Remodeling of the endothelial cell transcriptional program via paracrine and DNA-binding activities of MPO.

Myeloperoxidase (MPO) is an enzyme that functions in host defense. MPO is released into the vascular lumen by neutrophils during inflammation and may adhere and subsequently penetrate endothelial cells (ECs) coating vascular walls. We show that MPO enters the nucleus of ECs and binds chromatin independently of its enzymatic activity. MPO drives chromatin decondensation at its binding sites and enhances condensation at neighboring regions. It binds loci relevant for endothelial-to-mesenchymal transition (EndMT) and affects the migratory potential of ECs. Finally, MPO interacts with the RNA-binding factor ILF3 thereby affecting its relative abundance between cytoplasm and nucleus. This interaction leads to change in stability of ILF3-bound transcripts. MPO-knockout mice exhibit reduced number of ECs at scar sites following myocardial infarction, indicating reduced neovascularization. In summary, we describe a non-enzymatic role for MPO in coordinating EndMT and controlling the fate of endothelial cells through direct chromatin binding and association with co-factors.

The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo.

Poised enhancers (PEs) represent a genetically distinct set of distal regulatory elements that control the expression of major developmental genes. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally characterized in embryonic stem cells (ESC), it is currently unknown whether PEs are functionally conserved in vivo. Here, we show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that the interactions between PEs and their target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation within specific vertebrate clades, with only a few being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential roles during the induction of major developmental genes in vivo.

Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology.

Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance.

MAPRE2 mutations result in altered human cranial neural crest migration, underlying craniofacial malformations in CSC-KT syndrome.

Circumferential skin creases (CSC-KT) is a rare polymalformative syndrome characterised by intellectual disability associated with skin creases on the limbs, and very characteristic craniofacial malformations. Previously, heterozygous and homozygous mutations in MAPRE2 were found to be causal for this disease. MAPRE2 encodes for a member of evolutionary conserved microtubule plus end tracking proteins, the end binding (EB) family. Unlike MAPRE1 and MAPRE3, MAPRE2 is not required for the persistent growth and stabilization of microtubules, but plays a role in other cellular processes such as mitotic progression and regulation of cell adhesion. The mutations identified in MAPRE2 all reside within the calponin homology domain, responsible to track and interact with the plus-end tip of growing microtubules, and previous data showed that altered dosage of MAPRE2 resulted in abnormal branchial arch patterning in zebrafish. In this study, we developed patient derived induced pluripotent stem cell lines for MAPRE2, together with isogenic controls, using CRISPR/Cas9 technology, and differentiated them towards neural crest cells with cranial identity. We show that changes in MAPRE2 lead to alterations in neural crest migration in vitro but also in vivo, following xenotransplantation of neural crest progenitors into developing chicken embryos. In addition, we provide evidence that changes in focal adhesion might underlie the altered cell motility of the MAPRE2 mutant cranial neural crest cells. Our data provide evidence that MAPRE2 is involved in cellular migration of cranial neural crest and offers critical insights into the mechanism underlying the craniofacial dysmorphisms and cleft palate present in CSC-KT patients. This adds the CSC-KT disorder to the growing list of neurocristopathies.

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development.

The neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1-900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.

Modeling the Pathological Long-Range Regulatory Effects of Human Structural Variation with Patient-Specific hiPSCs.

The pathological consequences of structural variants disrupting 3D genome organization can be difficult to elucidate in vivo due to differences in gene dosage sensitivity between mice and humans. This is illustrated by branchiooculofacial syndrome (BOFS), a rare congenital disorder caused by heterozygous mutations within TFAP2A, a neural crest regulator for which humans, but not mice, are haploinsufficient. Here, we present a BOFS patient carrying a heterozygous inversion with one breakpoint located within a topologically associating domain (TAD) containing enhancers essential for TFAP2A expression in human neural crest cells (hNCCs). Using patient-specific hiPSCs, we show that, although the inversion shuffles the TFAP2A hNCC enhancers with novel genes within the same TAD, this does not result in enhancer adoption. Instead, the inversion disconnects one TFAP2A allele from its cognate enhancers, leading to monoallelic and haploinsufficient TFAP2A expression in patient hNCCs. Our work illustrates the power of hiPSC differentiation to unveil long-range pathomechanisms.

yylncT Defines a Class of Divergently Transcribed lncRNAs and Safeguards the T-mediated Mesodermal Commitment of Human PSCs.

Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs are preferentially encoded from the genomic loci of key developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns in vivo in mouse and in human embryos. Signifying their developmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, which localizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncT depletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions.

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Epigenomics-Based Identification of Major Cell Identity Regulators within Heterogeneous Cell Populations.

