Potentiation by the lithium ion of morphogenetic responses to a Xenopus inducing factor

We have cultured explants of Xenopus blastular animal cap tissue from embryos that had received an earlier treatment with LiCl and from their untreated siblings, in various concentrations of XTC-cell-derived mesoderm-inducing factor (XTC-MIF, Smith, 1987; Smith et al. 1988). The pretreatment with lithium that we used transforms later morphogenesis in the whole embryo to give radialized body forms with anterior/dorsal levels of structure grossly over-represented. In addition, animal caps from 'Li+' embryos were allowed to develop without exposure to in vitro MIF (Li+ controls) and compared with normal uninduced control explants, and explants were made from normal early blastulae but given various initial treatments with LiCl in culture. The results confirm that the lithium ion itself will not induce mesoderm in competent, animal cap tissue of Xenopus. It does, however, enhance the responsiveness of this tissue to XTC-MIF, in a way that parallels its recently reported effect in the case of another mesoderm inducer of different character, bFGF (Slack et al. 1988). The effects observed are sufficient to imply that the altered body pattern that follows lithium treatment, in whole embryos, could be caused by modulation of the responses to an unaltered pattern of in situ inductive stimuli. We also observe evidence that appreciable inductive signals reach animal pole tissue beyond the limits of mesoderm formation in normal development. Relatively low concentrations of MIF prevent the development of an epidermis-specific marker in dissociated blastular animal cap cells (Symes et al. 1988). When such experiments are repeated in relation to the lithium pretreatment of embryos, such treatment is seen to have sensitized the cell population, so that the MIF concentration range that assures complete suppression of the marker is reduced. The results are discussed in relation to induction considered as pattern formation.     

Inductive interactions in the spatial and temporal restriction of lens-forming potential in embryonic ectoderm of Xenopus laevis

The process of lens cell determination in amphibians is currently viewed as one involving a series of inductive interactions. On the basis of previous investigations, these interactions are thought to begin during gastrulation when the presumptive foregut endoderm and then the heart mesoderm come into contact with the presumptive lens ectoderm. This earlier period of induction is followed by the later interaction of the optic vesicle with the lens-forming ectoderm. Transplantation experiments were performed to determine the relative significance of the early and later periods of induction in the process of lens cell determination in the anuran Xenopus laevis. Various ectodermal tissues were transplanted either into the lens-forming region of open neural plate stage host embryos or over the newly formed optic vesicle of later neurula stage embryos. All transplanted tissues were labeled with the intracellular marker horseradish peroxidase to assess the exact origins of any induced lens structures. The results indicate that all nonneural ectodermal tissues have some lens-forming potential early during gastrulation; however, this potential is restricted to the lens-forming region, and perhaps nearby regions, later in development during the time of neurulation. Furthermore, the results show that the optic vesicle is not a substantial inductor of the lens in tissues that have not been previously exposed to the earlier series of inductive interactions that take place during gastrulation and neurulation. Since the optic vesicle does not appear to be a sufficient inductor of the lens, these earlier inductive interactions are, therefore, essential in the process of lens cell determination in Xenopus. These earlier inductive interactions lead to a steady increase in what may be called a lens-forming bias in the presumptive lens ectoderm during this period of development. The eventual loss in the ability of nonlens ventral ectoderm to respond to these lens inductors is presumably the result of other determinative processes that occur in this tissue.     

Analysis of competence: receptors for fibroblast growth factor in early Xenopus embryos

Xenopus ectodermal cells have previously been shown to respond to acidic and basic FGF by differentiating into mesodermal tissue. In the present study, ectodermal explants from Xenopus blastulae were shown to have high affinity binding sites for 125I-aFGF (Kd = 1.4 X 10(-10) M). The total number of sites, determined by Scatchard analysis, was 3 X 10(8) per explant (surface area of approximately 1 mm2). Two putative receptors of relative molecular mass 130,000 and 140,000 were identified by chemical crosslinking to 125I-aFGF. Both acidic and basic FGF, but not TGF beta 2, could compete for affinity labelling of these bands. The receptor density at the cell surface parallels the developmental competence of Xenopus animal pole cells to respond to FGF. Receptors are present at highest density in the marginal zone but are not restricted to cells in this region.     

Biophysical integration of effects of epidermal growth factor and fibronectin on fibroblast migration

Cell migration is regulated simultaneously by growth factors and extracellular matrix molecules. Although information is continually increasing regarding the relevant signaling pathways, there exists little understanding concerning how these pathways integrate to produce the biophysical processes that govern locomotion. Herein, we report the effects of epidermal growth factor (EGF) and fibronectin (Fn) on multiple facets of fibroblast motility: locomotion speed, membrane extension and retraction activity, and adhesion. A surprising finding is that EGF can either decrease or increase locomotion speed depending on the surface Fn concentration, despite EGF diminishing global cell adhesion at all Fn concentrations. At the same time, the effect of EGF on membrane activity varies from negative to positive to no-effect as Fn concentration and adhesion range from low to high. Taking these effects together, we find that EGF and Fn regulate fibroblast migration speed through integration of the processes of membrane extension, attachment, and detachment, with each of these processes being rate-limiting for locomotion in sequential regimes of increasing adhesivity. Thus, distinct biophysical processes are shown to integrate for overall cell migration responses to growth factor and extracellular matrix stimuli.     

