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V. Andrés’s laboratory is supported by Ministerio de Ciencia e Innovación (MICIN)/Agencia Estatal de Investigación (AEI) (grant PID2019-108489RBI00/AEI/10.13039/501100011033) with cofund from Fondo Social Europeo “El FSE invierte en tu futuro”. The CNIC is supported by the MICIN, the Instituto de Salud Carlos III, the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/ AEI/10.13039/501100011033). J. Díez is consultant to AstraZeneca, Bayer, and Vifor Pharma. Sí

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations-dolines-capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli. We thank R. Parton (Institute for Molecular Biosciences, Queensland), P. Pilch (Boston University School of Medicine) and L. Liu (Boston University School of Medicine) for kindly providing PTRFKO cells and reagents, S. Casas Tintó for kindly providing SH-Sy5y cells, P. Bassereau (Curie Institute, Paris) for kindly providing OT setup, V. Labrador Cantarero from CNIC microscopy Unit for helping with ImageJ analysis, O. Otto and M. Herbig for providing help with RTDC experiments, S. Berr and K. Gluth for technical assistance in cell culture, F. Steiniger for support in electron tomography, and A. Norczyk Simón for providing pCMV-FLAG-PTRF construct. This project received funding from the European Union Horizon 2020 Research and Innovation Programme through Marie Sklodowska-Curie grant 641639; grants from the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033): SAF2014-51876-R, SAF2017-83130-R co-funded by ‘ERDF A way of making Europe’, PID2020-118658RB-I00, PDC2021-121572-100 co-funded by ‘European Union NextGenerationEU/PRTR’, CSD2009- 0016 and BFU2016-81912-REDC; and the Asociación Española Contra el Cáncer foundation (PROYE20089DELP) all to M.A.d.P. M.A.d.P. is member of the Tec4Bio consortium (ref. S2018/NMT¬4443; Comunidad Autónoma de Madrid/FEDER, Spain), co-recipient with P.R.-C. of grants from Fundació La Marató de TV3 (674/C/2013 and 201936- 30-31), and coordinator of a Health Research consortium grant from Fundación Obra Social La Caixa (AtheroConvergence, HR20-00075). M.S.-A. is recipient of a Ramón y Cajal research contract from MCIN (RYC2020-029690-I). The CNIC Unit of Microscopy and Dynamic Imaging is supported by FEDER ‘Una manera de hacer Europa’ (ReDIB ICTS infrastructure TRIMA@CNIC, MCIN). We acknowledge the support from Deutsche Forschungsgemeinschaft through grants to M.M.K. (KE685/7-1) and B.Q. (QU116/6-2 and QU116/9-1). Work in D.N. laboratory was supported by grants from the European Union Horizon 2020 Research and Innovation Programme through Marie Sklodowska-Curie grant 812772 and MCIN (DPI2017-83721-P). Work in C.L. laboratory was supported by grants from Curie, INSERM, CNRS, Agence Nationale de la Recherche (ANR-17-CE13-0020-01) and Fondation ARC pour la Recherche (PGA1-RF20170205456). Work in P.R.-C. lab is funded by the MCIN (PID2019-110298GB-I00), the EC (H20 20-FETPROACT-01-2016-731957). Work in X.T. lab is funded by the MICIN (PID2021-128635NB-I00), ERC (Adv-883739) and La Caixa Foundation (LCF/PR/HR20/52400004; co-recipient with P.R.-C.). IBEC is recipient of a Severo Ochoa Award of Excellence from the MINECO. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MCIN and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). Sí

As evidenced by scintigraphy imaging, the prevalence of transthyretin (TTR) cardiac amyloidosis in heart failure patients with preserved ejection fraction (HFpEF) and left ventricular hypertrophy (LVH) ranges between 13% and 19%. The natural evolution of cardiac amyloidosis begins with the deposition of amyloid material in the myocardium, with LVH ensuing at later stages. With current imaging modalities, it is possible to detect TTR cardiac amyloidosis before the hypertrophic stage. The aim of this study was to determine the prevalence of TTR cardiac amyloidosis in HFpEF patients without LVH. The study prospectively enrolled patients admitted for HF with LV ejection fraction (LVEF) ≥ 50% and LV wall thickness