Cellular heterogeneity within embryonic and adult tissues is involved in multiple biological and pathological processes. Here, we present a simple epigenomic strategy that allows the functional dissection of cellular heterogeneity. By integrating H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data, we demonstrate that the presence of broad H3K27me3 domains at transcriptionally active genes reflects the heterogeneous expression of major cell identity regulators. Using dorsoventral patterning of the spinal neural tube as a model, the proposed approach successfully identifies the majority of previously known dorsoventral patterning transcription factors with high sensitivity and precision. Moreover, poorly characterized patterning regulators can be similarly predicted, as shown for ZNF488, which confers p1/p2 neural progenitor identity. Finally, we show that, as our strategy is based on universal chromatin features, it can be used to functionally dissect cellular heterogeneity within various organisms and tissues, thus illustrating its potential applicability to a broad range of biological and pathological contexts.

Retrograde migration of pectoral girdle muscle precursors depends on CXCR4/SDF-1 signaling.

In vertebrates, muscles of the pectoral girdle connect the forelimbs with the thorax. During development, the myogenic precursor cells migrate from the somites into the limb buds. Whereas most of the myogenic precursors remain in the limb bud to form the forelimb muscles, several cells migrate back toward the trunk to give rise to the superficial pectoral girdle muscles, such as the large pectoral muscle, the latissimus dorsi and the deltoid. Recently, this developing mode has been referred to as the "In-Out" mechanism. The present study focuses on the mechanisms of the "In-Out" migration during formation of the pectoral girdle muscles. Combining in ovo electroporation, tissue slice-cultures and confocal laser scanning microscopy, we visualize live in detail the retrograde migration of myogenic precursors from the forelimb bud into the trunk region by live imaging. Furthermore, we present for the first time evidence for the involvement of the chemokine receptor CXCR4 and its ligand SDF-1 during these processes. After microsurgical implantations of CXCR4 inhibitor beads in the proximal forelimb region of chicken embryos, we demonstrate with the aid of in situ hybridization and live-cell imaging that CXCR4/SDF-1 signaling is crucial for the retrograde migration of pectoral girdle muscle precursors. Moreover, we analyzed the MyoD expression in CXCR4-mutant mouse embryos and observed a considerable decrease in pectoral girdle musculature. We thus demonstrate the importance of the CXCR4/SDF-1 axis for the pectoral girdle muscle formation in avians and mammals.

Human ß-defensin-3 correlates with muscle fibre degeneration in idiopathic inflammatory myopathies.

Sporadic inclusion body myositis (sIBM) and polymyositis (PM) are characterized by muscle inflammation, with sIBM showing additional degenerative alterations. In this study we investigated human beta defensins and associated TLRs to elucidate the role of the innate immune system in idiopathic inflammatory myopathies (IIM), and its association with inflammatory and degenerative alterations. Expression levels of human beta-defensin (HBD)-1, HBD-2, HBD-3 and TLR2, 3, 4, 7 and 9 were analysed by quantitative real-time PCR in skeletal muscle tissue. Localization of HBD-3, collagen 6, dystrophin, CD8-positive T-cells, CD-68-positive macrophages, ß-amyloid, the autophagy marker LC3, and TLR3 were detected by immunofluorescence and co-localization was quantified. HBD-3 and all TLRs except for TLR9 were overexpressed in both IIM with significant overexpression of TLR3 in sIBM. HBD-3 showed characteristic intracellular accumulations near deposits of ß-amyloid, LC3 and TLR3 in sIBM, and was detected in inflammatory infiltrations and macrophages invading necrotic muscle fibres in both IIM. The characteristic intracellular localization of HBD-3 near markers of degeneration and autophagy, and overexpression of endosomal TLR3 in sIBM hint at different pathogenetic mechanisms in sIBM compared with PM. This descriptive study serves as a first approach to the role of the innate immune system in sIBM and PM.

A novel role of CXCR4 and SDF-1 during migration of cloacal muscle precursors.

The cloaca acts as a common chamber into which gastrointestinal and urogenital tracts converge in lower vertebrates. The distal end of the cloaca is guarded by a ring of cloacal muscles or sphincters, the equivalent of perineal muscles in mammals. It has recently been shown that the development of the cloacal musculature depends on hindlimb muscle formation. The signaling molecules responsible for the outward migration of hindlimb myogenic precursors are not known. Based on the expression studies for CXCR4 and SDF-1, we hypothesized a role of this signaling pair during cloacal muscle precursor migration. The aim of our study was to investigate the role of SDF-1/CXCR4 during cloacal muscle precursor migration in the chicken embryos. We show that SDF-1 is expressed in the cloacal region, and by experimentally manipulating the SDF-1/CXCR4 signaling, we can show that SDF-1 guides the migration of CXCR4-expressing cloacal muscle precursors.

