; Quintana, Juan A.; Martínez-de-Mena, Raquel; Castejón-Vega, Beatriz; Pun-Garcia, Andres; Través, Paqui G; Bonzon-Kulichenko, Elena; Garcia-Marques, Fernando; Cusso, Lorena; Alonso-Gonzalez, Noelia; González-Guerra, Andrés; Roche-Molina, Marta; Martin-Salamanca, Sandra; Crainiciuc, Georgiana; Guzman-Martinez, Gabriela; Larrazabal, Jagoba; Herrero-Galan, Elas; Alegre-Cebollada, Jorge; Lemke, Greg; Rothlin, Carla V; Jimenez-Borreguero, Luis J.; Reyes, Guillermo; Castrillo, Antonio; Desco, Manuel; Munoz-Canoves, Pura; Ibáñez, Borja; Torres, Miguel; Ng, Lai Guan; Priori, Silvia G.; Bueno, Hector; Vazquez, Jesus; Cordero, Mario D; Bernal, Juan Antonio; Enriquez, Jose Antonio; Hidalgo, Andres; Ministerio de Ciencia e Innovación (España); Unión Europea. Comisión Europea. European Research Council (ERC); Fondation Leducq; Instituto de Salud Carlos III; Fundación La Caixa; Comunidad de Madrid (España); Fundación La Marató TV3; Howard Hughes Medical Institute; Centro Nacional de Investigaciones Cardiovasculares Carlos III (España); Fundación ProCNICCardiomyocytes are subjected to the intense mechanical stress and metabolic demands of the beating heart. It is unclear whether these cells, which are long-lived and rarely renew, manage to preserve homeostasis on their own. While analyzing macrophages lodged within the healthy myocardium, we discovered that they actively took up material, including mitochondria, derived from cardiomyocytes. Cardiomyocytes ejected dysfunctional mitochondria and other cargo in dedicated membranous particles reminiscent of neural exophers, through a process driven by the cardiomyocyte's autophagy machinery that was enhanced during cardiac stress. Depletion of cardiac macrophages or deficiency in the phagocytic receptor Mertk resulted in defective elimination of mitochondria from the myocardial tissue, activation of the inflammasome, impaired autophagy, accumulation of anomalous mitochondria in cardiomyocytes, metabolic alterations, and ventricular dysfunction. Thus, we identify an immune-parenchymal pair in the murine heart that enables transfer of unfit material to preserve metabolic stability and organ function. VIDEO ABSTRACT.Publication A predictive model of asymmetric morphogenesis from 3D reconstructions of mouse heart looping dynamics(eLife Sciences Publications, 2017) Le Garrec, Jean-François; Domínguez, Jorge N; Desgrange, Audrey; Ivanovitch, Kenzo; Raphaël, Etienne; Bangham, J Andrew; Torres, Miguel; Coen, Enrico; Mohun, Timothy J; Meilhac, Sigolène M; Agence Nationale de la Recherche (Francia); Institut National de la Santé et de la Recherche Médicale (Francia); Institut Pasteur; Ministerio de Economía, Industria y Competitividad (España)How left-right patterning drives asymmetric morphogenesis is unclear. Here, we have quantified shape changes during mouse heart looping, from 3D reconstructions by HREM. In combination with cell labelling and computer simulations, we propose a novel model of heart looping. Buckling, when the cardiac tube grows between fixed poles, is modulated by the progressive breakdown of the dorsal mesocardium. We have identified sequential left-right asymmetries at the poles, which bias the buckling in opposite directions, thus leading to a helical shape. Our predictive model is useful to explore the parameter space generating shape variations. The role of the dorsal mesocardium was validated in Shh-/- mutants, which recapitulate heart shape changes expected from a persistent dorsal mesocardium. Our computer and quantitative tools provide novel insight into the mechanism of heart looping and the contribution of different factors, beyond the simple description of looping direction. This is relevant to congenital heart defects.Publication A Second Heart Field-Derived Vasculogenic Niche Contributes to Cardiac Lymphatics.