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Research in R.B.’s laboratory was supported by the European Research Council Starting Grant AngioGenesHD (638028) and Consolidator Grant AngioUnrestUHD (101001814), the CNIC Intramural Grant Program Severo Ochoa (11-2016-IGP-SEV-2015-0505), the Ministerio de Ciencia e Innovación (MCIN) (SAF2013-44329-P, RYC-2013- 13209, and SAF2017-89299-P) and ‘La Caixa’ Banking Foundation (HR19-00120). J.V.’s laboratory was supported by MCIN (PGC2018- 097019-B-I00 and PID2021-122348NB-I00) and La Caixa (HR17-00247 and HR22-00253). K.G.’s laboratory was supported by Knut and Alice Wallenberg Foundation (2020.0057) and Vetenskapsrådet (2021-04896). The CNIC is supported by Instituto de Salud Carlos III, MCIN, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MCIN/ AEI/10.13039/501100011033). Microscopy experiments were performed at the Microscopy and Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033 and FEDER ‘Una manera de hacer Europa’ (ICTS-2018-04-CNIC-16). M.F.-C. was supported by PhD fellowships from La Caixa (CX_E-2015-01) and Boehringer Ingelheim travel grants. S.M. was supported by the Austrian Science Fund (J4358). A.R. was supported by the Youth Employment Initiative (PEJD-2019-PRE/BMD-16990). L.G.-O. was supported by the Spanish Ministry of Economy and Competitiveness (PRE2018-085283). We thank S. Bartlett (CNIC) for English editing, as well as the members of the Transgenesis, Microscopy, Genomics, Citometry and Bioinformatic units at CNIC. We also thank F. Radtke (Swiss Institute for Experimental Cancer Research), R. H. Adams (Max Planck Institute for Molecular Biomedicine), F. Alt (Boston Children’s Hospital, Harvard Medical School), T. Honjo (Kyoto University Institute for Advanced Studies), I. Flores (CNIC), J. Lewis (Cancer Research UK London Research Institute), S. Habu (Tokai University School of Medicine), T. Gridley (Maine Health Institute for Research) and C. Brakebusch (Biotech Research and Innovation Centre) for sharing the Dll4floxed, Notch1floxed, Notch2floxed, Cdh5(PAC)-creERT2, Myc floxed, Rbpj floxed, p21−/−, Jag1floxed, Dll1floxed, Jag2floxed and Rac1floxed mice. Sí

The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers. This study was supported by grants PID2019-104776RB-I00 and PID2020-120326RB-I00, CB16/11/00399 (CIBER CV) financed by MCIN/AEI/10.13039/501100011033, a grant from the Fundación BBVA (Ref. BIO14_298), and a grant from Fundació La Marató de TV3 (Ref. 20153431) to J.L.d.l.P. M.S.-A. was supported by a PhD contract from the Severo Ochoa Predoctor-al Program (SVP-2014-068723) of the MCIN/AEI/10.13039/501100011033. J.R.G.-B. was supported by SEC/FEC-INV-BAS 21/021. A.R. was funded by grants from MCIN (PID2021123925OB-I00), TerCel (RD16/0011/0024), AGAUR (2017-SGR-899), and Fundació La Marató de TV3 (201534-30). J.M.P.-P. was supported by RTI2018-095410-B-I00 (MCIN) and PY2000443 (Junta de Andalucía). B.I. was supported by the European Commission (H2020-HEALTH grant No. 945118) and by MCIN (PID2019-107332RB-I00). DO’R was sup-ported by the Medical Research Council (MC-A658-5QEB0) and KAMcG by the British Heart Foundation (RG/19/6/34387, RE/18/4/34215). The cost of this publication was supported in part with funds from the European Regional Devel-opment Fund. The Centro Nacional de Investigaciones Cardiovasculares is sup-ported by the ISCIII, the MCIN, and the Pro Centro Nacional de Investigaciones Cardiovasculares Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020001041-S) financed by MCIN/AEI/10.13039/501100011033. For the purpose of open access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising. Sí

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

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).

