Four and a half domain 2 (FHL2) scaffolding protein is a marker of connective tissues of developing digits and regulates fibrogenic differentiation of limb mesodermal progenitors.

Four and a half LIM domain 2 (FHL2) is a multifunctional scaffolding protein of well-known function regulating cell signalling cascades and gene transcription in cancer tissues. However, its function in embryonic systems is poorly characterized. Here, we show that Fhl2 is involved in the differentiation of connective tissues of developing limb autopod. We show that Fhl2 exhibits spatially restricted and temporally dynamic expression around the tendons of developing digits, interphalangeal joint capsules, and fibrous peridigital tissue. Immunolabelling analysis of the skeletal progenitors identified a predominant, but not exclusive, cytoplasmic distribution of FHL2 being associated with focal adhesions and actin cytoskeleton. In the course of chondrogenic differentiation of cultures of limb skeletal progenitors, the expression of Fhl2 is down-regulated. Furthermore, cultures of skeletal progenitors overexpressing Fhl2 take on a predominant fibrogenic appearance. Both gain-of-function and loss-of-function experiments in the micromass culture assays revealed a positive transcriptional influence of Fhl2 in the expression of fibrogenic markers including Scleraxis, Tenomodulin, Tenascin C, ßig-h3, and Tgif1. We further show that the expression of Fhl2 is positively regulated by profibrogenic signals including Tgfß2, all-trans-retinoic acid, and canonical Wnt signalling molecules and negatively regulated by prochondrogenic factors of the bone morphogenetic protein family. Expression of Fhl2 is also regulated negatively in immobilized limbs, but this influence appears to be mediated by other connective tissue markers, such as Tgfßs and Scleraxis.

Analysis of the molecular cascade responsible for mesodermal limb chondrogenesis: Sox genes and BMP signaling.

Here, we have studied how Sox genes and BMP signaling are functionally coupled during limb chondrogenesis. Using the experimental model of TGFbeta1-induced interdigital digits, we dissect the sequence of morphological and molecular events during in vivo chondrogenesis. Our results show that Sox8 and Sox9 are the most precocious markers of limb cartilage, and their induction is independent and precedes the activation of BMP signaling. Sox10 appears also to cooperate with Sox9 and Sox8 in the establishment of the digit cartilages. In addition, we show that experimental induction of Sox gene expression in the interdigital mesoderm is accompanied by loss of the apoptotic response to exogenous BMPs. L-Sox5 and Sox6 are respectively induced coincident and after the expression of Bmpr1b in the prechondrogenic aggregate, and their activation correlates with the induction of Type II Collagen and Aggrecan genes in the differentiating cartilages. The expression of Bmpr1b precedes the appearance of morphological changes in the prechondrogenic aggregate and establishes a landmark from which the maintenance of the expression of all Sox genes and the progress of cartilage differentiation becomes dependent on BMPs. Moreover, we show that Ventroptin precedes Noggin in the modulation of BMP activity in the developing cartilages. In summary, our findings suggest that Sox8, Sox9, and Sox10 have a cooperative function conferring chondrogenic competence to limb mesoderm in response to BMP signals. In turn, BMPs in concert with Sox9, Sox6, and L-Sox5 would be responsible for the execution and maintenance of the cartilage differentiation program.

Expression of Sox8, Sox9 and Sox10 in the developing valves and autonomic nerves of the embryonic heart.

We describe the expression pattern of Sox8, Sox9 and Sox10 during the development of the chick embryo heart. These Sox genes constitute the group E of the large Sox family of transcription factors. We show that the expression of Sox8, Sox9 and Sox10 in the developing heart correlates with heart septation and with the differentiation of the connective tissue of the valve leaflets. Sox10 appears also as a specific marker of developing heart nerves. These findings fit with the occurrence of morphological and functional anomalies of the heart reported in humans deficient for Sox9 and Sox10.

Comparative analysis of the expression and regulation of Wnt5a, Fz4, and Frzb1 during digit formation and in micromass cultures.