Stromal-derived factor-1 (SDF-1) expression during early chick development.

Cell migration plays a fundamental role in a wide variety of biological processes including development, tissue repair and disease. These processes depend on directed cell migration along and through cell layers. Chemokines are small secretory proteins that exert their effects by activating a family of G-protein coupled receptors and have been shown to play numerous fundamental roles in the control of physiological and pathological processes during development and in adult tissues, respectively. Stromal-derived factor-1 (SDF-1/CXCL12), a ligand of the chemokine receptor, CXCR4, is involved in providing cells with directional cues as well as in controlling their proliferation and differentiation. Here we studied the expression pattern of SDF-1 in the developing chick embryo. We could detect a specific expression of SDF-1 in the ectoderm, the sclerotome, the intersomitic spaces and the developing limbs. The expression domains of SDF-1 reflect its role in somitic precursor migration and vessel formation in the limbs.

Expression of the avian gene cNOC2 encoding nucleolar complex associated protein 2 during embryonic development.

Genetic information that directs a cell during different phases of embryogenesis is locked up in the genome. Therein is contained the road map for growth, proliferation, differentiation and morphogenesis. The cellular transportation machinery plays a major role to ensure that all the components for transcription and translation are available at the right place at the right time. Nucleolar complex associated protein2 (NOC2) has a highly conserved UPF0120 domain, and is an element involved in ribosome transportation from the nucleoplasm to the cytoplasm. However, its gene expression pattern is still unknown. We chose the developing chick embryo to investigate the possible involvement of avian NOC2 (cNOC2) in developmental processes, particularly neurogenesis and myogenesis. For this purpose, we constructed a fragment of chicken cNOC2, which contains the UPF0120 domain coding sequence, into pDrive vector, and performed in situ hybridization on chicken embryos of different stages with this gene probe. A dynamic expression pattern of cNOC2 transcripts can be seen beginning as early as from stage HH7 until stage HH32. Using in situ hybridization we could detect that cNOC2 transcripts were expressed ubiquitously, but prominent expression could be found in the neural tissue, the somites and in the developing limbs. Comparison of cNOC2 gene expression with the proliferation marker gene cPCNA, muscle specific marker genes cMyf5 and cMyoD in single or double in situ hybridisation show that cNOC2 is expressed in the myotome, similar to cMyf5 and cMyoD, but not like cPCNA, which is hardly detectable in the myotome. Our results suggest that cNOC2 is involved in the development of neural tissue, somites and limbs.

Inhibitors of CXCR4 affect the migration and fate of CXCR4+ progenitors in the developing limb of chick embryos.

Chemokines and their receptors play major roles in numerous physiological and pathological processes during development and disease. CXCR4 is the most abundantly expressed chemokine receptor during development. In contrast to other chemokine receptors, CXCR4 binds and is activated exclusively by its ligand stromal derived factor-1 (SDF-1) or CXCL12. SDF-1 signaling has a wide range of effects on CXCR4-expressing cells depending on the cell type ranging from cell growth to adhesion, chemotaxis, and migration. CXCR4 also serves as a co-receptor for HIV-1 entry into T-cells and has been implicated in the pathogenesis of rheumatoid arthritis and cancer growth and invasion. Numerous inhibitors and antagonists of CXCR4 have been produced and are being tested for their efficiency to target its role in pathogenesis. Our initial expression analysis revealed that CXCR4 is expressed by the migrating myogenic and angiogenic precursors in the developing chick limb. In this study, we used the most specific peptidic inhibitors of CXCR4, T140 and its analog TN14003, to analyse the effect of blocking CXCR4/SDF-1 signaling on the undetermined bioptent migratory progenitors in the developing chick limb. Our results point to defects in migration and an altered differentiation program of these CXCR4-expressing progenitor pool in the limb.

Expression of chemokine receptor CXCR4 during chick embryo development.

The chemokine receptor CXCR4 plays a decisive role in physiological cell migration both in developmental processes and adult tissues; it has also been implicated in metastasis formation of different human cancers (Balkwill 2004) and in HIV pathogenesis (Murdoch 2000). Here we present the expression pattern of this important chemokine receptor CXCR4 in the chick embryo. A dynamic expression pattern can be detected beginning as early as the gastrulation stages until the observed stage of HH28. During gastrulation, expression was observed in the epiblast at the level of the primitive streak and in the endoderm. Later, expression was noticeable in the ventral foregut portal, developing somites, tail bud, neural tube, the intermediate mesoderm, Wolffian duct, the lateral plate mesoderm and the developing blood vessels. Our descriptive data suggest a role for CXCR4 in gastrulation and other morphogenetic events connected with angiogenesis and kidney development.