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3′ end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs., This work was funded by the Spanish government (grants BFU2017-84914-P and PID2020-115755GB-I00 to M.M.; BFU2016-74961-P and BFU2016-81887-REDT to J.L.G.-S.), the Andalusian government (grant BIO-396 to J.L.G.-S.), and the European Research Council (ERC; grant agreement 740041 to J.L.G.-S.). M.T. held Juan de la Cierva fellowships from the Spanish government (FJCI-2017-31791 and IJC2019-038897-I), R.R. and R.D.A. held FPU fellowships from the government, and J.V. was the recipient of a “La Caixa” fellowship. Work in the laboratory of J.L.G.-S. was supported by a María de Maetzu Unit of Excellence Grant (MDM-2016-0687) to the Department of Gene Regulation and Morphogenesis of the CABD. The CBMSO is supported by an institutional grant from the Fundación Ramon Areces, and the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). : With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2020-001041-S).

Germinal centers (GC) are microstructures where B cells that have been activated by antigen can improve the affinity of their B cell receptors and differentiate into memory B cells (MBCs) or antibody-secreting plasma cells. Here, we have addressed the role of activation-induced deaminase (AID), which initiates somatic hypermutation and class switch recombination, in the terminal differentiation of GC B cells. By combining single cell transcriptome and immunoglobulin clonal analysis in a mouse model that traces AID-experienced cells, we have identified a novel subset of late-prePB cells (L-prePB), which shares the strongest clonal relationships with plasmablasts (PBs). Mice lacking AID have various alterations in the size and expression profiles of transcriptional clusters. We find that AID deficiency leads to a reduced proportion of L-prePB cells and severely impairs transitions between the L-prePB and the PB subsets. Thus, AID shapes the differentiation fate of GC B cells by enabling PB generation from a prePB state. We thank all the members of the B lymphocyte Biology lab for helpful suggestions, Ana Rodriguez-Ronchel for the elaboration of the graphical abstract, Sonia Mur for technical assistance, Virginia G de Yebenes for critical reading of our manuscript, Julia Merkenschlager, Carlos Torroja, and Enrique Vazquez for their advice on single cell sequencing and analysis, the CNIC Flow Cytometry for assistance on cell analysis and separation and the CNIC Genomics Unit for single cell sequencing. We also thank Sergio Roa, Alicia G Arroyo, Salvador Iborra, and David Sancho for kindly sharing mouse lines and reagents with us. CG-E is supported by a fellowship awarded by La Caixa Espana in ~ 2017 and ASN is an FPI Severo Ochoa fellow (PRE2018-083475). AB, FS-C, and ARR are supported by CNIC. This project was funded by grants from the Spanish Ministerio de Econom ıa, Industria y Competitividad (SAF2016-75511-R), the Spanish Ministerio de Ciencia e Innovaci on (PID2019-106773RB-I00/AEI/10.13039/ 501100011033) and the “la Caixa” Banking Foundation under the project code HR17-00247 to ARR. FS-C is supported by the project RT2018-102084-B-I00 financed by MCIN/AEI/10.13039/5011000110033/ and by FEDER Una Manera de hacer Europa and by Ayuda EQC2021-007294-P financed by MCIN/AEI/ 10.13039/501100011033 and by the European Union NextGenerationEU/PRTR. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovacion (MCIN), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence, CEX2020-001041-S funded by MICIN/AEI/10.13039/ 501100011033. Sí