Previous studies have shown that three members of the Wnt signaling pathway, the ligand WNT5A, the receptor FZ4, and the Wnt antagonist FRZB1, are implicated in the formation and differentiation of the digits. In this study, we have attempted to establish a functional correlation between them by comparing their expression patterns and their regulation by the signals controlling proliferation and differentiation of the limb mesoderm during formation of the avian digits in vivo and in micromass cultures. In vivo Wnt5a and Fz4 are expressed in the undifferentiated mesoderm of the autopod and in the differentiating digit cartilages. In the undifferentiated mesoderm, the expression of both genes is regulated positively by FGFs and negatively by bone morphogenetic proteins (BMPs). As chondrogenic differentiation starts, Fz4 becomes intensely up-regulated in the aggregate and in the developing perichondrium, whereas transcripts of Wnt5a are excluded from the core of the aggregate but maintained in the surrounding mesenchyme and perichondrium. In addition, at this stage, the expression of both genes become positively regulated by BMPs. These changes in expression and regulation are coincident with the induction of Frzb1 in the chondrogenic aggregate, which is expressed under the positive control of BMPs. Our findings fit with a role of Wnt5a/Fz4 negatively regulating in vivo the initiation and progression of cartilage differentiation. In vitro, only Frzb1 is expressed and regulated in a manner resembling that observed in vivo. Wnt5a and Fz4 are both expressed in the differentiating mesenchyme of micromass cultures, but their expression is not significantly regulated by the addition of FGF-2 or BMP-7 to the culture medium.

Role of FGFs in the control of programmed cell death during limb development.

We have investigated the role of FGFs in the control of programmed cell death during limb development by analyzing the effects of increasing and blocking FGF signaling in the avian limb bud. BMPs are currently considered as the signals responsible for cell death. Here we show that FGF signaling is also necessary for apoptosis and that the establishment of the areas of cell death is regulated by the convergence of FGF- and BMP-mediated signaling pathways. As previously demonstrated, cell death is inhibited for short intervals (12 hours) after administration of FGFs. However, this initial inhibition is followed (24 hours) by a dramatic increase in cell death, which can be abolished by treatments with a BMP antagonist (Noggin or Gremlin). Conversely, blockage of FGF signaling by applying a specific FGF-inhibitor (SU5402) into the interdigital regions inhibits both physiological cell death and that mediated by exogenous BMPs. Furthermore, FGF receptors 1, 2 and 3 are expressed in the autopodial mesoderm during the regression of the interdigital tissue, and the expression of FGFR3 in the interdigital regions is regulated by FGFs and BMPs in the same fashion as apopotosis. Together our findings indicate that, in the absence of FGF signaling BMPs are not sufficient to trigger apoptosis in the developing limb. Although we provide evidence for a positive influence of FGFs on BMP gene expression, the physiological implication of FGFs in apoptosis appears to result from their requirement for the expression of genes of the apoptotic cascade. We have identified MSX2 and Snail as candidate genes associated with apoptosis the expression of which requires the combined action of FGFs and BMPs.

Expression of N-cadherin, N-CAM, fibronectin and tenascin is stimulated by TGF-beta1, beta2, beta3 and beta5 during the formation of precartilage condensations.