(Elsevier, 2020-02-10) Lioux, Ghislaine; Liu, Xiaolei; Temiño, Susana; Oxendine, Michael; Ayala, Estefanía; Ortega, Sagrario; Kelly, Robert G; Oliver, Guillermo; Torres, Miguel; Instituto de Salud Carlos III; Ministerio de Ciencia e Innovación (España); Comunidad de Madrid (España); Ministerio de Ciencia e Innovación. Centro de Excelencia Severo Ochoa (España)The mammalian heart contains multiple cell types that appear progressively during embryonic development. Advance in determining cardiac lineage diversification has often been limited by the unreliability of genetic tracers. Here we combine clonal analysis, genetic lineage tracing, tissue transplantation, and mutant characterization to investigate the lineage relationships between epicardium, arterial mesothelial cells (AMCs), and the coronary vasculature. We report a contribution of the second heart field (SHF) to a vasculogenic niche composed of AMCs and sub-mesothelial cells at the base of the pulmonary artery. Sub-mesothelial cells from this niche differentiate into lymphatic endothelial cells and, in close association with AMC-derived cells, contribute to and are essential for the development of ventral cardiac lymphatics. In addition, regionalized epicardial/mesothelial retinoic acid signaling regulates lymphangiogenesis, contributing to the niche properties. These results uncover a SHF vasculogenic contribution to coronary lymphatic development through a local niche at the base of the great arteries.Publication Analysis of the DNA-binding profile and function of TALE homeoproteins reveals their specialization and specific interactions with Hox genes/proteins(Elsevier, 2013-04-25) Penkov, Dmitry; Mateos San Martín, Daniel; Fernandez-Díaz, Luis C; Rossello, Catalina A; Torroja, Carlos; Sanchez-Cabo, Fatima; Warnatz, H J; Sultan, Marc; Yaspo, Marie L; Gabrieli, Arianna; Tkachuk, Vsevolod; Brendolan, Andrea; Blasi, Francesco; Torres, Miguel; Italian Association for Cancer Research; Ministero dell Università e della Ricerca (Italia); Ministerio de Economía y Competitividad (España); Comunidad de Madrid (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Fundación ProCNICThe interactions of Meis, Prep, and Pbx1 TALE homeoproteins with Hox proteins are essential for development and disease. Although Meis and Prep behave similarly in vitro, their in vivo activities remain largely unexplored. We show that Prep and Meis interact with largely independent sets of genomic sites and select different DNA-binding sequences, Prep associating mostly with promoters and housekeeping genes and Meis with promoter-remote regions and developmental genes. Hox target sequences associate strongly with Meis but not with Prep binding sites, while Pbx1 cooperates with both Prep and Meis. Accordingly, Meis1 shows strong genetic interaction with Pbx1 but not with Prep1. Meis1 and Prep1 nonetheless coregulate a subset of genes, predominantly through opposing effects. Notably, the TALE homeoprotein binding profile subdivides Hox clusters into two domains differentially regulated by Meis1 and Prep1. During evolution, Meis and Prep thus specialized their interactions but maintained significant regulatory coordination.Publication Axial skeleton anterior-posterior patterning is regulated through feedback regulation between Meis transcription factors and retinoic acid.(The Company of Biologists, 2021-01-04) López-Delgado, Alejandra C; Delgado, Irene; Cadenas, Vanessa; Sanchez-Cabo, Fatima; Torres, Miguel; Ministerio de Ciencia, Innovación y Universidades (España); Instituto de Salud Carlos III; Comunidad de Madrid (España); Fundación ProCNIC; Ministerio de Economía y Competitividad (España)Vertebrate axial skeletal patterning is controlled by co-linear expression of Hox genes and axial level-dependent activity of HOX protein combinations. MEIS transcription factors act as co-factors of HOX proteins and profusely bind to Hox complex DNA; however, their roles in mammalian axial patterning remain unknown. Retinoic acid (RA) is known to regulate axial skeletal element identity through the transcriptional activity of its receptors; however, whether this role is related to MEIS/HOX activity remains unknown. Here, we study the role of Meis in axial skeleton formation and its relationship to the RA pathway in mice. Meis elimination in the paraxial mesoderm produces anterior homeotic transformations and rib mis-patterning associated to alterations of the hypaxial myotome. Although Raldh2 and Meis positively regulate each other, Raldh2 elimination largely recapitulates the defects associated with Meis deficiency, and Meis overexpression rescues the axial skeletal defects in Raldh2 mutants. We propose a Meis-RA-positive feedback loop, the output of which is Meis levels, that is essential to establish anterior-posterior identities and patterning of the vertebrate axial skeleton.Publication Cardiomyocyte-Specific Wt1 Is Involved in Cardiac Metabolism and Response to Damage.