During cardiac development, cells from the precardiac mesoderm fuse to form the primordial heart tube, which then grows by addition of further progenitors to the venous and arterial poles. In the zebrafish, wilms tumor 1 transcription factor a (wt1a) and b (wt1b) are expressed in the pericardial mesoderm at the venous pole of the forming heart tube. The pericardial mesoderm forms a single layered mesothelial sheet that contributes to further the growth of the myocardium, and forms the proepicardium. Proepicardial cells are subsequently transferred to the myocardial surface and give rise to the epicardium, the outer layer covering the myocardium in the adult heart. wt1a/b expression is downregulated during the transition from pericardium to myocardium, but remains high in proepicardial cells. Here we show that sustained wt1 expression impaired cardiomyocyte maturation including sarcomere assembly, ultimately affecting heart morphology and cardiac function. ATAC-seq data analysis of cardiomyocytes overexpressing wt1 revealed that chromatin regions associated with myocardial differentiation genes remain closed upon wt1b overexpression in cardiomyocytes, suggesting that wt1 represses a myocardial differentiation program. Indeed, a subset of wt1a/b-expressing cardiomyocytes changed their cell adhesion properties, delaminated from the myocardial epithelium, and upregulated the expression of epicardial genes, as confirmed by in vivo imaging. Thus, we conclude that wt1 acts as a break for cardiomyocyte differentiation by repressing chromatin opening at specific genomic loci and that sustained ectopic expression of wt1 in cardiomyocytes can lead to their transformation into epicardial cells.

Here we explored the role of interleukin-1β (IL-1β) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1β monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1β/IL-1rn levels under steady-state, and that loss of repression of IL-1β signaling may underlie pre-leukemic lesion and AML progression.

Pluripotency is regulated by a network of transcription factors that maintain early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development is its expression restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog. Use of Nanog null ES cells further demonstrated a role for Nanog in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting neural regulatory networks by repressing Pou3f1 at the onset of gastrulation. This article has an associated First Person interview with the joint first authors of the paper., Summary: Nanog is a key factor for pluripotency, but we show that it has further roles during gastrulation by regulating lineage determinants such as Pou3f1.

The development of single cell transcriptome sequencing has allowed researchers the possibility to dig inside the role of the individual cell types in a plethora of disease scenarios. It also expands to the whole transcriptome what before was only possible for a few tenths of antibodies in cell population analysis. More importantly, it allows resolving the permanent question of whether the changes observed in a particular bulk experiment are a consequence of changes in cell type proportions or an aberrant behavior of a particular cell type. However, single cell experiments are still complex to perform and expensive to sequence making bulk RNA-Seq experiments yet more common. scRNA-Seq data is proving highly relevant information for the characterization of the immune cell repertoire in different diseases ranging from cancer to atherosclerosis. In particular, as scRNA-Seq becomes more widely used, new types of immune cell populations emerge and their role in the genesis and evolution of the disease opens new avenues for personalized immune therapies. Immunotherapy have already proven successful in a variety of tumors such as breast, colon and melanoma and its value in other types of disease is being currently explored. From a statistical perspective, single-cell data are particularly interesting due to its high dimensionality, overcoming the limitations of the "skinny matrix" that traditional bulk RNA-Seq experiments yield. With the technological advances that enable sequencing hundreds of thousands of cells, scRNA-Seq data have become especially suitable for the application of Machine Learning algorithms such as Deep Learning (DL). We present here a DL based method to enumerate and quantify the immune infiltration in colorectal and breast cancer bulk RNA-Seq samples starting from scRNA-Seq. Our method makes use of a Deep Neural Network (DNN) model that allows quantification not only of lymphocytes as a general population but also of specific CD8+, CD4Tmem, CD4Th and CD4Tregs subpopulations, as well as B-cells and Stromal content. Moreover, the signatures are built from scRNA-Seq data from the tumor, preserving the specific characteristics of the tumor microenvironment as opposite to other approaches in which cells were isolated from blood. Our method was applied to synthetic bulk RNA-Seq and to samples from the TCGA project yielding very accurate results in terms of quantification and survival prediction. This work was supported by the European Union’s Horizon 2020 research and innovation program under grant agreement number 633592 (Project APERIM: Advanced bioinformatics platform for personalized cancer immunotherapy). We thank Francesca Finotello and Zlatko Trajanoski for fruitful discussions and to the CNIC Bioinformatics Unit members for continuous support and work. The CNIC is supported by MEIC and the ProCNIC Foundation, and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505). Sí