Cardiovascular disease (CVD) is the leading cause of mortality in the world, with most CVD-related deaths resulting from myocardial infarction or stroke. The main underlying cause of thrombosis and cardiovascular events is atherosclerosis, an inflammatory disease that can remain asymptomatic for long periods. There is an urgent need for therapeutic and diagnostic options in this area. Atherosclerotic plaques contain autoantibodies, and there is a connection between atherosclerosis and autoimmunity. However, the immunogenic trigger and the effects of the autoantibody response during atherosclerosis are not well understood. Here we performed high-throughput single-cell analysis of the atherosclerosis-associated antibody repertoire. Antibody gene sequencing of more than 1,700 B cells from atherogenic Ldlr and control mice identified 56 antibodies expressed by in-vivo-expanded clones of B lymphocytes in the context of atherosclerosis. One-third of the expanded antibodies were reactive against atherosclerotic plaques, indicating that various antigens in the lesion can trigger antibody responses. Deep proteomics analysis identified ALDH4A1, a mitochondrial dehydrogenase involved in proline metabolism, as a target antigen of one of these autoantibodies, A12. ALDH4A1 distribution is altered during atherosclerosis, and circulating ALDH4A1 is increased in mice and humans with atherosclerosis, supporting the potential use of ALDH4A1 as a disease biomarker. Infusion of A12 antibodies into Ldlr mice delayed plaque formation and reduced circulating free cholesterol and LDL, suggesting that anti-ALDH4A1 antibodies can protect against atherosclerosis progression and might have therapeutic potential in CVD., Ministerio de Economía y Competitividad (SVP-2014-068289); P.D. was supported by an AECC grant (AIO 2012, Ayudas a Investigadores en Oncología 2012); A.S.-B. is a Juan de la Cierva researcher (IJC2018-035279-I); I.M.-F. was a fellow of the research training program funded by Ministerio de Economía y Competitividad (SVP-2014-068216); and A.R.R. and J.V. are supported by Centro Nacional de Investigaciones Cardiovasculares (CNIC). The project leading to these results has received funding from la Caixa Banking Foundation under the project code HR17-00247 and from SAF2016-75511-R and PID2019-106773RB-I00 grants to A.R.R. (Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016

Genomic instability, the unresolved accumulation of DNA variants, is hypothesized as one of the contributors to the natural aging process. We assessed the frequency of unresolved DNA damage reaching the transcriptome of the murine myocardium during the course of natural aging and in hearts from four distinct mouse models of premature aging with established aging-related cardiac dysfunctions. RNA sequencing and variant calling based on total RNA sequencing was compared between hearts from naturally aging mice, mice with cardiomyocyte-specific deficiency of Ercc1, a component of the DNA repair machinery, mice with reduced mitochondrial antioxidant capacity, Tert-deficient mice with reduced telomere length, and a mouse model of human Hutchinson-Gilford progeria syndrome (HGPS). Our results demonstrate that no enrichment in variants is evident in the naturally aging murine hearts until 2 y of age from the HGPS mouse model or mice with reduced telomere lengths. In contrast, a dramatic accumulation of variants was evident in Ercc1 cardiomyocyte-specific knockout mice with deficient DNA repair machinery, in mice with reduced mitochondrial antioxidant capacity, and in the intestine, liver, and lung of naturally aging mice. Our data demonstrate that genomic instability does not evidently contribute to naturally aging of the mouse heart in contrast to other organs and support the contention that the endogenous DNA repair machinery is remarkably active to maintain genomic integrity in cardiac cells throughout life. F.D.M. is supported by HS-BAFTA and Kootstra fellowships of Maastricht University and a CVON-ARENA-PRIME fellowship. L.M. is supported by the fund Innovative Medical Research of the University of Münster Medical School (RÜ121510). M.R.H. is supported by a Juan de la Cierva contract from the Spanish Ministerio de Ciencia, Innovación y Universidades (IJC2019-040798-I). R.M.N. is the beneficiary of a predoctoral contract from the Spanish Ministerio de Educación, Cultura y Deporte (FPU16/ 05027). V.A. is supported by the Spanish Ministerio de Ciencia e Innovación (PID2019-108489RB-I00) and the Instituto de Salud Carlos III (ISCIII) (AC16/ 00091) as member of the ERA-CVD JCT2016 EXPERT Network (European Union’s Horizon 2020 Framework Programme), with cofunding from the European Regional Development Fund (“Una manera de hacer Europa”). The Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) is supported by the Ministry for Research, Science and Innovation (MICIN), the ISCIII, the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence. C.B. was supported by the Deutsche Forschungsgemeinschaft (DFG) (BA5631/ 2-1). T.T. was supported by the European Research Council (ERC) Consolidator Grant LONGHEART, by ERA-CVD JCT2016 EXPERT, and the DFG (TH903/ 22-1). D.J.D., M.S., and L.J.D.W. acknowledge support from the Dutch CardioVascular Initiative: the Netherlands Heart Foundation, Dutch Federation of University Medical Centers, ZonMW, and the Royal Netherlands Academy of Sciences (CVON-ARENA-PRIME, CVON-RACE-V, CVON-PREDICT-2). B.S. acknowledges funding by the Netherlands Heart Foundation (Dr. Dekker 2014T105 and CVON-SHE-PREDICTS-HF) and a VIDI Award 917.14.363 from the Dutch Research Council (NWO). A.S.A. was funded by Association Française contres les Myopathies (AFM 18802). F.D.M., T.T., and L.J.D.W. are supported by ERA-CVD JCT2016 EXPERT. M.S. is funded by the DFG (RTG2220, Project 281125614) and Marie Skłodowska-Curie Grant Agreement 81371. L.J.D.W. was further supported by ERC Consolidator Grant 311549 CALMIRS, a VICI Award 918-156-47 from NWO and Marie Skłodowska-Curie Grant Agreements 813716 and 765274. Sí