Cell surface adhesion and extracellular matrix proteins are known to play a key role in the formation of cell condensations during skeletal development, and their formation is crucial for the expression of cartilage-specific genes. However, little is known about the relationship between adhesion molecules (N-cadherin and N-CAM), extracellular matrix proteins (fibronectin and tenascin) and TGF-beta1, TGF-beta2 and TGF-beta3 during in vitro precartilage condensations in mouse chondrogenesis. On these bases, we determined the participation of mammalian TGF-beta1, TGF-beta2 and TFG-beta3 and Xenopus TGF-beta5 on the expression of cell surface adhesion and extracellular matrix proteins during the formation of precartilage condensations. Also, we characterized the effects of TGF-betas on proteoglycan metabolism at different cellular densities in mouse embryonic limb bud mesenchymal cells. In TGF-beta1 and TGF-beta5-treated cultures, proteoglycan biosynthesis was higher than in controls, while there were no differences in proteoglycan catabolism, which caused the accumulation of cartilage extracellular matrix. When mesenchymal cells were seeded at three different cellular densities in the presence of TGF-betas, only high density cultures presented increased stimulation of proteoglycan biosynthesis, compared to low and intermediate densities. To determine whether the effect of TGF-betas on precartilage condensations is mediated through the expression of N-cadherin, N-CAM, fibronectin and tenascin, we evaluated their expression. Results showed that TGF-beta1, TGF-beta2, TGF-beta3, and TGF-beta5 differentially enhanced the expression of N-cadherin, N-CAM, fibronectin and tenascin in precartilage condensations, suggesting that TGF-beta isoforms play an important role in the establishment of cell-cell and cell-extracellular matrix interactions during precartilage condensations.

Clinical, morphological and biochemical features in the familial articular hypermobility syndrome (FAHS): a family study.

A female with clinical features of familial articular hypermobility syndrome (FAHS) and her family were studied. The subject showed generalized hypermobility, except for a painful shoulder which presented functional limitation with a diagnosis of painful shoulder syndrome. Biochemical studies demonstrated that collagen and glycosaminoglycans (GAGs) contents from skin biopsies of the subject and her family were almost normal. Nevertheless, the densitometric analysis of electrophoretic patterns showed differences in the relative proportions of their collagenous components. They were characterized by changes in type I and III collagens and the presence of type V collagen, in the subject, her father and brother. Also, they presented changes in the types of GAGs, when compared with those of normal skin. Morphological studies revealed a general disorganization of dermal components, a loose collagen network characterized by thick bundles. Also, besides cellular elements, the presence of an abundant darkly staining material was observed. Biochemical and morphological findings permit us to suggest a connective tissue defect, initially described in the FAHS, otherwise known as Ehlers Danlos syndrome (EDS) type XI.

Differential effects of transforming growth factors beta 1, beta 2, beta 3 and beta 5 on chondrogenesis in mouse limb bud mesenchymal cells.

The present study was performed to determine whether mammalian TGF-beta isoforms and Xenopus TGF-beta 5 elicit a differential chondrogenic response on mesenchymal cells during mouse limb development. Results showed that TGF-beta isoforms produced a distinct chondrogenic pattern depending on embryonic stage. When they were applied to 5 day micromass cultures of limb mesenchymal cells from embryonic stages 19, 20 and 21, a differential response to all four TGF-beta isoforms assayed was observed. By stage 19 the cells formed a uniform sheet of cartilage cells; by stage 20, mesenchymal cells were more responsive to TGF-beta 1 and TGF-beta 5 than at stages 19 and 21, showing an entire cell layer of chondrogenic cells with higher accumulation of extracellular matrix. The diminished effect of TGF-beta 2 and TGF-beta 3 at stages 20 and 21 was accompanied by a nodular pattern of chondrogenic cells rather than by a uniform sheet, as seen at stage 19. At stage 20 TGF-beta 1 and TGF-beta 5 enhanced the expression of sulfated proteoglycans, type II collagen, cartilage link protein and alkaline phosphatase activity. In contrast, TGF-beta 2 and TGF-beta 3 caused less expression in the same parameters. Only a transient exposure to TGF-beta isoforms at days 1 and 2 of culture stimulate chondrogenesis, indicating that TGF-beta isoforms could regulate chondrogenesis at early stages of chondrocyte differentiation. However, when TGF-beta isoforms were applied to low density cultures of mesenchymal cells, chondrogenesis was enhanced only by 25%, suggesting that TGF-beta isoforms enhanced cartilage differentiation to higher levels in micromass cultures than in situations in which little or no chondrogenic differentiation normally occurs.