(Multidisciplinary Digital Publishing Institute (MDPI), 2023-05-12) Díaz Del Moral, Sandra; Benaouicha, Maha; Villa Del Campo, Cristina; Torres, Miguel; Wagner, Nicole; Wagner, Kay-Dietrich; Muñoz-Chápuli, Ramón; Carmona, Rita; Ministerio de Economía, Industria y Competitividad (España); Instituto de Salud Carlos III; Unión Europea. Comisión Europea. H2020; Ministerio de Ciencia e Innovación (España)The Wilms tumor suppressor gene (Wt1) encodes a C2H2-type zinc-finger transcription factor that participates in transcriptional regulation, RNA metabolism, and protein-protein interactions. WT1 is involved in the development of several organs, including the kidneys and gonads, heart, spleen, adrenal glands, liver, diaphragm, and neuronal system. We previously provided evidence of transient WT1 expression in about 25% of cardiomyocytes of mouse embryos. Conditional deletion of Wt1 in the cardiac troponin T lineage caused abnormal cardiac development. A low expression of WT1 has also been reported in adult cardiomyocytes. Therefore, we aimed to explore its function in cardiac homeostasis and in the response to pharmacologically induced damage. Silencing of Wt1 in cultured neonatal murine cardiomyocytes provoked alterations in mitochondrial membrane potential and changes in the expression of genes related to calcium homeostasis. Ablation of WT1 in adult cardiomyocytes by crossing αMHCMerCreMer mice with homozygous WT1-floxed mice induced hypertrophy, interstitial fibrosis, altered metabolism, and mitochondrial dysfunction. In addition, conditional deletion of WT1 in adult cardiomyocytes increased doxorubicin-induced damage. These findings suggest a novel role of WT1 in myocardial physiology and protection against damage.Publication Cell Competition Promotes Phenotypically Silent Cardiomyocyte Replacement in the Mammalian Heart(Cell Press, 2014) Villa del Campo, Cristina; Claveria, Cristina; Sierra, Rocio; Torres, Miguel; Ministerio de Economía y Competitividad (España); Comunidad de Madrid (España); Fundación ProCNICHeterogeneous anabolic capacity in cell populations can trigger a phenomenon known as cell competition, through which less active cells are eliminated. Cell competition has been induced experimentally in stem/precursor cell populations in insects and mammals and takes place endogenously in early mouse embryonic cells. Here, we show that cell competition can be efficiently induced in mouse cardiomyocytes by mosaic overexpression of Myc during both gestation and adult life. The expansion of the Myc-overexpressing cardiomyocyte population is driven by the elimination of wild-type cardiomyocytes. Importantly, this cardiomyocyte replacement is phenotypically silent and does not affect heart anatomy or function. These results show that the capacity for cell competition in mammals is not restricted to stem cell populations and suggest that stimulated cell competition has potential as a cardio-myocyte-replacement strategy.Publication Cell tracing reveals a dorsoventral lineage restriction plane in the mouse limb bud mesenchyme.(The Company of Biologists, 2007-10) Arques, Carlos G; Doohan, Roisin; Sharpe, James; Torres, Miguel; Ministerio de Educación y Ciencia (España); International Human Frontier Science Program Organization; Unión Europea. Comisión Europea; Ministerio de Sanidad y Consumo (España); Fundación ProCNICRegionalization of embryonic fields into independent units of growth and patterning is a widespread strategy during metazoan development. Compartments represent a particular instance of this regionalization, in which unit coherence is maintained by cell lineage restriction between adjacent regions. Lineage compartments have been described during insect and vertebrate development. Two common characteristics of the compartments described so far are their occurrence in epithelial structures and the presence of signaling regions at compartment