Odd skipped-related 1 controls the pro-regenerative response of fibro-adipogenic progenitors

Skeletal muscle regeneration requires the coordinated interplay of diverse tissue-resident- and infiltrating cells. Fibro-adipogenic progenitors (FAPs) are an interstitial cell population that provides a beneficial microenvironment for muscle stem cells (MuSCs) during muscle regeneration. Here we show that the transcription factor Osr1 is essential for FAPs to communicate with MuSCs and infiltrating macrophages, thus coordinating muscle regeneration. Conditional inactivation of Osr1 impaired muscle regeneration with reduced myofiber growth and formation of excessive fibrotic tissue with reduced stiffness. Osr1-deficient FAPs acquired a fibrogenic identity with altered matrix secretion and cytokine expression resulting in impaired MuSC viability, expansion and differentiation. Immune cell profiling suggested a novel role for Osr1-FAPs in macrophage polarization. In vitro analysis suggested that increased TGFβ signaling and altered matrix deposition by Osr1-deficient FAPs actively suppressed regenerative myogenesis. In conclusion, we show that Osr1 is central to FAP function orchestrating key regenerative events such as inflammation, matrix secretion and myogenesis

Local retinoic acid signaling directs emergence of the extraocular muscle functional unit

Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments

Die Rolle des Bindegewebs-spezifischen Transkriptionsfaktors Odd skipped- related (Osr1) in der Musterbildung der Muskulatur

