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Biomedical subjects

S Pizette

Publications and source records attributed to S Pizette.

11 recordsLinked to original sources
PubMed2001

Early steps in limb patterning and chondrogenesis.

The interplay of a number of signalling molecules coordinates growth and patterning of the early embryonic vertebrate limb along the three axes. An unresolved question is how this information translates into proper positioning and patterning of the skeletal elements. More is known about how these elements develop. Cells first form precartilaginous condensations and then subsequently differentiate into chondrocytes. This provides a cartilage template that will ultimately be ossified to give rise to the bony skeleton. Several studies support a role for the bone morphogenetic proteins (BMPs) as extracellular signals that regulate early steps of limb chondrogenesis (cartilage formation). However, they have not clarified the step(s) at which BMPs act, and no genetic evidence is available to date. Here, we have used a retroviral vector to misexpress the BMP antagonist Noggin in the embryonic chick limb. We find that BMP signalling is necessary for the formation of precartilaginous condensations, their differentiation into chondrocytes, and for maintenance of chondrogenesis. These results also indicate that Noggin could be clinically useful to treat diseases involving ectopic cartilage formation.

Animals↗
PubMed2001

BMP controls proximodistal outgrowth, via induction of the apical ectodermal ridge, and dorsoventral patterning in the vertebrate limb.

Dorsoventral (DV) patterning of the vertebrate limb requires the function of the transcription factor Engrailed 1 (EN1) in the ventral ectoderm. EN1 restricts, to the dorsal half of the limb, the expression of the two genes known to specify dorsal pattern. Limb growth along the proximodistal (PD) axis is controlled by the apical ectodermal ridge (AER), a specialized epithelium that forms at the distal junction between dorsal and ventral ectoderm. Using retroviral-mediated misexpression of the bone morphogenetic protein (BMP) antagonist Noggin or an activated form of the BMP receptor in the chick limb, we demonstrate that BMP plays a key role in both DV patterning and AER induction. Thus, the DV and PD axes are linked by a common signal. Loss and gain of BMP function experiments show that BMP signaling is both necessary and sufficient to regulate EN1 expression, and consequently DV patterning. Our results also indicate that BMPs are required during induction of the AER. Manipulation of BMP signaling results in either disruptions in the endogenous AER, leading to absent or severely truncated limbs or the formation of ectopic AERs that can direct outgrowth. Moreover, BMP controls the expression of the MSX transcription factors, and our results suggest that MSX acts downstream of BMP in AER induction. We propose that the BMP signal bifurcates at the level of EN1 and MSX to mediate differentially DV patterning and AER induction, respectively.

Animals↗
PubMed2000

BMPs are required at two steps of limb chondrogenesis: formation of prechondrogenic condensations and their differentiation into chondrocytes.

Formation of the long bones requires a cartilage template. Cartilage formation (chondrogenesis) proceeds through determination of cells and their aggregation into prechondrogenic condensations, differentiation into chondrocytes, and later maturation. Several studies indicate that members of the bone morphogenetic protein (BMP) family promote cartilage formation, but the exact step(s) in which BMPs are involved during this process remains undefined. To resolve this issue, we have used a retroviral vector to misexpress the BMP antagonist Noggin in the embryonic chick limb. Unlike previous reports, we have characterized the resulting phenotype in depth, analyzing histological and early chondrogenic markers, as well as the patterns of cell death and proliferation. Misexpression of Noggin prior to the onset of chondrogenesis leads to the total absence of skeletal elements, as previously reported (J. Capdevila and R. L. Johnson, 1998, Dev. Biol. 197, 205-217). Noggin inhibits cartilage formation at two distinct steps. First, we demonstrate that mesenchymal cells do not aggregate into prechondrogenic condensations, and additional results suggest that these cells persist in an undifferentiated state. Second, we show that differentiation of chondroprogenitors into chondrocytes can also be blocked, concurrent with expanded expression of a presumptive joint region marker. In addition, we observed alterations in muscle and tendon morphogenesis, and the potential role of BMPs in these processes will be discussed. Our studies therefore provide in vivo evidence that BMPs are necessary for different steps of chondrogenesis: chondroprogenitor determination and/or condensation and subsequent differentiation into chondrocytes.