borders. Whereas Drosophila compartmental organization represents a background subdivision of embryonic fields that is not necessarily related to anatomical structures, vertebrate compartment borders described thus far coincide with, or anticipate, anatomical or cell-type discontinuities. Here, we describe a general method for clonal analysis in the mouse and use it to determine the topology of clone distribution along the three limb axes. We identify a lineage restriction boundary at the limb mesenchyme dorsoventral border that is unrelated to any anatomical discontinuity, and whose lineage restriction border is not obviously associated with any signaling center. This restriction is the first example in vertebrates of a mechanism of primordium subdivision unrelated to anatomical boundaries. Furthermore, this is the first lineage compartment described within a mesenchymal structure in any organism, suggesting that lineage restrictions are fundamental not only for epithelial structures, but also for mesenchymal field patterning. No lineage compartmentalization was found along the proximodistal or anteroposterior axes, indicating that patterning along these axes does not involve restriction of cell dispersion at specific axial positions.Publication ChIP-Seq and RNA-Seq Analyses Identify Components of the Wnt and Fgf Signaling Pathways as Prep1 Target Genes in Mouse Embryonic Stem Cells(Public Library of Science (PLOS), 2015) Laurent, Audrey; Calabrese, Manuela; Warnatz, Hans-Joerg; Yaspo, Marie-Laure; Tkachuk, Vsevolod; Torres, Miguel; Blasi, Francesco; Penkov, Dmitry; Fondazione Umberto Veronesi; Italian Association for Cancer Research; Fondazione Cariplo; Ministero della Salute (Italia); Max Planck Society; Unión Europea. Comisión Europea; Ministerio de Economía y Competitividad (España); Russian Foundation for Basic ResearchThe Prep1 (Pknox1) homeodomain transcription factor is essential at multiple stages of embryo development. In the E11.5 embryo trunk, we previously estimated that Prep1 binds about 3,300 genomic sites at a highly specific decameric consensus sequence, mainly governing basal cellular functions. We now show that in embryonic stem (ES) cells Prep1 binding pattern only partly overlaps that of the embryo trunk, with about 2,000 novel sites. Moreover, in ES cells Prep1 still binds mostly to promoters, as in total embryo trunk but, among the peaks bound exclusively in ES cells, the percentage of enhancers was threefold higher. RNA-seq identifies about 1800 genes down-regulated in Prep1(-/-) ES cells which belong to gene ontology categories not enriched in the E11.5 Prep1(i/i) differentiated embryo, including in particular essential components of the Wnt and Fgf pathways. These data agree with aberrant Wnt and Fgf expression levels in the Prep1(-/-) ES cells with a deficient embryoid bodies (EBs) formation and differentiation. Re-establishment of the Prep1 level rescues the phenotype.Publication Control of mouse limb initiation and antero-posterior patterning by Meis transcription factors.(Nature Publishing Group, 2021-05-25) Delgado, Irene; Giovinazzo, Giovanna; Temiño, Susana; Gauthier, Yves; Balsalobre, Aurelio; Drouin, Jacques; Torres, Miguel; Ministerio de Ciencia e Innovación (España); Fundación ProCNICMeis1 and Meis2 are homeodomain transcription factors that regulate organogenesis through cooperation with Hox proteins. Elimination of Meis genes after limb induction has shown their role in limb proximo-distal patterning; however, limb development in the complete absence of Meis function has not been studied. Here, we report that Meis1/2 inactivation in the lateral plate mesoderm of mouse embryos leads to limb agenesis. Meis and Tbx factors converge in this function, extensively co-binding with Tbx to genomic sites and co-regulating enhancers of Fgf10, a critical factor in limb initiation. Limbs with three deleted Meis alleles show proximal-specific skeletal hypoplasia and agenesis of posterior skeletal elements. This failure in posterior specification results from an early role of Meis factors in establishing the limb antero-posterior prepattern required for Shh activation. Our results demonstrate roles for Meis transcription factors in early limb development and identify their involvement in previously undescribed interaction networks that regulate organogenesis.Publication Cre recombinase microinjection for single-cell tracing and localised gene targeting.