Unlike the hearts of mammals, the adult zebrafish heart regenerates after injury. Heart cryoinjury in zebrafish triggers the formation of a fibrotic scar that gradually degrades, leading to regeneration. Midkine-a (Mdka) is a multifunctional cytokine that is activated after cardiac injury. Here, we investigated the role of mdka in zebrafish heart regeneration. We show that mdka expression was induced at 1-day post-cryoinjury (dpci) throughout the epicardial layer, whereas by 7 dpci expression had become restricted to the epicardial cells covering the injured area. To study the role of mdka in heart regeneration, we generated mdka-knock out (KO) zebrafish strains. Analysis of injured hearts showed that loss of mdka decreased endothelial cell proliferation and resulted in an arrest in heart regeneration characterized by retention of a collagenous scar. Transcriptional analysis revealed increases in collagen transcription and intense TGFβ signaling activity. These results reveal a critical role for mdka in fibrosis regulation during heart regeneration. This study was supported by Grants PID2019-104776RB-I00, CB16/11/00399 (CIBER CV) and RD16/0011/0021 (TERCEL) from the Spanish Ministry of Science, Innovation and Universities (MCIU) and Grants from the Fundación BBVA (Ref.: BIO14_298), Fundación La Marató (Ref.: 20153431) and the European Commission through CardioNeT (Ref.: 28600) from the European Commission to JLP. DG held a Ph.D. fellowship linked to the Grant CardioNeT grant. The cost of this publication was supported in part with funds from the European Regional Development Fund. The CNIC was supported by the Instituto de Salud Carlos III (ISCIII), the MCIU and the Pro CNIC Foundation, and is a Severo Ochoa Centre of Excellence (SEV-2015-0505). Sí

Conventional type 1 dendritic cells (cDC1s) are central to antitumor immunity and their presence in the tumor microenvironment associates with improved outcomes in patients with cancer. DNGR-1 (CLEC9A) is a dead cell-sensing receptor highly restricted to cDC1s. DNGR-1 has been involved in both cross-presentation of dead cell-associated antigens and processes of disease tolerance, but its role in antitumor immunity has not been clarified yet.

