Insight into Physiological and Biochemical Determinants of Salt Stress Tolerance in Tetraploid Citrus.

Citrus are classified as salt-sensitive crops. However, a large diversity has been observed regarding the trends of tolerance among citrus. In the present article, physiological and biochemical studies of salt stress tolerance were carried out according to the level of polyploidy of different citrus genotypes. We particularly investigated the impact of tetraploidy in trifoliate orange (Poncirus trifoliata (L.) Raf.) (PO4x) and Cleopatra mandarin (Citrus reshni Hort. Ex Tan.) (CL4x) on the tolerance to salt stress compared to their respective diploids (PO2x and CL2x). Physiological parameters such as gas exchange, ions contents in leaves and roots were analyzed. Roots and leaves samples were collected to measure polyphenol, malondialdehyde (MDA), ascorbate and H2O2 contents but also to measure the activities of enzymes involved in the detoxification of active oxygen species (ROS). Under control conditions, the interaction between genotype and ploidy allowed to discriminate different behavior in terms of photosynthetic and antioxidant capacities. These results were significantly altered when salt stress was applied when salt stress was applied. Contrary to the most sensitive genotype, that is to say the diploid trifoliate orange PO2x, PO4x was able to maintain photosynthetic activity under salt stress and had better antioxidant capacities. The same observation was made regarding the CL4x genotype known to be more tolerant to salt stress. Our results showed that tetraploidy may be a factor that could enhance salt stress tolerance in citrus.

Interaction between the Accumulation of Cadmium and Deoxynivalenol Mycotoxin Produced by Fusarium graminearum in Durum Wheat Grains.

Durum wheat is one of the cereal crops that accumulates the highest concentrations of cadmium (Cd) and deoxynivalenol (DON) mycotoxin in its grains, thereby affecting the safety of products made of durum wheat grains (pasta and semolina). This study investigates in planta the interaction between Cd and Fusarium graminearum, the main causal agent of DON accumulation in grains. A pot experiment was designed to characterize the response of durum wheat to F. graminearum infection at three levels of Cd exposure: 0.1, 2, and 10 mg Cd kg-1 soil, which showed that the accumulation of Cd and DON resulted from interacting processes. On the one hand, plant exposure to Cd reduced the concentration of DON in grains. The mitigating effect of Cd on DON accumulation was attributed to the restricted growth of F. graminearum, which could result from enhanced plant resistance to the fungal pathogen induced by Cd exposure. On the other hand, F. graminearum infection of durum wheat increased the Cd concentration in the grains. The promoting effect of Fusarium infection on Cd accumulation was attributed to decoupling of the allocation of Cd and photoassimilates to the grains and to the reduced strength of the grain sink for photoassimilates caused by the fungus. Provided that this result is confirmed in field conditions, it suggests that in Cd-contaminated soils, particular attention should be paid to agronomic practices that affect Fusarium head blight disease to avoid further increase in the risk of exceeding the regulatory limit set by the European Union for Cd in durum wheat.

TMEM8C-mediated fusion is regionalized and regulated by NOTCH signalling during foetal myogenesis.

The location and regulation of fusion events within skeletal muscles during development remain unknown. Using the fusion marker myomaker (Mymk), named TMEM8C in chicken, as a readout of fusion, we identified a co-segregation of TMEM8C-positive cells and MYOG-positive cells in single-cell RNA-sequencing datasets of limbs from chicken embryos. We found that TMEM8C transcripts, MYOG transcripts and the fusion-competent MYOG-positive cells were preferentially regionalized in central regions of foetal muscles. We also identified a similar regionalization for the gene encoding the NOTCH ligand JAG2 along with an absence of NOTCH activity in TMEM8C+ fusion-competent myocytes. NOTCH function in myoblast fusion had not been addressed so far. We analysed the consequences of NOTCH inhibition for TMEM8C expression and myoblast fusion during foetal myogenesis in chicken embryos. NOTCH inhibition increased myoblast fusion and TMEM8C expression and released the transcriptional repressor HEYL from the TMEM8C regulatory regions. These results identify a regionalization of TMEM8C-dependent fusion and a molecular mechanism underlying the fusion-inhibiting effect of NOTCH in foetal myogenesis. The modulation of NOTCH activity in the fusion zone could regulate the flux of fusion events.