(The Company of Biologists, 2023-02-15) Sendra, Miquel; de Dios Hourcade, Juan; Temiño, Susana; Sarabia, Antonio J; Ocaña, Oscar H; Domínguez, Jorge N; Torres, Miguel; Ministerio de Ciencia e Innovación (España); Fundación La Caixa; Unión Europea. Comisión Europea. H2020; Centro Nacional de Investigaciones Cardiovasculares Carlos III (España)Tracing and manipulating cells in embryos are essential to understand development. Lipophilic dye microinjections, viral transfection and iontophoresis have been key to map the origin of the progenitor cells that form the different organs in the post-implantation mouse embryo. These techniques require advanced manipulation skills and only iontophoresis, a demanding approach of limited efficiency, has been used for single-cell labelling. Here, we perform lineage tracing and local gene ablation using cell-permeant Cre recombinase (TAT-Cre) microinjection. First, we map the fate of undifferentiated progenitors to the different heart chambers. Then, we achieve single-cell recombination by titrating the dose of TAT-Cre, which allows clonal analysis of nascent mesoderm progenitors. Finally, injecting TAT-Cre to Mycnflox/flox embryos in the primitive heart tube revealed that Mycn plays a cell-autonomous role in maintaining cardiomyocyte proliferation. This tool will help researchers identify the cell progenitors and gene networks involved in organ development, helping to understand the origin of congenital defects.Publication Dental Epithelial Stem Cells Express the Developmental Regulator Meis1(Frontiers Media, 2019) Sanz-Navarro, Maria; Delgado, Irene; Torres, Miguel; Mustonen, Tuija; Michon, Frederic; Rice, David P; Minerva Foundation; University of Helsinki (Finlandia); Finlands Akademi (Finlandia)MEIS1 is a key developmental regulator of several organs and participates in stem cell maintenance in different niches. However, despite the murine continuously growing incisor being a well described model for the study of adult stem cells, Meis1 has not been investigated in a dental context. Here, we uncover that Meis1 expression in the tooth is confined to the epithelial compartment. Its expression arises during morphogenesis and becomes restricted to the mouse incisor epithelial stem cell niche, the labial cervical loop. Meis1 is specifically expressed by Sox2+ stem cells, which give rise to all dental epithelial cell lineages. Also, we have found that Meis1 in the incisor is coexpressed with potential binding partner Pbx1 during both embryonic and adult stages. Interestingly, Meis2 is present in different areas of the forming tooth and it is not expressed by dental epithelial stem cells, suggesting different roles for these two largely homologous genes. Additionally, we have established the expression patterns of Meis1 and Meis2 during tongue, hair, salivary gland and palate formation. Finally, analysis of Meis1-null allele mice indicated that, similarly, to SOX2, MEIS1 is not essential for tooth initiation, but might have a role during adult incisor renewal.Publication Dissecting the Complexity of Early Heart Progenitor Cells.(Multidisciplinary Digital Publishing Institute (MDPI), 2021-12-26) Sendra, Miquel; Domínguez, Jorge N; Torres, Miguel; Ocaña, Oscar H; Ministerio de Ciencia e Innovación (España); Instituto de Salud Carlos III; Unión Europea. Comisión Europea; Fundación La Caixa; Fundación ProCNICEarly heart development depends on the coordinated participation of heterogeneous cell sources. As pioneer work from Adriana C. Gittenberger-de Groot demonstrated, characterizing these distinct cell sources helps us to understand congenital heart defects. Despite decades of research on the segregation of lineages that form the primitive heart tube, we are far from understanding its full complexity. Currently, single-cell approaches are providing an unprecedented level of detail on cellular heterogeneity, offering new opportunities to decipher its functional role. In this review, we will focus on three key aspects of