Correction to: Solving patients with rare diseases through programmatic reanalysis of genome-phenome data. Eur J Hum Genet. 2021 Sep;29(9):1466-1469. doi: 10.1038/s41431-021-00934-6. PMID: 34393220 Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP's Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics. The Solve-RD project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 779257. Data were analysed using the RD‐Connect Genome‐Phenome Analysis Platform, which received funding from EU projects RD‐Connect, Solve-RD and EJP-RD (grant numbers FP7 305444, H2020 779257, H2020 825575), Instituto de Salud Carlos III (grant numbers PT13/0001/0044, PT17/0009/0019; Instituto Nacional de Bioinformática, INB) and ELIXIR Implementation Studies. We acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya. We also acknowledge the support of the Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement and the Co-financing by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) with funds from the European Regional Development Fund (ERDF) corresponding to the 2014-2020 Smart Growth Operating Program. Sí

14 pags. 5 figs., 2 tabs., Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Variants in MYBPC3, the gene encoding cardiac myosin-binding protein C (cMyBP-C), are the leading cause of HCM. However, the pathogenicity status of hundreds of MYBPC3 variants found in patients remains unknown, as a consequence of our incomplete understanding of the pathomechanisms triggered by HCM-causing variants. Here, we examined 44 nontruncating MYBPC3 variants that we classified as HCM-linked or nonpathogenic according to cosegregation and population genetics criteria. We found that around half of the HCM-linked variants showed alterations in RNA splicing or protein stability, both of which can lead to cMyBP-C haploinsufficiency. These protein haploinsufficiency drivers associated with HCM pathogenicity with 100% and 94% specificity, respectively. Furthermore, we uncovered that 11% of nontruncating MYBPC3 variants currently classified as of uncertain significance in ClinVar induced one of these molecular phenotypes. Our strategy, which can be applied to other conditions induced by protein loss of function, supports the idea that cMyBP-C haploinsufficiency is a fundamental pathomechanism in HCM., A.-C. acknowledges funding from the Ministerio de Ciencia e Innovación (MCIN) throughgrants BIO2014-54768-P; BIO2017-83640-P/MINECO/AEI/FEDER, UE; EIN2019-102966 and RYC-2014-16604, the European Research Area Network on Cardiovascular Diseases (ERA-CVD/ISCIII, Spain MINOTAUR, AC16/00045), and the Comunidad de Madrid (consortium Tec4Bio-CM, S2018/NMT-4443, FEDER). TheCNIC is supported by the ISCIII, MCIN, and the Pro CNIC Foundation and was a Severo Ochoa Center of Excellence (SEV-2015-0505). We acknowledge funding from ISCIII to the Centro de Investigación Biomédica en Red (CB16/11/00425). L. S. acknowl-edges funding from MCIN (BFU2015-63571-P). J. S. acknowledgesfunding from AEI (PID2019-107293GB-I00), Gobierno de Aragón(E45_17R), and ERDF-InterregV-A POCTEFA, Spain (PIREPRED-EFA086/15). C. S.-C. is the recipient of an FPI-SO predoctoral fellowship BES-2016-076638. M. R. P. was the recipient of a Ph D fellowship from the Italian Ministry of Education, Universities and Research, Italy. H. G.-C. is the recipient of an FPU16/04232 doctoralcontract from MCIN.

Bone morphogenetic protein (Bmp) signaling is critical for organismal development and homeostasis. To elucidate Bmp2 function in the vascular/hematopoietic lineages we generated a new transgenic mouse line in which ectopic Bmp2 expression is controlled by the Tie2 promoter. Tie2CRE/+;Bmp2tg/tg mice develop aortic valve dysfunction postnatally, accompanied by pre-calcific lesion formation in valve leaflets. Remarkably, Tie2CRE/+;Bmp2tg/tg mice develop extensive soft tissue bone formation typical of acquired forms of heterotopic ossification (HO) and genetic bone disorders, such as Fibrodysplasia Ossificans Progressiva (FOP). Ectopic ossification in Tie2CRE/+;Bmp2tg/tg transgenic animals is accompanied by increased bone marrow hematopoietic, fibroblast and osteoblast precursors and circulating pro-inflammatory cells. Transplanting wild-type bone marrow hematopoietic stem cells into lethally irradiated Tie2CRE/+;Bmp2tg/tg mice significantly delays HO onset but does not prevent it. Moreover, transplanting Bmp2-transgenic bone marrow into wild-type recipients does not result in HO, but hematopoietic progenitors contribute to inflammation and ectopic bone marrow colonization rather than to endochondral ossification. Conversely, aberrant Bmp2 signaling activity is associated with fibroblast accumulation, skeletal muscle fiber damage, and expansion of a Tie2+ fibro-adipogenic precursor cell population, suggesting that ectopic bone derives from a skeletal muscle resident osteoprogenitor cell origin. Thus, Tie2CRE/+;Bmp2tg/tg mice recapitulate HO pathophysiology, and might represent a useful model to investigate therapies seeking to mitigate disorders associated with aberrant extra-skeletal bone formation., Grants PID2019-104776RB-I00, SAF2016-78370-R, CB16/11/00399 (CIBER CV), and RD16/0011/0021 (TERCEL) from the Spanish Ministry of Science, Innovation, and Universities (MCIU) to JLDLP. The cost of this publication was supported in part with FEDER funds. The CNIC is supported by the MCIU and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