Thrombin inactivates acidic fibroblast growth factor but not basic fibroblast growth factor

Incubation of bovine brain derived acidic fibroblast growth factor (aFGF) with bovine or human thrombin, 0.5 NIH unit/mL, for 24 h at 37 degrees C results in cleavage of the mitogen, generating a 14-kilodalton fragment which has significantly reduced affinity for immobilized heparin as compared to aFGF, and is at least 50-fold less potent at stimulating mitogenesis. In addition, an 18 amino acid peptide, aFGF(123-140), is generated, identifying one of the thrombin cleavage sites as the Arg-122/Thr-123 bond. The peptide, aFGF(123-140), is neither mitogenic itself nor an inhibitor of the mitogenic activity of aFGF. The cleavage of aFGF by thrombin is inhibited by heparin (50 micrograms/mL) and is completely blocked by the irreversible thrombin inhibitors D-Phe-Pro-Arg chloromethyl ketone and hirudin. Incubation of aFGF with 50 units/mL thrombin at 37 degrees C results in rapid cleavage of the mitogen into several fragments. In contrast, incubation of bovine brain derived basic fibroblast growth factor with 1 unit/mL thrombin for 24 h, or 50 units/mL thrombin for 6 h, does not result in significant cleavage of mitogen. The results show that the C-terminal region of aFGF is of functional importance in both mitogenesis and heparin binding. Most importantly, a novel role for anionic heparin-binding growth factors and their fragments is indicated in physiologic and pathologic situations associated with thrombin generation.     

Skeletal effects of systemic treatment with basic fibroblast growth factor

Systemic treatment of intact and ovariectomized rats with basic fibroblast growth factor (bFGF) has strong bone anabolic effects. These effects include marked increases in osteoblast number and activity along cancellous and endocortical bone surfaces, which result in accumulation of osteoid and augmentation of cancellous and cortical bone mass after only short-term treatment with bFGF. Osteoclast surface is markedly decreased in bFGF-treated rats, but this finding may be secondary to the extensive osteoid surface in these animals. Some undesirable skeletal effects of the growth factor include impaired bone mineralization and formation of structurally inferior woven bone. The lack of a bone anabolic response to bFGF at skeletal sites with fatty marrow and along the periosteal surface of cortical bone is also disappointing. Despite these disadvantages, bFGF stimulates cancellous bone formation to such a great extent that it may eventually be considered for use in patients with severe osteoporosis who are unresponsive to conventional therapies, provided that local delivery of the growth factor to bone can be achieved to avoid systemic side effects.     

Conversion of a paracrine fibroblast growth factor into an endocrine fibroblast growth factor

FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.     

Cysteine-rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3

Expression of the cysteine-rich fibroblast growth factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the secretion of co-expressed FGF3. By using a column retention assay and affinity chromatography, we demonstrate that at physiological salt concentrations FGF3 binds with strong affinity to CFR in vivo and in vitro. Furthermore, to show that FGF3 binds to CFR in vivo, truncation mutants of CFR with changed subcellular distributions were shown to cause a similar redistribution of FGF3. Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the Golgi apparatus, we show here that in COS-1 cells a substantial proportion of CFR is secreted. This is due to a carboxyl-terminal proteolytic cleavage that releases the intraluminal portion of the protein for secretion. However, the apparent size of the integral membrane and secreted CFR appears similar, since the loss of protein mass is balanced by a gain of complex carbohydrates. The released CFR is associated with the extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may control its biological activity by regulating its secretion.     

碱性成纤维细胞生长因子与肿瘤

碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF) 作为多种细胞的致裂源,与肿瘤的关系近几年来受到关注,本文概述了有关bFGF与肿瘤关系的研究进展。     

The pleiotropic effects of fibroblast growth factor receptors in mammalian development

In recent years the study of fibroblast growth factor receptors (FGFRs) in normal development and human genetic disorders has increased our understanding of some complex cellular processes. At least fifteen genetic disorders result from mutations within FGFR genes including skeletal dysplasias such as Apert syndrome and achondroplasia. In vitro experiments and the generation of animal models indicate that these mutations result in activation of the receptors and that FGFRs act as negative regulators of bone growth. FGFRs also play a role in wound healing and cancer. In this article, we review the expression of FGFRs in human development, the phenotypes resulting from FGFR mutations, and recent data identifying pathways downstream of the activated receptors.     

Over-expression of fibroblast growth factors in Xenopus embryos.