early heart morphogenesis: First, the segregation of myocardial and endocardial lineages, which yields an early lineage diversification in cardiac development; second, the signaling cues driving differentiation in these progenitor cells; and third, the transcriptional heterogeneity of cardiomyocyte progenitors of the primitive heart tube. Finally, we discuss how single-cell transcriptomics and epigenomics, together with live imaging and functional analyses, will likely transform the way we delve into the complexity of cardiac development and its links with congenital defects.Publication ESC-Track: A computer workflow for 4-D segmentation, tracking, lineage tracing and dynamic context analysis of ESCs(Future Medicine, 2017-05) Fernandez-de-Manuel, Laura; Diaz-Diaz, Covadonga; Jimenez-Carretero, Daniel; Torres, Miguel; Montoya, Maria; Ministerio de Economía y Competitividad (España); Unión Europea. Comisión Europea; Comunidad de Madrid (España); Fundación ProCNIC; Instituto de Salud Carlos IIIEmbryonic stem cells (ESCs) can be established as permanent cell lines, and their potential to differentiate into adult tissues has led to widespread use for studying the mechanisms and dynamics of stem cell differentiation and exploring strategies for tissue repair. Imaging live ESCs during development is now feasible due to advances in optical imaging and engineering of genetically encoded fluorescent reporters; however, a major limitation is the low spatio-temporal resolution of long-term 3-D imaging required for generational and neighboring reconstructions. Here, we present the ESC-Track (ESC-T) workflow, which includes an automated cell and nuclear segmentation and tracking tool for 4-D (3-D + time) confocal image data sets as well as a manual editing tool for visual inspection and error correction. ESC-T automatically identifies cell divisions and membrane contacts for lineage tree and neighborhood reconstruction and computes quantitative features from individual cell entities, enabling analysis of fluorescence signal dynamics and tracking of cell morphology and motion. We use ESC-T to examine Myc intensity fluctuations in the context of mouse ESC (mESC) lineage and neighborhood relationships. ESC-T is a powerful tool for evaluation of the genealogical and microenvironmental cues that maintain ESC fitness.Publication Exercise triggers ARVC phenotype in mice expressing a disease-causing mutated version of human plakophilin-2.(Elsevier, 2015-04-14) Cruz, Francisco M; Sanz-Rosa, David; Roche-Molina, Marta; García-Prieto, Jaime; García-Ruiz, José M; Pizarro, Gonzalo; Jiménez-Borreguero, Luis J; Torres, Miguel; Bernad, Antonio; Ruíz-Cabello, Jesús; Fuster, Valentín; Ibáñez, Borja; Bernal, Juan ABACKGROUND Exercise has been proposed as a trigger for arrhythmogenic right ventricular cardiomyopathy (ARVC) phenotype manifestation; however, research is hampered by the limited availability of animal models in which disease-associated mutations can be tested. OBJECTIVES This study evaluated the impact of exercise on ARVC cardiac manifestations in mice after adeno-associated virus (AAV)-mediated gene delivery of mutant human PKP2, which encodes the desmosomal protein plakophilin-2. METHODS We developed a new model of cardiac tissue-specific transgenic-like mice on the basis of AAV gene transfer to test the potential of a combination of a human PKP2 mutation and endurance training to trigger an ARVC-like phenotype. RESULTS Stable cardiac expression of mutant PKP2 (c.2203C>T), encoding the R735X mutant protein, was achieved 4 weeks after a single AAV9-R735X intravenous injection. High-field cardiac magnetic resonance over a 10-month postinfection follow-up did not detect an overt right ventricular (RV) phenotype in nonexercised (sedentary) mice. In contrast, endurance exercise training (initiated 2 weeks after AAV9-R735X injection) resulted in clear RV dysfunction that resembled the ARVC phenotype (impaired global RV systolic function and RV regional wall motion abnormalities on cardiac magnetic resonance). At the histological level, RV samples from endurance-trained R735X-infected mice displayed connexin 43 delocalization at intercardiomyocyte gap junctions, a change not observed in sedentary