Correction: Galenic lab-on-a-chip concept for lipid nanocapsules production.

Correction for 'Galenic Lab-on-a-Chip concept for lipid nanocapsules production' by Nicolas Rolley et al., Nanoscale, 2021, 13, 11899-11912, DOI: 10.1039/D1NR00879J.

Unexpected contribution of fibroblasts to muscle lineage as a mechanism for limb muscle patterning.

Positional information driving limb muscle patterning is contained in connective tissue fibroblasts but not in myogenic cells. Limb muscles originate from somites, while connective tissues originate from lateral plate mesoderm. With cell and genetic lineage tracing we challenge this model and identify an unexpected contribution of lateral plate-derived fibroblasts to the myogenic lineage, preferentially at the myotendinous junction. Analysis of single-cell RNA-sequencing data from whole limbs at successive developmental stages identifies a population displaying a dual muscle and connective tissue signature. BMP signalling is active in this dual population and at the tendon/muscle interface. In vivo and in vitro gain- and loss-of-function experiments show that BMP signalling regulates a fibroblast-to-myoblast conversion. These results suggest a scenario in which BMP signalling converts a subset of lateral plate mesoderm-derived cells to a myogenic fate in order to create a boundary of fibroblast-derived myonuclei at the myotendinous junction that controls limb muscle patterning.

Galenic Lab-on-a-Chip concept for lipid nanocapsules production.

The continuous production of drug delivery systems assisted by microfluidics has drawn a growing interest because of the high reproducibility, low batch-to-batch variations, narrow and controlled particle size distributions and scale-up ease induced by this kind of processes. Besides, microfluidics offers opportunities for high throughput screening of process parameters and the implementation of process characterization techniques as close to the product as possible. In this context, we propose to spotlight the GALECHIP concept through the development of an instrumented microfluidic pilot considered as a Galenic Lab-on-a-Chip to formulate nanomedicines, such as lipid nanocapsules (LNCs), under controlled process conditions. In this paper we suggest an optimal rational development in terms of chip costs and designs. First, by using two common additive manufacturing techniques, namely fused deposition modelling and multi-jet modelling to prototype customized 3D microfluidic devices (chips and connectors). Secondly, by manufacturing transparent Silicon (Si)/Glass chips with similar channel geometries but obtained by a new approach of deep reactive ion etching (DRIE) technology suitable with in situ small angle X-ray scattering characterizations. LNCs were successfully produced by a phase inversion composition (PIC) process with highly monodispersed sizes from 25 nm to 100 nm and formulated using chips manufactured by 3D printing and DRIE technologies. The transparent Si/Glass chip was also used for the small angle X-ray scattering (SAXS) analysis of the LNC formulation with the PIC process. The 3D printing and DRIE technologies and their respective advantages are discussed in terms of cost, easiness to deploy and process developments in a GALECHIP point of view.

Core-Shell Pure Collagen Threads Extruded from Highly Concentrated Solutions Promote Colonization and Differentiation of C3H10T1/2 Cells.

The elaboration of scaffolds able to efficiently promote cell differentiation toward a given cell type remains challenging. Here, we engineered dense type I collagen threads with the aim of providing scaffolds with specific morphological and mechanical properties for C3H10T1/2 mesenchymal stem cells. Extrusion of pure collagen solutions at different concentrations (15, 30, and 60 mg/mL) in a PBS 5× buffer generated dense fibrillated collagen threads. For the two highest concentrations, threads displayed a core-shell structure with a marked fibril orientation of the outer layer along the longitudinal axis of the threads. Young's modulus and ultimate tensile stress as high as 1 and 0.3 MPa, respectively, were obtained for the most concentrated collagen threads without addition of any cross-linkers. C3H10T1/2 cells oriented themselves with a mean angle of 15-24° with respect to the longitudinal axis of the threads. Cells penetrated the 30 mg/mL scaffolds but remained on the surface of the 60 mg/mL ones. After three weeks of culture, cells displayed strong expression of the tendon differentiation marker Tnmd, especially for the 30 mg/mL threads. These results suggest that both the morphological and mechanical characteristics of collagen threads are key factors in promoting C3H10T1/2 differentiation into tenocytes, offering promising levers to optimize tissue engineering scaffolds for tendon regeneration.