Pluripotency is regulated by a network of transcription factors that maintains early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development its expression is restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses the anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog. Use of Nanog null ES cells further demonstrated a role for Nanog in repressing anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting the neural fate by repressing Pou3f1 at the onset of gastrulation.

© The Author(s) 2019., Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC. iTRAQ analysis confirmed differential expression of a substantial proportion of those proteins in CPC relative to MSC, and systems biology analysis defined a clear overrepresentation of several categories related to enhanced angiogenic potential. The CPC plasma membrane compartment comprised 1,595 proteins, including a minimal signature of 167 proteins preferentially or exclusively expressed by CPC. CDH5 (VE-cadherin), OX2G (OX-2 membrane glycoprotein; CD200), GPR4 (G protein-coupled receptor 4), CACNG7 (calcium voltage-gated channel auxiliary subunit gamma 7) and F11R (F11 receptor; junctional adhesion molecule A; JAM-A; CD321) were selected for validation. Their differential expression was confirmed both in expanded CPC batches and in early stages of isolation, particularly when compared against cardiac fibroblasts. Among them, GPR4 demonstrated the highest discrimination capacity between all cell lineages analyzed., This study was supported by funding from the European Commission (HEALTH-2009_242038), and by grants to AB from the Spanish Ministry of Science and Innovation (SAF2012-34327; SAF2015-70882-R), the Research Program of the Comunidad Autónoma de Madrid (S2011/BMD-2420), the Instituto de Salud Carlos III (RETICS-RD12/0019/0018), and grants from the Portuguese Foundation for Science and Technology (PTDC/ BBB-BIO/1414) to PMA. iNOVA4Health - UID/Multi/04462/2013, fnancially supported by FCT/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement is also acknowledged. JL Abad, I Palacios and LR Borlado were employees of Coretherapix; Coretherapix is part of Tigenix Group since July 2015.

The Myc family of oncogenic transcription factors regulates myriad cellular functions. Myc proteins contain a basic region/helix‐loop‐helix/leucine zipper domain that mediates DNA binding and heterodimerization with its partner Max. Among the Myc proteins, c‐Myc is the most widely expressed and relevant in primary B lymphocytes. There is evidence suggesting that c‐Myc can perform some of its functions in the absence of Max in different cellular contexts. However, the functional in vivo interplay between c‐Myc and Max during B lymphocyte differentiation is not well understood. Using in vivo and ex vivo models, we show that while c‐Myc requires Max in primary B lymphocytes, several key biological processes, such as cell differentiation and DNA replication, can initially progress without the formation of c‐Myc/Max heterodimers. We also describe that B lymphocytes lacking Myc, Max, or both show upregulation of signaling pathways associated with the B‐cell receptor. These data suggest that c‐Myc/Max heterodimers are not essential for the initiation of a subset of important biological processes in B lymphocytes, but are required for fine‐tuning the initial response after activation.

Álvarez-Prado et al. report a detailed map of AID-induced off-target mutations and identify molecular features that predict gene mutability. They identify a novel AID hotspot and demonstrate that base excision and mismatch repair back up each other to repair most AID deamination events., Activation-induced deaminase (AID) initiates antibody diversification in germinal center (GC) B cells through the deamination of cytosines on immunoglobulin genes. AID can also target other regions in the genome, triggering mutations or chromosome translocations, with major implications for oncogenic transformation. However, understanding the specificity of AID has proved extremely challenging. We have sequenced at very high depth >1,500 genomic regions from GC B cells and identified 275 genes targeted by AID, including 30 of the previously known 35 AID targets. We have also identified the most highly mutated hotspot for AID activity described to date. Furthermore, integrative analysis of the molecular features of mutated genes coupled to machine learning has produced a powerful predictive tool for AID targets. We also have found that base excision repair and mismatch repair back up each other to faithfully repair AID-induced lesions. Finally, our data establish a novel link between AID mutagenic activity and lymphomagenesis.