mice. CONCLUSIONS The introduction of the PKP2 R735X mutation into mice resulted in an exercise-dependent ARVC phenotype. The R735X mutation appears to function as a dominant-negative variant. This novel system for AAV-mediated introduction of a mutation into wild-type mice has broad potential for study of the implication of diverse mutations in complex cardiomyopathies.Publication Gadd45g is essential for primary sex determination, male fertility and testis development(Public Library of Science (PLOS), 2013) Johnen, Heiko; González-Silva, Laura; Carramolino, Laura; Flores, Juana Maria; Torres, Miguel; Salvador, Jesús M; Unión Europea. Comisión Europea; Unión Europea. Comisión Europea. European Research Council (ERC); Instituto de Salud Carlos III; Ministerio de Ciencia e Innovación (España)In humans and most mammals, differentiation of the embryonic gonad into ovaries or testes is controlled by the Y-linked gene SRY. Here we show a role for the Gadd45g protein in this primary sex differentiation. We characterized mice deficient in Gadd45a, Gadd45b and Gadd45g, as well as double-knockout mice for Gadd45ab, Gadd45ag and Gadd45bg, and found a specific role for Gadd45g in male fertility and testis development. Gadd45g-deficient XY mice on a mixed 129/C57BL/6 background showed varying degrees of disorders of sexual development (DSD), ranging from male infertility to an intersex phenotype or complete gonadal dysgenesis (CGD). On a pure C57BL/6 (B6) background, all Gadd45g(-/-) XY mice were born as completely sex-reversed XY-females, whereas lack of Gadd45a and/or Gadd45b did not affect primary sex determination or testis development. Gadd45g expression was similar in female and male embryonic gonads, and peaked around the time of sex differentiation at 11.5 days post-coitum (dpc). The molecular cause of the sex reversal was the failure of Gadd45g(-/-) XY gonads to achieve the SRY expression threshold necessary for testes differentiation, resulting in ovary and Müllerian duct development. These results identify Gadd45g as a candidate gene for male infertility and 46,XY sex reversal in humans.Publication Growth and Morphogenesis during Early Heart Development in Amniotes(Multidisciplinary Digital Publishing Institute (MDPI), 2017-11-22) Ivanovitch, Kenzo; Esteban, Isaac; Torres, Miguel; Ministerio de Economía, Industria y Competitividad (España); Instituto de Salud Carlos III; European Molecular Biology Organization; International Human Frontier Science Program Organization; Fundación ProCNICIn this review, we will focus on the growth and morphogenesis of the developing heart, an aspect of cardiovascular development to which Antoon Moorman and colleagues have extensively contributed. Over the last decades, genetic studies and characterization of regionally regulated gene programs have provided abundant novel insights into heart development essential to understand the basis of congenital heart disease. Heart morphogenesis, however, is inherently a complex and dynamic three-dimensional process and we are far from understanding its cellular basis. Here, we discuss recent advances in studying heart morphogenesis and regionalization under the light of the pioneering work of Moorman and colleagues, which allowed the reinterpretation of regional gene expression patterns under a new morphogenetic framework. Two aspects of early heart formation will be discussed in particular: (1) the initial formation of the heart tube and (2) the formation of the cardiac chambers by the ballooning process. Finally, we emphasize that in addition to analyses based on fixed samples, new approaches including clonal analysis, single-cell sequencing, live-imaging and quantitative analysis of the data generated will likely lead to novel insights in understanding early heart tube regionalization and morphogenesis in the near future.Publication Imaging Approaches and the Quantitative Analysis of Heart Development.