Screening of Wood/Forest and Vine By-Products as Sources of New Drugs for Sustainable Strategies to Control Fusarium graminearum and the Production of Mycotoxins.

Fusarium graminearum is a fungal pathogen that can colonize small-grain cereals and maize and secrete type B trichothecene (TCTB) mycotoxins. The development of environmental-friendly strategies guaranteeing the safety of food and feed is a key challenge facing agriculture today. One of these strategies lies on the promising capacity of products issued from natural sources to counteract crop pests. In this work, the in vitro efficiency of sixteen extracts obtained from eight natural sources using subcritical water extraction at two temperatures was assessed against fungal growth and TCTB production by F. graminearum. Maritime pine sawdust extract was shown to be extremely efficient, leading to a significant inhibition of up to 89% of the fungal growth and up to 65% reduction of the mycotoxin production by F. graminearum. Liquid chromatography/mass spectrometry analysis of this active extract revealed the presence of three families of phenolics with a predominance of methylated compounds and suggested that the abundance of methylated structures, and therefore of hydrophobic compounds, could be a primary factor underpinning the activity of the maritime pine sawdust extract. Altogether, our data support that wood/forest by-products could be promising sources of bioactive compounds for controlling F. graminearum and its production of mycotoxins.

Investigating the Efficiency of Hydroxycinnamic Acids to Inhibit the Production of Enniatins by Fusarium avenaceum and Modulate the Expression of Enniatins Biosynthetic Genes.

Enniatins (ENNs) that belong to the group of emerging mycotoxins are widespread contaminants of agricultural commodities. There is currently insufficient evidence to rule out health concerns associated with long-term exposure to ENNs and efforts must be strengthened to define a control strategy. While the potential of plant compounds to counteract the contamination with legislated mycotoxins has been reported, little remains known regarding ENNs. The present study evidenced for the first time the efficiency of hydroxycinnamic acids to inhibit the fungal growth and ENNs yield by Fusarium avenaceum. Notably, 0.5 mM of exogenous ferulic, caffeic, and p-coumaric acids led to a drastic reduction of ENNs synthesis in pH4 broths, with ferulic acid being the most potent. The ENNs production inhibitory activity of ferulic acid was shown to be associated with a significant down-regulation of the expression of ENNs biosynthetic genes. To further investigate the bioactivity of ferulic acid, its metabolic fate was characterized in fungal broths and the capacity of F. avenaceum to metabolize it through a C2-cleavage type degradation was demonstrated. Overall, our data support the promising use of ferulic acid in ENNs control strategies, either as part of an environmentally friendly plant-care product or as a biomarker of plant resistance.

Synthesis of PDMS-b-POEGMA Diblock Copolymers and Their Application for the Thermoresponsive Stabilization of Water-Supercritical Carbon Dioxide Emulsions.

In this study, PDMS13-b-POEGMAx diblock copolymers consisting of a CO2-philic poly(dimethylsiloxane) (PDMS) block connected to a thermosensitive hydrophilic poly(oligoethylene glycol methacrylate) (POEGMA) block were synthesized by reversible addition-fragmentation chain-transfer (RAFT) radical polymerization. Their ability to decrease the water-supercritical CO2 (scCO2) interfacial tension (γ) and to stabilize water-scCO2 emulsions was investigated using an original homemade device developed in the laboratory. This device is able to control the pressure from 1 to 250 bar and the temperature from 40 to 80 °C. It was implemented with 2 visualization windows, a drop tensiometer and a remote optical head for dynamic light scattering (DLS) measurements. These experiments revealed that PDMS-b-POEGMA decreased γ down to 1-2 mN/m and was the most efficient at high pressure (250 bar) and low temperature (40 °C) where PDMS and POEGMA blocks exhibited the highest affinity for their respective phase. The diblock copolymers were shown to stabilize water-scCO2 emulsions. Moreover, the thermosensitive behavior of the POEGMA block in water (with a lower critical solubility temperature around 65 °C) resulted in the formation of temperature-responsive emulsions that could reversibly switch at 100 bar from stable at 40 °C to unstable at 80 °C. These results were rationalized based on the solubility of each individual block of the copolymers in water and scCO2 as a function of temperature and pressure.