Heart failure (HF) is a major health and economic burden in developed countries. It has been proposed that the pathogenesis of HF may involve the action of mitochondria. We evaluate three different mouse models of HF: tachycardiomyopathy, HF with preserved left ventricular (LV) ejection fraction (LVEF), and LV myocardial ischemia and hypertrophy. Regardless of whether LVEF is preserved, our results indicate that the three models share common features: an increase in mitochondrial reactive oxygen species followed by ultrastructural alterations in the mitochondrial cristae and loss of mitochondrial integrity that lead to cardiomyocyte death. We show that the ablation of the mitochondrial protease OMA1 averts cardiomyocyte death in all three murine HF models, and thus loss of OMA1 plays a direct role in cardiomyocyte protection. This finding identifies OMA1 as a potential target for preventing the progression of myocardial damage in HF associated with a variety of etiologies.

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).

Left ventricular noncompaction (LVNC) causes prominent ventricular trabeculations and reduces cardiac systolic function. The clinical presentation of LVNC ranges from asymptomatic to heart failure. We show that germline mutations in human MIB1 (mindbomb homolog 1), which encodes an E3 ubiquitin ligase that promotes endocytosis of the NOTCH ligands DELTA and JAGGED, cause LVNC in autosomal-dominant pedigrees, with affected individuals showing reduced NOTCH1 activity and reduced expression of target genes. Functional studies in cells and zebrafish embryos and in silico modeling indicate that MIB1 functions as a dimer, which is disrupted by the human mutations. Targeted inactivation of Mib1 in mouse myocardium causes LVNC, a phenotype mimicked by inactivation of myocardial Jagged1 or endocardial Notch1. Myocardial Mib1 mutants show reduced ventricular Notch1 activity, expansion of compact myocardium to proliferative, immature trabeculae and abnormal expression of cardiac development and disease genes. These results implicate NOTCH signaling in LVNC and indicate that MIB1 mutations arrest chamber myocardium development, preventing trabecular maturation and compaction. 28.054 JCR (2013) Q1, 2/291 Biochemistry & molecular biology, 3/185 Cell biology, 1/122 Medicine, research & experimental

Embryonic pluripotency in the mouse is established and maintained by a gene-regulatory network under the control of a core set of transcription factors that include octamer-binding protein 4 (Oct4; official name POU domain, class 5, transcription factor 1, Pou5f1), sex-determining region Y (SRY)-box containing gene 2 (Sox2), and homeobox protein Nanog. Although this network is largely conserved in eutherian mammals, very little information is available regarding its evolutionary conservation in other vertebrates. We have compared the embryonic pluripotency networks in mouse and chick by means of expression analysis in the pregastrulation chicken embryo, genomic comparisons, and functional assays of pluripotency-related regulatory elements in ES cells and blastocysts. We find that multiple components of the network are either novel to mammals or have acquired novel expression domains in early developmental stages of the mouse. We also find that the downstream action of the mouse core pluripotency factors is mediated largely by genomic sequence elements nonconserved with chick. In the case of Sox2 and Fgf4 , we find that elements driving expression in embryonic pluripotent cells have evolved by a small number of nucleotide changes that create novel binding sites for core factors. Our results show that the network in charge of embryonic pluripotency is an evolutionary novelty of mammals that is related to the comparatively extended period during which mammalian embryonic cells need to be maintained in an undetermined state before engaging in early differentiation events.

Human mitochondrial DNA (mtDNA) shows extensive within-population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic, metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation, insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.