Animals↗
PubMed1999

BMPs negatively regulate structure and function of the limb apical ectodermal ridge.

The apical ectodermal ridge (AER), a transient specialized epithelium at the distal limb tip, is essential for vertebrate embryonic limb outgrowth along the proximodistal axis. Among all the molecules expressed in the AER, only the Fibroblast Growth Factors (FGFs) have been shown to substitute for its function in limb outgrowth. After specification of the skeletal progenitors is complete, the AER regresses, having fulfilled its function. However, the cellular processes underlying AER regression remain largely unclear, and the molecular ones, totally unknown. Members of the Bone Morphogenetic Protein (BMP) family are expressed in the AER throughout its life and in the mesenchyme. Our studies using misexpression of Noggin, a BMP inhibitor, reveal an unsuspected role for BMPs in the negative regulation of Fgf expression and AER function. We find that BMPs limit limb outgrowth by promoting AER regression, as BMP inhibition results in persistence of the AER, prolonged Fgf expression and excess soft-tissue growth. In addition, the Noggin misexpression studies uncover an earlier role for BMPs in repression of AER function. Noggin overexpression results in extension of the AER anteriorly and loss of AER asymmetry. We show that overall the AER becomes taller, and its anterior half becomes more similar to a normal posterior AER. In addition, Fgf4 transcripts, which are usually restricted to the posterior half of the AER, are now also expressed anteriorly. Moreover, ectopicFgf4 expression is induced independently of Sonic Hedgehog, contrary to current models of Fgf4 regulation in the limb. Our studies also provide insight into the activity of the hypothesized apical ectodermal maintenance factor (AEMF), which is thought to maintain the tall shape of the posterior part of the AER. Our work shows that the AER is negatively regulated by BMP.

Animals↗
PubMed1996

FGF6 modulates the expression of fibroblast growth factor receptors and myogenic genes in muscle cells.

Fgf6 is the only known member of the FGF family whose expression is restricted to the muscle cell lineage during development, suggesting it may have a role in myogenesis. Muscle satellite cells but not C2 myoblast cells were found to express Fgf6. We have used purified recombinant FGF6 protein to explore the effect of this factor on C2 cells in culture. FGF6 stimulated the proliferation of C2 myoblasts and, in combination with heparin, induced their morphological transformation. FGF6, added at 5 ng/ml and in the presence of heparin, increased the expression of a subset of muscle cell differentiation markers. In contrast, at 25 ng/ml, it down-regulated the expression of myogenic markers and myogenic transcription factors examined and delayed differentiation into myotubes of C2 cells. It also up-regulated the expression of FgfR1 and had an opposite effect on FgfR4. These results suggest that intramuscular FGF6 concentrations could influence the proliferation and differentiation processes taking place during development.

Animals↗
PubMed1996

Structure and developmental expression of mouse Garp, a gene encoding a new leucine-rich repeat-containing protein.

Proteins with leucine-rich repeats (LRR) constitute a large family of molecules playing a role in protein-protein interactions and signal transduction. They are involved in various cellular processes in different species. We characterized the organization and pattern of expression of the mouse Garp gene. It is composed of two coding exons, expressed as a major 4.3 kb mRNA, and encodes a putative LRR transmembrane protein with an extracellular region almost entirely made of 20 repeats, and a short intracytoplasmic region. The mouse GARP deduced amino-acid sequence is highly similar to that of the human protein. The Garp gene is expressed in various areas in the mid-gestation developing embryo, including skin, lens fibre cells, nasal cavity, smooth and skeletal muscles, lung, and megakaryocytes of the fetal liver. In the adult it is expressed in the megakaryocytes of the spleen and in endothelial cells of the placenta. The data suggests that GARP might be involved in platelet-endothelium interactions.