Contents Acknowledgements 1 Abstract 2 Zusammenfassung 3 Résumé 4 Introduction 4.1 Embryological origins of skeletal muscles 4.1.1 Genetic networks controlling myogenesis 4.1.2 Generic program 4.2 Genetic cascades underlying skeletal muscle specification differ depending on muscle position in the embryo 4.2.1 Head Muscles 4.2.2 Axial Muscles 4.2.3 Limb muscles 4.3 Extrinsic signals regulating axial muscle formation 4.4 Limb muscle patterning 4.4.1 Migration 4.4.2 Establishment of the ventral and dorsal muscle masses 4.4.3 Limb muscle splitting 4.5 Lateral plate mesoderm derived tissues and their roles in muscle patterning 4.5.1 Cartilage and muscle interactions 4.5.2 Tendon and muscle interactions 4.5.3 Muscle connective tissue and muscle interactions 4.6 Odd-skipped related gene 1 (Osr1) 4.7 Chemokines signaling molecules orchestrate tissue formation 4.8 Extracellular matrix involvement in myogenesis 4.9 Aims of the study 5 Material 5.1 Instruments 5.2 Chemicals 5.3 Buffers 5.4 Kits 5.5 Plasmids 5.6 Antibodies 5.7 Bacteria 5.8 Primer 5.9 Imaging software 5.10 Other software 5.11 Internet resources 5.12 Mouse lines 6 Methods 6.1 Molecular Biological Methods 6.1.1 Isolation of genomic DNA 6.1.2 Total RNA isolation 6.1.3 cDNA synthesis 6.1.4 Synthesis of digoxigenin labeled RNA-transcript 6.1.5 Polymerase chain reaction (PCR) 6.1.6 Sanger sequencing 6.2 Preparation of animal tissue 6.2.1 Fixation of prepared embryonic tissue 6.3 Histological methods 6.3.1 Paraffin embedding and sectioning 6.3.2 Cryo-embedding and sectioning 6.3.3 In situ hybridization (cryosections) 6.3.4 Immunohistochemistry 6.3.5 Whole-mount immunohistochemistry 6.3.6 Oil Red O staning 6.4 Cell culture methods 6.4.1 Extraction and culturing of primary embryonic cells 6.4.2 Immunocytochemistry (ICC) 6.5 Biochemical Methods 6.5.1 Total Protein isolation and protein concentration 6.5.2 SDS PAGE 6.5.3 Western blot (WB) 6.6 Statistical analyses 7 Results 7 7.1 Characterization of Osr1 expression and cell lineage fate during mouse embryonic development 7.1.1 Expression pattern of Osr1 during embryonic limb development . 7.1.2 Contribution of Osr1 cells to limb tissues 7.2 Lack of Osr1 in Osr1GCE/GCE mutants leads to muscle defects 7.2.1 Muscle patterning defects in Osr1GCE/GCE embryos 7.2.2 Myofiber disorganization in Osr1GCE/GCE embryos 7.2.3 Formation of ectopic muscles and ectopically located muscle progenitors in Osr1GCE/GCE embryos 7.2.4 Correlation of Osr1 expression with phenotypic changes in muscle patterning 7.3 Transcriptome analysis of embryonic Osr1+ cells 7.3.1 RNA-sequencing of Osr1+ sorted cells 7.3.2 Gene ontology analysis of deregulated genes 7.4 Osr1 is required for connective tissue identity in the embryo 7.5 Extracellular matrix impairment in Osr1GCE/GCE embryos 7.5.1 Osr1 is required for the correct production and organization of structural components of the extracellular matrix 7.5.2 Basal lamina disruption in muscles fibers of Osr1GCE/GCE embryos 7.6 Osr1GCE/GCE embryos display defects in tendon formation 7.7 Impaired Cxcl12/Cxcr4 axis in muscle progenitors of Osr1GCE/GCE embryos 7.8 ReducedproliferativecapacitiesofmuscleprogenitorscellsinOsr1GCE/GCE mutants 7.9 Increased apoptosis of myogenic cells in the limb of Osr1GCE/GCE embryos 7.10 Reduced number of myogenic cells in the limb of Osr1GCE/GCE embryos 7.11 MyoblastsexhibitimpairedterminaldifferentiationinOsr1GCE/GCE mutants 7.12 DifferentialfusionimpairmentsofmyogeniccellsinOsr1GCE/GCE embryos in vitro and in vivo 7.13 Patterning defects are preceded by muscle progenitor mislocation 8 Discussion 8.1 Osr1 expression and potential during mouse development 8.1.1 Osr1 labels connective tissue cells associated with skeletal muscle during mouse limb development 8.1.2 Contribution of Osr1+ connective tissue cells to fetal tissues 8.2 Osr1 involvement in skeletal muscle formation 8.2.1 Muscle patterning impairments in Osr1 knockout mice 8.2.2 Muscle patterning defects are preceded by limited and mislocated myogenic cells 8.3 Defects in tendon formation 8.4 Transcriptome analyses of Osr1GCE/+ and Osr1GCE/GCE sorted cells 8.4.1 Maintenance of connective tissue identity 8.4.2 ImportanceofOsr1+connectivetissuecellsinextracellularmatrix formation 8.4.3 Osr1+ connective tissue cells and secreted molecules 9 Future work 10 References Supplementary Material List of figures List of tables List of publicationsThe musculoskeletal system allows body motion. Despite the distinct mesodermal origins of its components, the development of muscle, connective tissue (CT) and bone is highly coordinated. Osr1 encodes a zinc-finger transcription factor expressed in muscle CT in limbs. The aim of the PhD thesis was to elucidate Osr1 function in the non-cell autonomous regulation of mouse limb muscle formation. Genetic lineage tracing revealed that Osr1+ cells are progenitors for several CTs, including muscle, dermal and lung CTs, but also for smooth muscle and brown adipocytes. Comprehensive phenotypic analysis of skeletal muscles in E13.5Osr1GCE/GCE mouseembryos revealed impaired muscle formation. Transcriptomic analysis highlighted two major molecular characteristics caused by the lack of Osr1 activity. First,Osr1 activelyrepressed the expression of genesassociated with cartilage and tendondevelopment, suggesting that Osr1 confers a muscle connective tissue identity. Second, Osr1 positively regulated the expression of components of the extracellular matrix (ECM). In addition to the decrease of ECM components, numerous signaling molecules were significantly down-regulated in Osr1-deficient cells of mutant embryos. This highlights the function of Osr1+ resident connective tissue cells in limb muscle formation. It also establishes that Osr1 regulates the transcription of ECM components in limb muscle CT. Lastly, it suggests that Osr1 exerts its function via chemokines and secreted factors to ensure proper muscle development.Das muskuloskeletale System ist essentiell für die Fähigkeit zur Fortbewegung. Dieses aus mehreren Komponenten bestehende System erfordert eine koordinierte Morphogenese während der Entwicklung. Das Osr1 (Odd skipped-related 1) Gen kodiert für einen ZinkFinger Transkriptionsfaktor der im Muskel-Bindegewebe der Extremität im Huhn, wie auch in der Maus exprimiert ist. Das Ziel dieser Arbeit war, die Funktion von Osr1 in der nicht-Zell-autonomen Regulation der Muskelentwicklung in der Extremität der Maus zu analysieren. Durchgeführte Analysen der Osr1 Zelllinie zeigten, dass Osr1 Zellen Vorläufer für verschiedene Arten von Bindegewebe sind, darunter das Muskelbindegewebe, das Bindegewebe der Dermis oder retikuläre Fibroblasten der Lunge. Außerdem wurde gefunden, dass Osr1 Zellen Vorläufer für glatte Muskelzellen darstellen und für braune Fettzellen. Eine komprehensive Analyse des Muskelphänotyps in Osr1-defizienten Mäusen(Osr1GCE/GCE) zeigte klare Defekte in der lokalen Musterbildung. Durch eine Transkriptomanalyse konnte gezeigt werden, dass zwei Hauptaspekte durch das Fehlen von Osr1 betroffen waren. Zum einen wird Osr1 benötigt, um die Aktivität von Genen, die mit der Entwicklung von Knorpel- und Sehnenzellen assoziiert sind, zu reprimieren. Dies suggeriert, dass Osr1 an der Festlegung einer zellulären "Bindegewebsidentitätïn den mesenchymalen Vorläufern der Extremität beteiligt ist. Zum anderen wird Osr1 benötigt, um Gene der muskulären extrazellulären Matrix zu aktivieren. Zusätzlich zu einer Reduktion der Matrixkomponenten, zeigten zahlreiche Gene für Signalmoleküle eine Herunterregulation in Osr1+ Zellen aus Osr1GCE/GCE Embryos. Zusammengefasst zeigen diese Daten eine funktionelle Rolle der Osr1 BindegewebsZellpopulation im Prozess der Muskelentwicklung der Säugetier- Extremität. Osr1 scheint in diesem Zusammenhang die Transkription von extrazellulären Matrixkomponenten positiv zu regulieren. Schließlich suggerieren die Daten, dass Osr1 einen Teil seiner Funktion auch darüber bewerkstelligt, Chemokine und andere sekretierte Faktoren zu induzieren, welche die Musterbildung der Muskeln bestimmen