Ventricular chambers are essential for the rhythmic contraction and relaxation occurring in every heartbeat throughout life. Congenital abnormalities in ventricular chamber formation cause severe human heart defects. How the early trabecular meshwork of myocardial fibres forms and subsequently develops into mature chambers is poorly understood. We show that Notch signalling first connects chamber endocardium and myocardium to sustain trabeculation, and later coordinates ventricular patterning and compaction with coronary vessel development to generate the mature chamber, through a temporal sequence of ligand signalling determined by the glycosyltransferase manic fringe (MFng). Early endocardial expression of MFng promotes Dll4-Notch1 signalling, which induces trabeculation in the developing ventricle. Ventricular maturation and compaction require MFng and Dll4 downregulation in the endocardium, which allows myocardial Jag1 and Jag2 signalling to Notch1 in this tissue. Perturbation of this signalling equilibrium severely disrupts heart chamber formation. Our results open a new research avenue into the pathogenesis of cardiomyopathies.

et al., Microphthalmos is a rare congenital anomaly characterized by reduced eye size and visual deficits of variable degree. Sporadic and hereditary microphthalmos have been associated with heterozygous mutations in genes fundamental for eye development. Yet,many cases are idiopathic or await the identification of molecular causes. Here we show that haploinsufficiency of Meis1, which encodes a transcription factor with evolutionarily conserved expression in the embryonic trunk, brain and sensory organs, including the eye, causes microphthalmic traits and visual impairment in adultmice. By combining analysis of Meis1 loss-offunction and conditional Meis1 functional rescue with ChIP-seq and RNA-seq approaches we show that, in contrast to its preferential association with Hox-Pbx BSs in the trunk, Meis1 binds to Hox/Pbxindependent sites during optic cup development. In the eye primordium, Meis1 coordinates, in a dose-dependent manner, retinal proliferation and differentiation by regulating genes responsible for human microphthalmia and components of the Notch signaling pathway. In addition, Meis1 is required for eye patterning by controlling a set of eye territory-specific transcription factors, so that in Meis1−/− embryos boundaries among the different eye territories are shifted or blurred.We propose that Meis1 is at the core of a genetic network implicated in eye patterning/microphthalmia, and represents an additional candidate for syndromic cases of these ocular malformations., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2010-16031; BFU2013-43213-P, supported by FEDER Funds], the CIBERER, Instituto de Salud Carlos III (ISCIII) to P.B. and an Institutional Grant from the Fundación Ramon Areces; by MINECO [BFU2012-31086] and ISCIII [RD12/0019/0005] to M.T.; by Comunidad Autonoma de Madrid (CAM) [S2010/BMD-2315] to P.B. and M.T.; by MINECO [BFU2013-41322-P] and Andalusian Government [BIO-396] to J.L.G.-S.; and by MINECO [BFU2014-55738-REDT] to P.B., J.L.G.-S. and F.C. M.G.-L. was supported by a grant from the CONACyT. O.B. is supported by an Australian Research Council Discovery Early Career Researcher Award-DECRA [DE140101962]. The CNIC is supported by MINECO and the Pro-CNIC Foundation.

J.B.S. Haldane proposed in 1947 that the male germline may be more mutagenic than the female germline. Diverse studies have supported Haldane's contention of a higher average mutation rate in the male germline in a variety of mammals, including humans. Here we present, to our knowledge, the first direct comparative analysis of male and female germline mutation rates from the complete genome sequences of two parent-offspring trios. Through extensive validation, we identified 49 and 35 germline de novo mutations (DNMs) in two trio offspring, as well as 1,586 non-germline DNMs arising either somatically or in the cell lines from which the DNA was derived. Most strikingly, in one family, we observed that 92% of germline DNMs were from the paternal germline, whereas, in contrast, in the other family, 64% of DNMs were from the maternal germline. These observations suggest considerable variation in mutation rates within and between families.

Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin− MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin+ cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP+ Pdgfrα− cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation. DOI: http://dx.doi.org/10.7554/eLife.03696.001, eLife digest During the earliest phases of development, the embryo is formed by groups of stem cells that can develop into all the different types of tissue in the body—from bones to brain tissue. Later in life, small stockpiles of adult stem cells are found in various tissues and provide a reservoir of new cells available for replacing old or damaged cells. The most important source of blood stem cells is the bone marrow, which produces and stores cells that are capable of developing into blood and immune system cells. These processes are assisted by different bone marrow cells called stromal cells, which create a specialized local environment or ‘niche’. But are the stromal stem cells that form the skeleton the same ones that form this niche during development? Or do the various types of stromal stem cells develop from distinct groups of cells in the embryo? Furthermore, it is unclear which cells guide blood stem cells towards the forming bones. Other types of cells, including some of the cells of the nervous system, can communicate with the stem cells in the adult marrow and influence their behavior. This led scientists to wonder whether the stem cells in the bone marrow niche and the cells that communicate with them developed from the same type of embryonic stem cell. Isern et al. tracked down the developmental origins of different types of bone marrow stromal stem cells by examining the bone marrow from the long bones (for example, the bones in the leg) of unborn and infant mice. It turns out that not all stromal stem cells in the developing bone marrow are alike. In fact, one pool of stromal stem cells forms the skeleton and loses stem cell activity in the process. In contrast, a different population of stromal stem cells develops from the same group of embryonic cells that gives rise to the cells of the nervous system. The stromal stem cells in this second group function as a niche to recruit and store the incoming blood stem cells and retain their stem cell activity throughout life. The findings of Isern et al. help to explain why the nervous system is able to communicate with stem cells in the adult marrow, and provide a model for understanding how stem cell niches in organs that contain nerve tissue are established. DOI: http://dx.doi.org/10.7554/eLife.03696.002

Alternative splicing is the main mechanism governing protein diversity. The recent developments in RNA-Seq technology have enabled the study of the global impact and regulation of this biological process. However, the lack of standardized protocols constitutes a major bottleneck in the analysis of alternative splicing. This is particularly important for the identification of exon-exon junctions, which is a critical step in any analysis workflow. Here we performed a systematic benchmarking of alignment tools to dissect the impact of design and method on the mapping, detection and quantification of splice junctions from multi-exon reads. Accordingly, we devised a novel pipeline based on TopHat2 combined with a splice junction detection algorithm, which we have named FineSplice. FineSplice allows effective elimination of spurious junction hits arising from artefactual alignments, achieving up to 99% precision in both real and simulated data sets and yielding superior F1 scores under most tested conditions. The proposed strategy conjugates an efficient mapping solution with a semi-supervised anomaly detection scheme to filter out false positives and allows reliable estimation of expressed junctions from the alignment output. Ultimately this provides more accurate information to identify meaningful splicing patterns. FineSplice is freely available at https://sourceforge.net/p/finesplice/. European Union’s FP7 [ERG-239158 to E.L.-P., CardioNeT-ITN-289600 to E.L.-P., S.A.C. and P.J.R.B.]; Spanish Ministry of Science and Innovation [BFU2009-10016, SAF2012-31451 to E.L.-P.]; Regional Government of Madrid [2010-BMD-2321 ‘Fibroteam’ to E.L.-P.]; Spanish Ministry of Economy and Competitiveness and by the Pro-CNIC Foundation (towards CNIC); National Institute for Health Research Cardiovascular BRU at the Royal Brompton & Harefield NHS Foundation Trust and Imperial College London (to S.A.C. and P.J.B.). Funding for open access charge: European Union’s FP7 [ERG-239158, CardioNeT-ITN289600 to E.L.-P.] Sí

The Hedgehog (Hh) family of signaling molecules function as organizers in many morphogenetic processes. Hh signaling requires cholesterol in both signal-generating and -receiving cells, and it requires the tumor suppressor Patched (Ptc) in receiving cells in which it plays a negative role. Ptc both blocks the Hh pathway and limits the spread of Hh. Sequence analysis suggests that it has 12 transmembrane segments, 5 of which are homologous to a conserved region that has been identified in several proteins involved in cholesterol homeostasis and has been designated the sterol-sensing domain (SSD). In the present study, we show that a Ptc mutant with a single amino acid substitution in the SSD induces target gene activation in a ligand-independent manner. This mutant PtcSSD protein shows dominant-negative activity in blocking Hh signaling by preventing the downregulation of Smoothened (Smo), a positive effector of the Hh pathway. Despite its dominant-negative activity, the mutant Ptc protein functioned like the wild-type protein in sequestering and internalizing Hh. In addition, we show that PtcSSD preferentially accumulates in endosomes of the endocytic compartment. All these results suggest a role of the SSD of Ptc in mediating the vesicular trafficking of Ptc to regulate Smo activity.