RationaleThe rupture of the fibrous cap in atherosclerotic lesions is the underlying cause of most thrombi leading to heart attack and a frequent cause of stroke. Caps are produced by smooth muscle cells (SMCs) that are recruited to the subendothelial space. We hypothesized that the recruitment mechanisms are likely common to embryonic artery development, which relies prominently on Notch signaling to form the subendothelial layers of medial SMCs.ObjectiveTo analyze the causal roles of the Notch signaling pathway in SMCs for atherogenesis and cap formation.Methods and ResultsNotch elements involved in arterial media development were found expressed in regions of fibrous cap in mouse plaques. To assess the causal role of Notch signaling in cap formation, we studied atherosclerosis in mice in which the Notch pathway was inactivated specifically in SMCs by conditional knockout of the essential effector transcription factor RBPJ. No major effects were observed on plaque size, but the presence of cap SMCs was significantly reduced. Lineage tracing revealed that the accumulation of SMC-derived plaque cells in the cap region was unaltered but that Notch-defective cells failed to re-acquire the SMC phenotype in the cap. To analyze whether the accumulation of SMC-derived cells in atherogenesis requires down-regulation of Notch signaling, we studied atherosclerosis in mice with constitutive Notch signaling in SMCs achieved by conditional expression of the Notch intracellular domain. Forced Notch signaling inhibited the ability of medial SMCs to contribute to plaque cells, including both cap SMCs and osteochondrogenic cells, and significantly reduced atherosclerosis development.ConclusionsSequential loss and gain of Notch signaling is needed to build the cap SMC population. The shared mechanisms with embryonic arterial media assembly suggest that the fibrous cap forms as a neo-media that restores the connection between endothelium and stabilizing SMCs, which is transiently disrupted by atherogenesis.

In germinal centres (GC) mature B cells undergo intense proliferation and immunoglobulin gene modification before they differentiate into memory B cells or long-lived plasma cells (PC). GC B-cell-to-PC transition involves a major transcriptional switch that promotes a halt in cell proliferation and the production of secreted immunoglobulins. Here we show that the CCCTC-binding factor (CTCF) is required for the GC reaction in vivo, whereas in vitro the requirement for CTCF is not universal and instead depends on the pathways used for B-cell activation. CTCF maintains the GC transcriptional programme, allows a high proliferation rate, and represses the expression of Blimp-1, the master regulator of PC differentiation. Restoration of Blimp-1 levels partially rescues the proliferation defect of CTCF-deficient B cells. Thus, our data reveal an essential function of CTCF in maintaining the GC transcriptional programme and preventing premature PC differentiation., Activated B cells differentiate into antibody-producing plasma cells in the germinal centre in secondary lymphoid organs. Here the authors show that this differentiation process and related transcription programs are modulated by the transcription factor CTCF, partly by suppressing the premature expression of Blimp-1.