Aging↗
PubMed1995

A diphtheria toxin/fibroblast growth factor 6 mitotoxin selectively kills fibroblast growth factor receptor-expressing cell lines.

The fibroblast growth factors (FGFs) constitute a family of nine polypeptides implicated in a number of physiological and pathological processes. They bind to at least three types of cell surface molecules, including low and high affinity receptor families. The role of FGFs and their receptors in human tumorigenesis has been suspected but not formally proven. FGF6 is an oncogene encoding a precursor protein of 208 amino acids that has been shown to bind to FGF receptors. Its normal function has not been identified, but its restricted pattern of expression suggests a role in muscle development or function. We have constructed, produced, and purified a diphtheria toxin/FGF6 mitotoxin that selectively kills FGF receptor-expressing cells. Interestingly, at least two cell lines that normally respond to FGF6 have been found resistant to DT/FGF6, suggesting that FGF6 acts on these cells through a transduction pathway that does not involve FGF receptor.

3T3 Cells↗
PubMed1994

The human and mouse fibroblast growth factor 6 (FGF6) genes and their products: possible implication in muscle development.

FGF6 is structurally very similar to the other members of the FGF gene family, and particularly to the FGF4 gene, which was instrumental in its isolation. Its longest open reading frame encodes a 208 amino acid residues long protein, both in man and in the mouse. It is expressed as a 4.8 kb transcript in skeletal muscle. In developing muscle, expression starts at the myotomal stage and culminates in differentiated fetal muscle masses. In culture, FGF6 protein is mitogenic and has a transforming capacity for fibroblasts. It represses the terminal differentiation of myoblasts. Action of FGF6 could be mediated by the FGFR4 receptor, which binds FGF6 and whose gene is also expressed in developing skeletal muscle.

Animals↗
PubMed1993

Expression of the Fgf6 gene is restricted to developing skeletal muscle in the mouse embryo.

Fgf6, a member of the Fibroblast Growth Factor (FGF) family, is developmentally regulated and its expression is highly restricted in the adult. To gain further insight into the role of Fgf6, we studied its expression during embryogenesis using RNA in situ hybridization. Fgf6 expression is restricted to developing skeletal muscle. Fgf6 transcripts are first detected in the somites at 9.5 days post-conceptus, and expression continues in developing skeletal muscles up to at least 16.5 days post-conceptus. Fgfr4 is a putative receptor for FGF6. Its pattern of expression during myogenesis overlaps that of Fgf6, but both genes are not expressed in exactly the same population of cells. In addition, recombinant FGF6 protein is able to repress the terminal differentiation of myoblasts in culture, providing additional support to the concept that FGF6 plays an important role in myogenesis.

Animals↗
PubMed1991

Production and functional characterization of human recombinant FGF-6 protein.

The fibroblast growth factor (FGF) gene family to date comprises seven members and has been implicated in a wide range of physiological and biological processes, including angiogenesis, morphogenesis, and tumorigenesis. The FGFs are mitogens for a broad range of cells of various embryological origins and can act as differentiation factors. The FGFs can bind to tyrosine kinase and non-tyrosine kinase transmembrane receptors; the physiological basis for this is still unknown. In order to study more thoroughly the activities of FGF-6, we have constructed a bacterial expression vector by inserting FGF-6 complementary DNA sequences into the T7 RNA polymerase-based pET3a vector. The resulting construct is able to drive the expression of a high amount of FGF-6 protein in Escherichia coli, which can be solubilized and purified through heparin-Sepharose chromatography and high salt elution. The purified FGF-6 protein displays a strong mitogenic activity on BALB/c 3T3 cells and is able to morphologically transform these cells. By contrast, adult bovine aortic endothelial cells, which normally require the presence of FGF-2 for their growth, show only a limited mitogenic response that is highly dependent on heparin concentration.

3T3 Cells↗