Mechanical and molecular parameters that influence the tendon differentiation potential of C3H10T1/2 cells in 2D- and 3D-culture systems.

One of the main challenges relating to tendons is to understand the regulators of the tendon differentiation program. The optimum culture conditions that favor tendon cell differentiation have not been identified. Mesenchymal stem cells present the ability to differentiate into multiple lineages in cultures under different cues ranging from chemical treatment to physical constraints. We analyzed the tendon differentiation potential of C3H10T1/2 cells, a murine cell line of mesenchymal stem cells, upon different 2D- and 3D-culture conditions. We observed that C3H10T1/2 cells cultured in 2D conditions on silicone substrate were more prone to tendon differentiation, assessed with the expression of the tendon markers Scx, Col1a1 and Tnmd as compared to cells cultured on plastic substrate. The 3D-fibrin environment was more favorable for Scx and Col1a1 expression compared to 2D cultures. We also identified TGFß2 as a negative regulator of Tnmd expression in C3H10T1/2 cells in 2D and 3D cultures. Altogether, our results provide us with a better understanding of the culture conditions that promote tendon gene expression and identify mechanical and molecular parameters upon which we could act to define the optimum culture conditions that favor tenogenic differentiation in mesenchymal stem cells.

Genome-wide strategies identify downstream target genes of chick connective tissue-associated transcription factors.

Connective tissues support organs and play crucial roles in development, homeostasis and fibrosis, yet our understanding of their formation is still limited. To gain insight into the molecular mechanisms of connective tissue specification, we selected five zinc-finger transcription factors - OSR1, OSR2, EGR1, KLF2 and KLF4 - based on their expression patterns and/or known involvement in connective tissue subtype differentiation. RNA-seq and ChIP-seq profiling of chick limb micromass cultures revealed a set of common genes regulated by all five transcription factors, which we describe as a connective tissue core expression set. This common core was enriched with genes associated with axon guidance and myofibroblast signature, including fibrosis-related genes. In addition, each transcription factor regulated a specific set of signalling molecules and extracellular matrix components. This suggests a concept whereby local molecular niches can be created by the expression of specific transcription factors impinging on the specification of local microenvironments. The regulatory network established here identifies common and distinct molecular signatures of limb connective tissue subtypes, provides novel insight into the signalling pathways governing connective tissue specification, and serves as a resource for connective tissue development.

Mycotoxin Biosynthesis and Central Metabolism Are Two Interlinked Pathways in Fusarium graminearum, as Demonstrated by the Extensive Metabolic Changes Induced by Caffeic Acid Exposure.