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. We thank Cristina Villa del Campo, Ester de la Cruz, and other members of the Torres group for stimulating discussions and suggestions. We thank members of the Microscopy, Genomics, Bioinformatics, Transgenesis, and Animal Facility CNIC units for excellent support. This work was supported by grants PGC2018-096486-B-I00 and RD16/0011/0019 (ISCIII) from the Spanish Ministry of Science, Innovation, and Universities and grant S2017/BMD3875 from the Comunidad de Madrid to M.T., grant ITN-CARDIONET-ref.289600 from EU to G.L., and by grant BFU2015-71376-R to S.O. The CNIC is supported by the Ministerio de Ciencia, Innovacio´ n y Universidades, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). This work was also supported by NIH grant RO1HL073402 to G.O. and by AHA grant 18CDA34110356 to X.L. Sí

Vascular co-option by brain metastasis-initiating cells has been demonstrated as a critical step in organ colonization. The physical interaction between the cancer cell and the endothelial cell is mediated by integrins and L1CAM and could be involved in aggressive growth but also latency and immune evasion. The key involvement of vascular co-option in brain metastasis has created an emerging field that aims to identify critical targets as well as effective inhibitors with the goal of preventing brain metastases. Research in the Brain Metastasis Group is supported by MINECO-Retos SAF2017-89643-R (M.V.), Bristol-Myers Squibb-Melanoma Research Alliance Young Investigator Award (498103) (M.V.), Beug Foundation's Prize for Metastasis Research (M.V.), Fundacion Ramon Areces (CIVP19S8163) (M. V.), Worldwide Cancer Research (19-0177) (M.V.), H2020-FETOPEN (828972) (M.V.), CLIP Award Cancer Research Institute (54545) (M.V.), and AECC Coordinated Translational Groups 2017 (GCTRA16015SEOA) (M.V.). P.G-G. is the recipient of La Caixa Foundation (ID100010434) and European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement (No. 713673) PhD Program Fellowship (LCF/BQ/IN17/11620028). M.V. is a Ramon y Cajal Investigator (RYC-2013-13365) and EMBO YIP (4053). No

et al., Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development., This work was funded by the Spanish Ministerio de Economia y Competitividad (grants BFU2011-23083 and BFU2014-54608-P to MM, BFU2013-41322-P to JLGS and BFU2014-58449-JIN to JJT), the Comunidad Autónoma de Madrid (grant CELLDD-CM to MM), the Andalusian regional government (grant BIO-396 to JLGS), the Universidad Pablo de Olavide (postdoctoral grant to JJT). PDO is funded by the Heart and Stroke Foundation of Canada (G-17-0018613), Operational Funds from the Hospital for Sick Children, the Natural Sciences and Engineering Research Council of Canada (NSERC) (500865 to PDO), and the Canadian Institutes of Health Research (CIHR) (PJT-149046). The CNIC is supported by the Spanish Ministerio de Economia y Competitividad and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

The mammalian epiblast is formed by pluripotent cells able to differentiate into all tissues of the new individual. In their progression to differentiation, epiblast cells and their in vitro counterparts, embryonic stem cells (ESCs), transit from naive pluripotency through a differentiation-primed pluripotent state. During these events, epiblast cells and ESCs are prone to death, driven by competition between Myc-high cells (winners) and Myc-low cells (losers). Using live tracking of Myc levels, we show that Myc-high ESCs approach the naive pluripotency state, whereas Myc-low ESCs are closer to the differentiation-primed state. In ESC colonies, naive cells eliminate differentiating cells by cell competition, which is determined by a limitation in the time losers are able to survive persistent contact with winners. In the mouse embryo, cell competition promotes pluripotency maintenance by elimination of primed lineages before gastrulation. The mechanism described here is relevant to mammalian embryo development and induced pluripotency. We thank B. Sleckman for the GFP-Myc mice, the CNIC Microscopy unit for the live confocal analysis, the CNIC Genomics and Bioinformatics units for the RNA-seq analysis, J.C. Alonso for video editing, and M. Manzanares, J.A. Enrı´quez, and members of the Torres lab for helpful comments on the manuscript. This work was supported by grants BFU2015-71519-P and RD16/0011/0019 (ISCIII) from the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) and grant P2010/BMD-2315 from the Madrid Regional Government to M.T., and grants (BIO2014-62200-EXP) and (PIFIS-2016) from MEIC to M.C.M. C.D.D. was supported by an FPU grant from the Spanish Ministry of Education, Culture and Sports. The CNIC is supported by the Spanish MEIC and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505). Sí