(Multidisciplinary Digital Publishing Institute (MDPI), 2023-03-29) Raiola, Morena; Sendra, Miquel; Torres, Miguel; Ministerio de Ciencia e Innovación (España); Unión Europea. Comisión Europea. H2020; Fundación La Caixa; Fundación ProCNICHeart morphogenesis is a complex and dynamic process that has captivated researchers for almost a century. This process involves three main stages, during which the heart undergoes growth and folding on itself to form its common chambered shape. However, imaging heart development presents significant challenges due to the rapid and dynamic changes in heart morphology. Researchers have used different model organisms and developed various imaging techniques to obtain high-resolution images of heart development. Advanced imaging techniques have allowed the integration of multiscale live imaging approaches with genetic labeling, enabling the quantitative analysis of cardiac morphogenesis. Here, we discuss the various imaging techniques used to obtain high-resolution images of whole-heart development. We also review the mathematical approaches used to quantify cardiac morphogenesis from 3D and 3D+time images and to model its dynamics at the tissue and cellular levels.Publication Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis(2017) Ivanovitch, Kenzo; Termino, Susana; Torres, Miguel; Ministerio de Economía y Competitividad (España); Instituto de Salud Carlos III; European Molecular Biology Organization; International Human Frontier Science Program OrganizationDuring vertebrate heart development, two progenitor populations, first and second heart fields (FHF, SHF), sequentially contribute to longitudinal subdivisions of the heart tube (HT), with the FHF contributing the left ventricle and part of the atria, and the SHF the rest of the heart. Here, we study the dynamics of cardiac differentiation and morphogenesis by tracking individual cells in live analysis of mouse embryos. We report that during an initial phase, FHF precursors differentiate rapidly to form a cardiac crescent, while limited morphogenesis takes place. In a second phase, no differentiation occurs while extensive morphogenesis, including splanchnic mesoderm sliding over the endoderm, results in HT formation. In a third phase, cardiac precursor differentiation resumes and contributes to SHF-derived regions and the dorsal closure of the HT. These results reveal tissue-level coordination between morphogenesis and differentiation during HT formation and provide a new framework to understand heart development.Publication Lymphoangiocrine signals promote cardiac growth and repair.(Nature Publishing Group, 2020-12) Liu, Xiaolei; De la Cruz, Ester; Gu, Xiaowu; Balint, Laszlo; Oxendine-Burns, Michael; Terrones, Tamara; Ma, Wanshu; Kuo, Hui-Hsuan; Lantz, Connor; Bansal, Trisha; Thorp, Edward; Burridge, Paul; Jakus, Zoltán; Herz, Joachim; Cleaver, Ondine; Torres, Miguel; Oliver, Guillermo; Ministerio de Educación (España); European Molecular Biology Organization; Ministerio de Ciencia e Innovación (España); National Institutes of Health (Estados Unidos)Recent studies have suggested that lymphatics help to restore heart function after cardiac injury1-6. Here we report that lymphatics promote cardiac growth, repair and cardioprotection in mice. We show that a lymphoangiocrine signal produced by lymphatic endothelial cells (LECs) controls the proliferation and survival of cardiomyocytes during heart development, improves neonatal cardiac regeneration and is cardioprotective after myocardial infarction. Embryos that lack LECs develop smaller hearts as a consequence of reduced cardiomyocyte proliferation and increased cardiomyocyte apoptosis. Culturing primary mouse cardiomyocytes in LEC-conditioned medium increases cardiomyocyte proliferation and survival, which indicates that LECs produce lymphoangiocrine signals that control cardiomyocyte homeostasis. Characterization of the LEC secretome identified the extracellular protein reelin (RELN) as a key component of this process. Moreover, we report that LEC-specific Reln-null mouse embryos develop smaller hearts, that RELN is required for efficient heart repair and function after neonatal myocardial infarction, and that cardiac delivery of RELN using collagen patches improves heart function in adult mice after myocardial infarction by a cardioprotective effect. These results highlight a lymphoangiocrine role of LECs during cardiac development and injury response, and identify RELN as an important mediator of this function.