© 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev161208. doi:10.1242/dev.161208 TECHNIQUES AND RESOURCES RESEARCH ARTICLE Genome-wide strategies identify downstream target genes of chick connective tissue-associated transcription factors Mickael Orgeur1,2,3, Marvin Martens3, Georgeta Leonte2,4, Sonya Nassari3, Marie-Ange Bonnin3, Stefan T. Börno2, Bernd Timmermann2, Jochen Hecht2,5,6,7, Delphine Duprez3,*,‡ and Sigmar Stricker1,2,*,‡ ABSTRACT part of the musculoskeletal system. Muscle CT is pivotal for the Connective tissues support organs and play crucial roles in mechanical properties of muscle and is structurally continuous with development, homeostasis and fibrosis, yet our understanding of their the tendons, which finally transmit force to the skeleton. formation is still limited. To gain insight into the molecular mechanisms Dysregulation of CT homeostasis leads to fibrosis, which is of connective tissue specification, we selected five zinc-finger observed during pathological tissue repair or healing processes transcription factors – OSR1, OSR2, EGR1, KLF2 and KLF4 – based and in cancer (Kalluri, 2016). Although fibrosis is a common on their expression patterns and/or known involvement in connective research subject, normal CT formation during development remains tissue subtype differentiation. RNA-seq and ChIP-seq profiling of chick poorly investigated. limb micromass cultures revealed a set of common genes regulated by The appendage of vertebrate embryos is an excellent model all five transcription factors, which we describe as a connective tissue system for analysing tissue differentiation and cellular interactions core expression set. This common core was enriched with genes during development. In limbs, cells forming the skeleton, as well as associated with axon guidance and myofibroblast signature, including regular and irregular CTs, are derived from the lateral plate fibrosis-related genes. In addition, each transcription factor regulated a mesoderm, whereas myogenic cells originate from the somites specific set of signalling molecules and extracellular matrix components. (Chevallier et al., 1977; Christ et al., 1977). Classical embryological This suggests a concept whereby local molecular niches can be created experiments have shown that limb patterning is dependent on lateral by the expression of specific transcription factors impinging on the plate-derived CTs that provide instructive cues to guarantee correct specification of local microenvironments. The regulatory network muscle, nerve and vessel formation (Gaut and Duprez, 2016; established here identifies common and distinct molecular signatures Kardon, 1998; Lance-Jones and Dias, 1991; Michaud et al., 1997). of limb connective tissue subtypes, provides novel insight into the The nature of these cues is so far mostly elusive, but it is assumed signalling pathways governing connective tissue specification, and that a key role is played by specific ECM in combination with serves as a resource for connective tissue development. locally produced paracrine signalling factors (Hasson, 2011; Nassari et al., 2017a). Altogether, CT cells appear to be key KEY WORDS: Chicken model, Limb development, Connective tissue players creating local microenvironments that contain permissive differentiation, Extracellular matrix, Signalling pathways, and/or instructive cues for organ patterning. Transcription factors, Transcriptional regulatory network Specification, differentiation and function taken by a progenitor cell involve dramatic transcriptional and finally phenotypic changes INTRODUCTION that differ for each cell/tissue type. Lineage-specific genetic Connective tissue (CT) is one of the main components of the body programmes ensure fine-tuning between the repression and the essential for supporting tissues and organs, in part by the production expression of a given set of genes in response to extrinsic and of tissue-specific extracellular matrix (ECM). The term CT gathers intrinsic signals at a specific location and/or at a precise time (Heinz together an ensemble of tissues, including specialized CT (cartilage et al., 2015). Progenitor cells are specified and induced to and bone), soft CT (adipose tissue and vasculature) and dense CT. differentiate along a certain lineage upon activation of lineage- Dense CT can be divided into regular CT (tendon and ligament) and specific key transcription factors (TFs) that drive specific irregular CT (loose CT surrounding or within organs such as muscle transcriptional programmes (Spitz and Furlong, 2012). As a CT) (Nassari et al., 2017a). Regular and irregular CTs are an integral consequence, the differentiating progenitor cells express lineage- specific genes that reinforce lineage commitment, as well as 1Freie Universität Berlin, Institute of Chemistry and Biochemistry, Thielallee 63, providing unique characteristics to the specific cell type and the 14195 Berlin, Germany. 