Tendon formation and repair rely on specific combinations of transcription factors, growth factors, and mechanical parameters that regulate the production and spatial organization of type I collagen. Here, we investigated the function of the zinc finger transcription factor EGR1 in tendon formation, healing, and repair using rodent animal models and mesenchymal stem cells (MSCs). Adult tendons of Egr1 -/-mice displayed a deficiency in the expression of tendon genes, including Scx, Col1a1, and Col1a2, and were mechanically weaker compared with their WT littermates. EGR1 was recruited to the Col1a1 and Col2a1 promoters in postnatal mouse tendons in vivo. Egr1 was required for the normal gene response following tendon injury in a mouse model of Achilles tendon healing. Forced Egr1 expression programmed MSCs toward the tendon lineage and promoted the formation of in vitro-engineered tendons from MSCs. The application of EGR1-producing MSCs increased the formation of tendon-like tissues in a rat model of Achilles tendon injury. We provide evidence that the ability of EGR1 to promote tendon differentiation is partially mediated by TGF-β2. This study demonstrates EGR1 involvement in adult tendon formation, healing, and repair and identifies Egr1 as a putative target in tendon repair strategies.

The molecular signals driving tendon development are not fully identified. We have undertaken a transcriptome analysis of mouse limb tendon cells that were isolated at different stages of development based on scleraxis (Scx) expression. Microarray comparisons allowed us to establish a list of genes regulated in tendon cells during mouse limb development. Bioinformatics analysis of the tendon transcriptome showed that the two most strongly modified signalling pathways were TGF-β and MAPK. TGF-β/SMAD2/3 gain-and loss-offunction experiments in mouse limb explants and mesenchymal stem cells showed that TGF-β signalling was sufficient and required via SMAD2/3 to drive mouse mesodermal stem cells towards the tendon lineage ex vivo and in vitro. TGF-β was also sufficient for tendon gene expression in late limb explants during tendon differentiation. FGF does not have a tenogenic effect and the inhibition of the ERK MAPK signalling pathway was sufficient to activate Scx in mouse limb mesodermal progenitors and mesenchymal stem cells.

The molecules involved in vertebrate tendon formation during development remain largely unknown. To date, only two DNA-binding proteins have been identified as being involved in vertebrate tendon formation, the basic helix-loop-helix transcription factor Scleraxis and, recently, the Mohawk homeobox gene. We investigated the involvement of the early growth response transcription factors Egr1 and Egr2 in vertebrate tendon formation. We established that Egr1 and Egr2 expression in tendon cells was correlated with the increase of collagen expression during tendon cell differentiation in embryonic limbs. Vertebrate tendon differentiation relies on a muscle-derived FGF (fibroblast growth factor) signal. FGF4 was able to activate the expression of Egr genes and that of the tendon-associated collagens in chick limbs. Egr gene misexpression experiments using the chick model allowed us to establish that either Egr gene has the ability to induce de novo expression of the reference tendon marker scleraxis, the main tendon collagen Col1a1, and other tendon-associated collagens Col3a1, Col5a1, Col12a1, and Col14a1. Mouse mutants for Egr1 or Egr2 displayed reduced amounts of Col1a1 transcripts and a decrease in the number of collagen fibrils in embryonic tendons. Moreover, EGR1 and EGR2 trans-activated the mouse Col1a1 proximal promoter and were recruited to the tendon regulatory regions of this promoter. These results identify EGRs as novel DNA-binding proteins involved in vertebrate tendon differentiation by regulating type I collagen production.Vertebrate tendons are specialized dense connective tissues mainly composed of collagens that connect muscle to bone.Tendon repair following injuries or during aging is a clinical challenge because tendons are only repaired slowly and partially. The establishment of new strategies for tendon repair is prevented by a limited understanding of tendon development and consequently awaits a better understanding of tendon development. An important goal is to understand the molecular mechanisms involved in regulating collagen expression, production, and assembly during tendon development to be able to design new therapies for tendon repair.Tendons consist of elongated fibroblasts named tenocytes that produce an abundant extracellular matrix composed of Ͼ90% collagen. The major collagen component of mature tendon is type I collagen, a heterotrimeric molecule composed of two ␣1 chains and one ␣2 chain that are genetically distinct and encoded by Col1a1 and Col1a2, respectively (1-3). Type I collagen molecules self-assemble into highly organized parallel collagen fibrils and then into fibers, which provide the tensile strength of tendon. Other collagens, such as the fibrillar collagens III and V and the nonfibrillar collagens named FACITs (fibril-associated collagens with interrupted triple helices), and collagens XII and XIV are important for collagen fibril formation, growth, and integrity of tendons. In addition to collagens, tendons contain various matrix components contributing to the prop...

The molecular programme underlying tendon development has not been fully identified. Interactions with components of the musculoskeletal system are important for limb tendon formation. Limb tendons initiate their development independently of muscles; however, muscles are required for further tendon differentiation. We show that both FGF/ERK MAPK and TGFβ/SMAD2/3 signalling pathways are required and sufficient for SCX expression in chick undifferentiated limb cells, whereas the FGF/ERK MAPK pathway inhibits Scx expression in mouse undifferentiated limb mesodermal cells. During differentiation, muscle contraction is required to maintain SCX, TNMD and THBS2 expression in chick limbs. The activities of FGF/ERK MAPK and TGFβ/SMAD2/3 signalling pathways are decreased in tendons under immobilisation conditions. Application of FGF4 or TGFβ2 ligands prevents SCX downregulation in immobilised limbs. TGFβ2 but not FGF4 prevent TNMD and THBS2 downregulation under immobilisation conditions. We did not identify any intracellular crosstalk between both signalling pathways in their positive effect on SCX expression. Independently of each other, both FGF and TGFβ promote tendon commitment of limb mesodermal cells and act downstream of mechanical forces to regulate tendon differentiation during chick limb development.