Fusarium graminearum is a major plant pathogen that causes devastating diseases of cereals and produces type B trichothecene (TCTB) mycotoxins in infected grains. A comprehensive understanding of the molecular and biochemical mechanisms underlying the regulation of TCTB biosynthesis is required for improving strategies to control the TCTB contamination of crops and ensuring that these strategies do not favor the production of other toxic metabolites by F. graminearum Elucidation of the association of TCTB biosynthesis with other central and specialized processes was the focus of this study. Combined 1H nuclear magnetic resonance (1H NMR) and liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS) analyses were used to compare the exo- and endometabolomes of F. graminearum grown under toxin-inducing and -repressing caffeic acid conditions. Ninety-five metabolites were putatively or unambiguously identified, including 26 primary and 69 specialized metabolites. Our data demonstrated that the inhibition of TCTB production induced by caffeic acid exposure was associated with significant changes in the secondary and primary metabolism of F. graminearum, although the fungal growth was not affected. The main metabolic changes were an increase in the accumulation of several polyketides, including toxic ones, alterations in the tricarboxylic organic acid cycle, and modifications in the metabolism of several amino acids and sugars. While these findings provide insights into the mechanisms that govern the inhibition of TCTB production by caffeic acid, they also demonstrate the interdependence between the biosynthetic pathway of TCTB and several primary and specialized metabolic pathways. These results provide further evidence of the multifaceted role of TCTB in the life cycle of F. graminearumIMPORTANCEFusarium graminearum is a major plant pathogen that causes devastating diseases of cereal crops and produces type B trichothecene (TCTB) mycotoxins in infected grains. The best way to restrict consumer exposure to TCTB is to limit their production before harvest, which requires increasing the knowledge on the mechanisms that regulate their biosynthesis. Using a metabolomics approach, we investigated the interconnection between the TCTB production pathway and several fungal metabolic pathways. We demonstrated that alteration in the TCTB biosynthetic pathway can have a significant impact on other metabolic pathways, including the biosynthesis of toxic polyketides, and vice versa. These findings open new avenues for identifying fungal targets for the design of molecules with antimycotoxin properties and therefore improving sustainable strategies to fight against diseases caused by F. graminearum Our data further demonstrate that analyses should consider all fungal toxic metabolites rather than the targeted family of mycotoxins when assessing the efficacy of control strategies.

The chemokines CXCL12 and CXCL14 differentially regulate connective tissue markers during limb development.

Connective tissues (CT) support and connect organs together. Understanding the formation of CT is important, as CT deregulation leads to fibrosis. The identification of CT specific markers has contributed to a better understanding of CT function during development. In developing limbs, Osr1 transcription factor is involved in the differentiation of irregular CT while the transcription factor Scx labels tendon. In this study, we show that the CXCL12 and CXCL14 chemokines display distinct expression pattern in limb CT during chick development. CXCL12 positively regulates the expression of OSR1 and COL3A1, a collagen subtype of irregular CT, while CXCL14 activates the expression of the tendon marker SCX. We provide evidence that the CXCL12 effect on irregular CT involves CXCR4 receptor and vessels. In addition, the expression of CXCL12, CXCL14 and OSR genes is suppressed by the anti-fibrotic BMP signal. Finally, mechanical forces, known to be involved in adult fibrosis, control the expression of chemokines, CT-associated transcription factors and collagens during limb development. Such unexpected roles of CXCL12 and CXCL14 chemokines during CT differentiation can contribute to a better understanding of the fibrosis mechanisms in adult pathological conditions.

Variability of composition and effects of essential oils from Rhanterium adpressum Coss. & Durieu against mycotoxinogenic Fusarium strains.

The antifungal potency of the essential oils of Rhanterium adpressum was evaluated against four mycotoxigenic strains of the genus Fusarium. The essential oils were obtained, separately, by hydro-distillation of the aerial parts of R. adpressum (leaves and flowers). The parts were collected during the period of bloom (3 months) for 3 years. The GC-MS analysis revealed thirty-six compounds for the essential oils, divided into four classes of chemical compounds, with variable percentages according to the month of extraction. The monoterpene hydrocarbons form the main class in these oils. On the other hand, the highest percentages of the oxygenated compounds are observed in the samples collected during the month of May. The direct contact method was used to evaluate the antifungal activity of the essential oils. The activity can be attributed to their relatively high composition of oxygenated monoterpenes. Flowers extract showed strong inhibitory activity, with very interesting concentrations of IC50 and MIC for both tests on solid and liquid medium. The effect of these oils on the production of type B trichothecenes (TCTBs) was evaluated, showing a significant inhibitory effect on TCTBs production, for both extracts (leaves and flowers). The rates of inhibition were 66-97 and 76-100% of FX, 3-ADON and 15-ADON, respectively. The inhibition of fungal biomass and the production of TCTBs depended on the used concentration of the essential oils. These results suggest that the essential oils from R. adpressum are able to control the growth of the tested strains and their subsequent production of TCTB mycotoxins.

EGR1 Regulates Transcription Downstream of Mechanical Signals during Tendon Formation and Healing.