2Max Planck Institute for Molecular Genetics, Ihnestr. 63- tissue it gives rise to. Although master TFs governing cell type- 73, 14195 Berlin, Germany. 3Sorbonne Universités, UPMC Univ. Paris 06, CNRS UMR 7622, Inserm U1156, IBPS-Developmental Biology Laboratory, 9 Quai Saint- specific gene expression programmes have been identified for Bernard, 75005 Paris, France. 4Freie Universität Berlin, Institute of Biology, Königin- cartilage (SOX9), bone (RUNX2) and muscle (MYF5, MYF6, Luise-Str. 1-3, 14195 Berlin, Germany. 5Berlin-Brandenburg Center for MYOD) development (Braun and Gautel, 2011; Kronenberg, Regenerative Therapies (BCRT), CharitéUniversitatsmedizin, Augustenburger Platz 1, 13353 Berlin, Germany. 6Centre for Genomic Regulation (CRG), The 2003), knowledge is sparse for dense CT. CT is mostly identified Barcelona Institute for Science and Technology, Dr. Aiguader 88, 08003 Barcelona, by gene/protein expression associated with CT function. Irregular Spain. 7Universitat Pompeu Fabra (UPF), 08002 Barcelona, Spain. CT is associated with type-III and -VI collagens, whereas regular *These authors contributed equally to this work CT (tendon/ligament) is characterized by the expression of ‡Authors for correspondence ([email protected]; structural and functional components such as type-I and -XII [email protected]) collagens or tenomodulin (TNMD) (Gaut and Duprez, 2016; Huang D.D., 0000-0003-0248-7417; S.S., 0000-0002-7174-5363 et al., 2015). Few TFs specific to CT lineages have been identified. Scleraxis (SCX) is to date the best marker for tendon cells; however, Received 2 November 2017; Accepted 24 February 2018 it is not necessary for development of most tendons (Murchison DEVELOPMENT 1 TECHNIQUES AND RESOURCES Development (2018) 145, dev161208. doi:10.1242/dev.161208 et al., 2007; Schweitzer et al., 2001). Early growth response 1 CT, whereas EGR1, KLF2 and KLF4 are expressed in different (EGR1) is involved in type-I collagen production in chick and regions of regular CT in developing chick limbs. mouse developing tendons (Lejard et al., 2011). Moreover, forced expression of EGR1 is sufficient to induce the expression of tendon- CT-associated TFs influence differentiation of limb associated genes in murine mesenchymal stem cells (Guerquin et al., mesenchymal progenitors 2013). TBX4 and TBX5 are expressed in limb irregular CT, and To analyse the functionality of the five TFs in relation to CT TCF4 (TCF7L2) is expressed in irregular CT associated with muscle, differentiation, we chose the chick micromass (chMM) explant but they have no obvious role in CT differentiation (Hasson et al., model (Fig. 2A). In this three-dimensional culture model, limb bud 2010; Kardon et al., 2003). In contrast, odd-skipped related 1 and 2 cells behave close to the in vivo situation and differentiate into the (OSR1 and OSR2) are expressed and involved in irregular CT mesenchymal lineages observed in native limb buds (Ahrens et al., differentiation during chick and mouse limb development (Nassari 1979). We tested the ability of the five TFs to shift cell et al., 2017b; Stricker et al., 2006, 2012; Vallecillo-García et al., differentiation in chMM cultures by their overexpression with 2017). Moreover, TBX4, TBX5, TCF4 and OSR1 are involved in the RCAS replication-competent retroviruses. This retroviral system non-cell-autonomous regulation of muscle patterning by CT (Hasson allows for high transfection efficiency with mild overexpression of et al., 2010; Mathew et al., 2011; Vallecillo-García et al., 2017). genes of interest, which has been shown to be in a physiological Here, we present a comprehensive analysis of five selected zinger range compared with endogenous expression in native limb buds, finger TFs addressing the molecular mechanisms underlying CT and has been tested for cell differentiation, transcriptome and differentiation and function during chick limb development. OSR1/ chromatin-binding analyses previously (Ibrahim et al., 2013). 2 and EGR1 were chosen based on their demonstrated contribution Owing to the absence of specific antibodies targeting each of the in irregular and regular CT development, respectively. KLF2 and selected chicken TFs, we used the triple-FLAG (3F) tag, which was KLF4 (Krüppel-like factor 2 and 4) were chosen based on their fused to the C terminus of the coding sequence (CDS) of each TF. expression patterns in CT associated with tendons, although their Overexpression of the recombinant TFs was monitored by role in limb development has not yet been determined. We immunohistochemistry