Thyroid hormone receptors (TR) act as activators of transcription in the presence of the thyroid hormone (T 3 ) and as repressors in its absence. While many in vitro approaches have been used to study the molecular mechanisms of TR action, their physiological relevance has not been addressed. Here we investigate how TR regulates gene expression during vertebrate postembryonic development by using T 3 -dependent amphibian metamorphosis as a model. Earlier studies suggest that TR acts as a repressor during premetamorphosis when T 3 is absent. We hypothesize that corepressor complexes containing the nuclear receptor corepressor (N-CoR) are key factors in this TR-dependent gene repression, which is important for premetamorphic tadpole growth. To test this hypothesis, we isolated Xenopus laevis N-CoR (xN-CoR) and showed that it was present in pre-and metamorphic tadpoles. Using a chromatin immunoprecipitation assay, we demonstrated that xN-CoR was recruited to the promoters of T 3 response genes during premetamorphosis and released upon T 3 treatment, accompanied by a local increase in histone acetylation. Furthermore, overexpression of a dominant-negative N-CoR in tadpole tail muscle led to increased transcription from a T 3 -dependent promoter. Our data indicate that N-CoR is recruited by unliganded TR to repress target gene expression during premetamorphic animal growth, an important process that prepares the tadpole for metamorphosis.Combinatorial actions of transcription factors are critical for coordinating development, cell homeostasis, and physiology (31). Nuclear receptors represent a large class of transcription factors implicated in the control of many important biological processes (29,31). This superfamily includes receptors for thyroid hormone (T 3 , or 3,5,3Ј-triiodothyronine), vitamin D, retinoids, steroids, and components of lipid metabolism, as well as a number of orphan receptors (29). One of the most striking developmental processes involving nuclear receptors is amphibian metamorphosis.The metamorphic transformation involves degeneration of larval tissues through apoptosis with concurrent proliferation and differentiation of adult cell types (6, 48). Metamorphosis requires the induction of a number of gene cascades at a precise developmental time point (47, 49). In Xenopus laevis, the switch to the metamorphic program is entirely T 3 dependent (6, 48) but only occurs when the tadpole has grown to a suitable size and stage. This suggests that a molecular repressor mechanism must be in place during the growth period to repress metamorphosis-inducing genes.Our working hypothesis was that this repressor mechanism, vital to correct physiological development, would involve certain corepressors. This hypothesis was derived from the results of a series of elegant in vitro and ex vivo experiments that show liganded as well as unliganded thyroid hormone receptors (TRs) to be capable of binding to target genes through cisacting DNA sequences known as thyroid hormone response elements (T 3 REs). In the absen...

In higher vertebrates, the primordium of the nervous system, the neural tube, is shaped along the rostrocaudal axis through two consecutive, radically different processes referred to as primary and secondary neurulation. Failures in neurulation lead to severe anomalies of the nervous system, called neural tube defects (NTDs), which are among the most common congenital malformations in humans. Mechanisms causing NTDs in humans remain ill-defined. Of particular interest, the thoracolumbar region, which encompasses many NTD cases in the spine, corresponds to the junction between primary and secondary neurulations. Elucidating which developmental processes operate during neurulation in this region is therefore pivotal to unraveling the etiology of NTDs. Here, using the chick embryo as a model, we show that, at the junction, the neural tube is elaborated by a unique developmental program involving concerted movements of elevation and folding combined with local cell ingression and accretion. This process ensures the topological continuity between the primary and secondary neural tubes while supplying all neural progenitors of both the junctional and secondary neural tubes. Because it is distinct from the other neurulation events, we term this phenomenon junctional neurulation. Moreover, the planarcell-polarity member, Prickle-1, is recruited specifically during junctional neurulation and its misexpression within a limited time period suffices to cause anomalies that phenocopy lower spine NTDs in human. Our study thus provides a molecular and cellular basis for understanding the causality of NTD prevalence in humans and ascribes to Prickle-1 a critical role in lower spinal cord formation.

Delivery of ASCs in poloxamer addresses multiple facets of the complexity of nerve/muscle unit regeneration, representing a promising avenue for further study. Muscle Nerve 58: 251-260, 2018.

Although the transcription factor EGR1 is known as NGF1-A, TIS8, Krox24, zif/268, and ZENK, it still has many fewer names than biological functions. A broad range of signals induce Egr1 gene expression via numerous regulatory elements identified in the Egr1 promoter. EGR1 is also the target of multiple post-translational modifications, which modulate EGR1 transcriptional activity. Despite the myriad regulators of Egr1 transcription and translation, and the numerous biological functions identified for EGR1, the literature reveals a recurring theme of EGR1 transcriptional activity in connective tissues, regulating genes related to the extracellular matrix. Egr1 is expressed in different connective tissues, such as tendon (a dense connective tissue), cartilage and bone (supportive connective tissues), and adipose tissue (a loose connective tissue). Egr1 is involved in the development, homeostasis, and healing processes of these tissues, mainly via the regulation of extracellular matrix. In addition, Egr1 is often involved in the abnormal production of extracellular matrix in fibrotic conditions, and Egr1 deletion is seen as a target for therapeutic strategies to fight fibrotic conditions. This generic EGR1 function in matrix regulation has little-explored implications but is potentially important for tendon repair.