BACKGROUND: Tendon is a mechanical tissue that transmits forces generated by muscle to bone in order to allow body motion. The molecular pathways that sense mechanical forces during tendon formation, homeostasis and repair are not known. EGR1 is a mechanosensitive transcription factor involved in tendon formation, homeostasis and repair. We hypothesized that EGR1 senses mechanical signals to promote tendon gene expression. METHODOLOGY/PRINCIPAL FINDINGS: Using in vitro and in vivo models, we show that the expression of Egr1 and tendon genes is downregulated in 3D-engineered tendons made of mesenchymal stem cells when tension is released as well as in tendon homeostasis and healing when mechanical signals are reduced. We further demonstrate that EGR1 overexpression prevents tendon gene downregulation in 3D-engineered tendons when tension is released. Lastly, ultrasound and microbubbles mediated EGR1 overexpression prevents the downregulation of tendon gene expression during tendon healing in reduced load conditions. CONCLUSION/SIGNIFICANCE: These results show that Egr1 expression is sensitive to mechanical signals in tendon cells. Moreover, EGR1 overexpression prevents the downregulation of tendon gene expression in the absence of mechanical signals in 3D-engineered tendons and tendon healing. These results show that EGR1 induces a transcriptional response downstream of mechanical signals in tendon cells and open new avenues to use EGR1 to promote tendon healing in reduced load conditions.

TGFß and FGF promote tendon progenitor fate and act downstream of muscle contraction to regulate tendon differentiation during chick limb development.

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 TGFß/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 TGFß/SMAD2/3 signalling pathways are decreased in tendons under immobilisation conditions. Application of FGF4 or TGFß2 ligands prevents SCX downregulation in immobilised limbs. TGFß2 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 TGFß promote tendon commitment of limb mesodermal cells and act downstream of mechanical forces to regulate tendon differentiation during chick limb development.

Muscle contraction is required to maintain the pool of muscle progenitors via YAP and NOTCH during fetal myogenesis.

The importance of mechanical activity in the regulation of muscle progenitors during chick development has not been investigated. We show that immobilization decreases NOTCH activity and mimics a NOTCH loss-of-function phenotype, a reduction in the number of muscle progenitors and increased differentiation. Ligand-induced NOTCH activation prevents the reduction of muscle progenitors and the increase of differentiation upon immobilization. Inhibition of NOTCH ligand activity in muscle fibers suffices to reduce the progenitor pool. Furthermore, immobilization reduces the activity of the transcriptional co-activator YAP and the expression of the NOTCH ligand JAG2 in muscle fibers. YAP forced-activity in muscle fibers prevents the decrease of JAG2 expression and the number of PAX7+ cells in immobilization conditions. Our results identify a novel mechanism acting downstream of muscle contraction, where YAP activates JAG2 expression in muscle fibers, which in turn regulates the pool of fetal muscle progenitors via NOTCH in a non-cell-autonomous manner.

Is the Fgap1 mediated response to oxidative stress chemotype dependent in Fusarium graminearum?

This study aims to compare the role of the transcription factor Fgap1 in oxidative stress response for two Fusarium graminearum strains belonging to the two chemotypes DON/ADON and NIV/FX. While the response to H2O2 was shown to be chemotype dependent, an opposite result was observed for diamide: whatever the chemotype, the global level of TCTB (i.e. trichothecene B) production was strongly increased by the treatment with diamide. Fgap1 was shown to be involved in this regulation for both chemotypes. Our data show that the response to diamide is mediated by Fgap1 whatever the chemotype of the F. graminearum strains. However, the NIV/FX chemotype has developed higher antioxidant capacities in response to oxidative stress. But when this capacity is overwhelmed by an increment in the H2O2 level, the NIV/FX strains also responds by an increase in toxin accumulation.

Transcriptomic analysis of mouse limb tendon cells during development.

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-ß and MAPK. TGF-ß/SMAD2/3 gain- and loss-of-function experiments in mouse limb explants and mesenchymal stem cells showed that TGF-ß signalling was sufficient and required via SMAD2/3 to drive mouse mesodermal stem cells towards the tendon lineage ex vivo and in vitro. TGF-ß 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.