Odd skipped-related 1 (Osr1) identifies muscle-interstitial fibro-adipogenic progenitors (FAPs) activated by acute injury

Fibro-adipogenic progenitors (FAPs) are resident mesenchymal progenitors in adult skeletal muscle that support muscle repair, but also give rise to fibrous and adipose infiltration in response to disease and chronic injury. FAPs are identified using cell surface markers that do not distinguish between quiescent FAPs and FAPs actively engaged in the regenerative process. We have shown previously that FAPs are derived from cells that express the transcription factor Osr1 during development. Here we show that adult FAPs express Osr1 at low levels and frequency, however upon acute injury FAPs reactivate Osr1 expression in the injured tissue. Osr1+ FAPs are enriched in proliferating and apoptotic cells demonstrating that Osr1 identifies activated FAPs. In vivo genetic lineage tracing shows that Osr1+ activated FAPs return to the resident FAP pool after regeneration as well as contribute to adipocytes after glycerol-induced fatty degeneration. In conclusion, reporter LacZ or eGFP-CreERt2 expression from the endogenous Osr1 locus serves as marker for FACS isolation and tamoxifen-induced manipulation of activated FAPs. Keywords: Skeletal muscle, Fibro-adipogenic progenitors, Mesenchymal progenitors, Muscle interstitium, Muscle regeneratio

A local subset of mesenchymal cells expressing the transcription factor Osr1 orchestrates lymph node initiation

During development, lymph node (LN) initiation is coordinated by lymphoid tissue organizer (LTo) cells that attract lymphoid tissue inducer (LTi) cells at strategic positions within the embryo. The identity and function of LTo cells during the initial attraction of LTi cells remain poorly understood. Using lineage tracing, we demonstrated that a subset of Osr1-expressing cells was mesenchymal LTo progenitors. By investigating the heterogeneity of Osr1+ cells, we uncovered distinct mesenchymal LTo signatures at diverse anatomical locations, identifying a common progenitor of mesenchymal LTos and LN-associated adipose tissue. Osr1 was essential for LN initiation, driving the commitment of mesenchymal LTo cells independent of neural retinoic acid, and for LN-associated lymphatic vasculature assembly. The combined action of chemokines CXCL13 and CCL21 was required for LN initiation. Our results redefine the role and identity of mesenchymal organizer cells and unify current views by proposing a model of cooperative cell function in LN initiation

Odd skipped-related 1 identifies a population of embryonic fibro-adipogenic progenitors regulating myogenesis during limb development

Fibro-adipogenic progenitors (FAPs) form part of interstitial muscle connective tissue (MCT) in adults but the origin of this non-myogenic lineage is unclear. Here, the authors show that Odd skipped related 1 (Osr1) in mice marks embryonic MCT, giving rise to FAPs, and loss of Osr1 in the limb causes muscle defects

Enhancer hijacking at the ARHGAP36 locus is associated with connective tissue to bone transformation

: Heterotopic ossification is a disorder caused by abnormal mineralization of soft tissues in which signaling pathways such as BMP, TGFβ and WNT are known key players in driving ectopic bone formation. Identifying novel genes and pathways related to the mineralization process are important steps for future gene therapy in bone disorders. In this study, we detect an inter-chromosomal insertional duplication in a female proband disrupting a topologically associating domain and causing an ultra-rare progressive form of heterotopic ossification. This structural variant lead to enhancer hijacking and misexpression of ARHGAP36 in fibroblasts, validated here by orthogonal in vitro studies. In addition, ARHGAP36 overexpression inhibits TGFβ, and activates hedgehog signaling and genes/proteins related to extracellular matrix production. Our work on the genetic cause of this heterotopic ossification case has revealed that ARHGAP36 plays a role in bone formation and metabolism, outlining first details of this gene contributing to bone-formation and -disease