Nature 535, 561–565 (2016); doi:10.1038/nature18618 In this Letter, the number of mice monitored in Fig. 1a should have stated: ‘n = 31 BL/6C57 and n = 29 BL/6NZB’, rather than: ‘n = 31 per genotype (19 males, 12 females)’. In addition, in Extended Data Table 1, the non-synonymous mutation at nucleotide position 9985 (mt-Nd4l) was inadvertently omitted.

Zebrafish (Danio rerio) is a popular vertebrate model organism largely deployed using outbred laboratory animals. The nonisogenic nature of the zebrafish as a model system offers the opportunity to understand natural variations and their effect in modulating phenotype. In an effort to better characterize the range of natural variation in this model system and to complement the zebrafish reference genome project, the whole genome sequence of a wild zebrafish at 39-fold genome coverage was determined. Comparative analysis with the zebrafish reference genome revealed approximately 5.2 million single nucleotide variations and over 1.6 million insertion–deletion variations. This dataset thus represents a new catalog of genetic variations in the zebrafish genome. Further analysis revealed selective enrichment for variations in genes involved in immune function and response to the environment, suggesting genome-level adaptations to environmental niches. We also show that human disease gene orthologs in the sequenced wild zebrafish genome show a lower ratio of nonsynonymous to synonymous single nucleotide variations.

The 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. F.B. was supported by grants from the AIRC (Associazione Italiana Ricerche sul Cancro, 8929), Ministero dell’Universita e Ricerca (MERIT; EU FP7 Prepobedia, MIUR-FIRB RBNE08NKH7), the Cariplo Foundation, and the Italian Ministry of Health. M.T. was supported by grants RD06/0010/0008 and BFU2009-08331/BMC from the Spanish Ministerio de Economia y Competitividad (MINECO). D.M.S.M. was supported by a fellowship from the Consejerı´a de Educacio´ n de la Comunidad de Madrid and the European Social Fund. D.P. and V.T. were supported by grant 02.740.11.0872 from the Russian Ministry of Education and Science. D.P. was supported by grant 12-04-01659-a from the Russian Foundation for Basic Research. A.B. was supported by Associazione Italiana Ricerca sul Cancro start-up grant #4780. The CNIC is supported by the MINECO and the pro-CNIC Foundation. IFOM is supported by the FIRC (Fondazione Italiana Ricerche sul Cancro). Sí

Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. Here, we have investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. We describe that Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a longrange gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Iglike and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. We also show that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium., This work was supported by Grants BFU2005-04183 and BFU2008-03320/BMC and by Consolider Program CDS 2007-00008 from the Spanish MICINN, by Marie Curie RTN FP6 (RTN 035528-2) and FP7 (ITN 238186) projects, and by an institutional grant from the Fundación Areces to I.G. It was also financially supported by fellowships awarded by the Junta para la Ampliación de Estudios-Consejo Superior de Investigaciones Cientificas program (to N.G. and A.C.), a Juan de la Cierva fellowship from the Spanish MICINN (to A.B.), a Marie Curie RTN 035528-2 FP6 contract (to E.M.), a contract from the Spanish MICINN (to L.D.), and the senior researcher Programa Amarouto from Severo Ochoa Fondation program of the Comunidad Autónoma de Madrid (G.A.)

The activities of the Hedgehog (Hh) protein family are central to the growth and patterning of developing tissues and organs in many different organisms. Hh proteins are secreted ligands synthesized in discrete regions. The receptor of Hh is Patched (Ptc) and it is expressed in the cells close to the source of Hh. Ptc binds the ligand and transduces a signal which is modulated depending on the context and the concentration of Hh received. Hh and several molecular components of the pathway were first identified and characterized in Drosophila, providing relevant milestones to our understanding on how the Hh signal is transduced. However, important gaps in the pathway still need to be elucidated. Some of these gaps converge on the Ptc receptor and its intriguing mechanisms of Hh reception and signal transduction. Mutations of Ptc that prevail both in animal and human populations are giving some clues on crucial aspects of its function. Patients bearing mutated forms of Ptc suffer a variety of serious diseases. Molecular and cellular studies in Drosophila have given us a clue of the function of Ptc receptor such as the normal topology and/or sorting of the receptor. Thus, a widened knowledge of the function of Ptc might help to design specific therapies for these disorders. This chapter focuses on recent advances that shed some light on how Ptc may operate in the cell.

The Hedgehog (Hh) family of morphogenetic proteins has important instructional roles in metazoan development. Despite Hh being modified by Ct-cholesterol and Nt-palmitate adducts, Hh migrates far from its site of synthesis and programs cellular outcomes, depending on its local concentrations. We show that in the receiving cells of the Drosophilawing imaginal disc, lipid-unmodified Hh spreads across many more cell diameters than the wild type and this spreading leads to the activation of low but not high threshold responses. Unlipidated Hh forms become internalized through the apical plasma membrane, while wild-type Hh enters through the basolateral cell surface - in all cases via a dynamin-dependent mechanism. Full activation of the Hh pathway and the spread of Hh throughout the extracellular matrix depend on the ability of lipid-modified Hh to interact with heparan sulfate proteoglycans (HSPG). However, neither Hh-lipid modifications nor HSPG function are required to activate the targets that respond to low levels of Hh. All these data show that the interaction of lipid-modified Hh with HSPG is important both for precise Hh spreading through the epithelium surface and for correct Hh reception.

Morphogens are molecules that spread from localized sites of production, specifying distinct cell outcomes at different concentrations. Members of the Hedgehog (Hh) family of signaling molecules act as morphogens in different developmental systems. If we are to understand how Hh elicits multiple responses in a temporally and spatially specific manner, the molecular mechanism of Hh gradient formation needs to be established. Moreover, understanding the mechanisms of Hh signaling is a central issue in biology, not only because of the role of Hh in morphogenesis, but also because of its involvement in a wide range of human diseases. Here, we review the mechanisms affecting the dynamics of Hh gradient formation, mostly in the context of Drosophila wing development, although parallel findings in vertebrate systems are also discussed. © 2005 Wiley Periodicals, Inc., I3P contract awarded by the CSIC

The Hedgehog (Hh) morphogenetic gradient controls multiple developmental patterning events in Drosophila and vertebrates. Patched (Ptc), the Hh receptor, restrains both Hh spreading and Hh signaling. We report how endocytosis regulates the concentration and activity of Hh in the wing imaginal disc. Our studies show that Ptc limits the Hh gradient by internalizing Hh through endosomes in a dynamin-dependent manner, and that both Hh and Ptc are targeted to lysosomal degradation. We also found that the ptc14 mutant does not block Hh spreading, as it has a failure in endocytosis. However, this mutant protein is able to control the expression of Hh target genes as the wild-type protein, indicating that the internalization mediated by Ptc is not required for signal transduction. In addition, we noted that both in this mutant and in those not producing Ptc protein, Hh still occurred in the endocytic vesicles of Hh-receiving cells, suggesting the existence of a second, Ptc independent, mechanism of Hh internalization, Spanish MCYT (BMC2002-03839); Comunidad Autónoma de Madrid (08.6/0045/2001.2); Fundación Areces.; I3P fellowship awarded by the CSIC

Nature 496, 498–503 (2013); doi:10.1038/nature12111 In this Letter, five authors were inadvertently omitted: Sharmin Begum and Christine Lloyd from the Wellcome Trust Sanger Institute, and Christa Lanz, Gunter Raddatz and Stephan C. Schuster from the Max Planck Institute for Developmental Biology. David Elliot was incorrectly listed as David Eliot, Beverley Mortimore was incorrectly listed as Beverly Mortimer, and James D.