A number of forms of fibroblast growth factor (FGF) were over-expressed within Xenopus embryos by injection of synthetic FGF mRNAs into fertilized eggs. Injected embryos showed abnormalities in development which were mainly secondary to a disruption of gastrulation movements. The effects observed after injection of bFGF mRNA, however, were much less severe than those observed after injection of an altered form of bFGF mRNA which differs only by the addition of a signal sequence for secretion, or of another member of the FGF family, kFGF, which is normally efficiently secreted. All forms of FGF caused the induction of mesoderm in animal cap explants isolated from blastulae, but the amount of bFGF mRNA required to induce the formation of significant levels of mesoderm was higher by a factor of over a hundred than that of the FGFs which contain a signal sequence for secretion. Over-expressed bFGF accumulated in the nuclei of blastulae but did not necessarily cause mesoderm formation. These results show that FGFs must be secreted from the cells in which they are synthesised in order to act efficiently as mesoderm inducing factors and suggest that bFGF itself, which does not contain a signal sequence for secretion, is unlikely to be directly involved in mesoderm induction during early embryonic development.     

Fibroblast growth factor signalling and regional specification of the pharyngeal ectoderm

Branchial arch development involves dynamic interactions between neural crest cells as well as ectodermal, endodermal and mesodermal cell populations. Despite their importance and evolutionary conservation, the intercellular interactions guiding the early development of the branchial arches are still poorly understood. We have here studied fibroblast growth factor (FGF) signalling in early pharyngeal development. In mice homozygous for a hypomorphic allele of Fgfr1, neural crest cells migrating from the hindbrain mostly fail to enter the second branchial arch. This defect is non-cell-autonomous suggesting that Fgfr1 provides a permissive environment for neural crest cell migration. Here we demonstrate localized down-regulation of the expression of the FGF responsive gene, Sprouty1 in the epithelium covering the presumptive second branchial arch of hypomorphic Fgfr1 mutants. This appears to result in a failure to establish an ectodermal signalling center expressing Fgf3 and Fgf15. We also studied differentiation of the ectoderm in the second branchial arch region. Development of the geniculate placode as well as the VIIth cranial ganglion is affected in Fgfr1 hypomorphs. Our results suggest that Fgfr1 is important for localized signalling in the pharyngeal ectoderm and consequently for normal tissue interactions in the developing second branchial arch.     

Effects of platelet-derived growth factor and fibroblast growth factor on free intracellular calcium and mitogenesis

Although increased free intracellular calcium (Cai) may be one of the main regulators of cell growth and differentiation, studies in cell populations have implied that not all growth factors produce Cai increases. In order to examine in more detail whether Cai increases were related to mitogenesis, we used digital image analysis of intracellular Fura-2 fluorescence to measure Cai in individual BALB/c 3T3 cells stimulated with either platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF). We found that PDGF induced larger and more prolonged Cai increases than FGF did, but that both growth factors induced an initial rapid increase in Cai (less than 2 min) followed by a later sustained increase (greater than 20 min). Only the prolonged Cai increase required extracellular calcium. Following PDGF treatment (1-8 units/ml), the percentage of cells with a large peak Cai increase (greater than twofold) correlated with the percentage of cells made competent (subsequent growth in 1% platelet-poor-plasma). In contrast, purified bovine basic FGF (200-800 pg/ml) and recombinant human acidic FGF (10-300 ng/ml) produced peak Cai increases that were not directly correlated with mitogenesis. In addition, concentrations of intracellular Quin 2 that inhibited Cai transients also inhibited PDGF stimulation but not FGF stimulation of mitogenesis. Thus, Cai increases are necessary for mitogenesis in BALB/c 3T3 cells stimulated by PDGF, but not that stimulated by FGF.     

Dura mater biology: autocrine and paracrine effects of fibroblast growth factor 2

The dura mater, the outermost layer of the meninges, is thought to be essential for calvarial morphogenesis, postnatal suture fusion, and osseous repair of calvarial defects. Despite numerous studies illustrating the fundamental role of the dura mater, there is little information about the autocrine and paracrine mechanisms regulating dural cell biology during calvarial ossification. Previous work conducted in the authors' laboratory demonstrated that non-suture-associated dural cells from 6-day-old rat pups expressed high levels of fibroblast growth factor 2 (FGF-2), whereas dural cells from 60-day-old adult rats expressed very little FGF-2. Because young mammals can successfully heal large calvarial defects, the authors sought to investigate the autocrine and/or paracrine effects of FGF-2 on the proliferation, gene expression, and alkaline phosphatase production of dural cells.Cultures of non-suture-associated dural cells were established from 6-day-old Sprague-Dawley rat pups and then stimulated with recombinant human FGF-2 (rhFGF-2; 10 ng/ml). Dural cells stimulated with rhFGF-2 proliferated significantly faster than untreated dural cells at 24 hours (2.1 x 10(5) +/- 3.2 x 10(4) versus 1.1 x 10(5) +/- 1.8 x 10(4), p < or = 0.001) and 48 hours (2.3 x 10(5) +/- 4.2 x 10(4) versus 1.2 x 10(5) +/- 1.3 x 10(4), p < or = 0.001). Moreover, dural cells stimulated with rhFGF-2 expressed 7-fold more proliferating cell nuclear antigen than did control cultures. Treatment with rhFGF-2 increased dural cell expression of genes important for skeletal repair: FGF-2 (7-fold), transforming growth factor beta 1 (3-fold), transforming growth factor beta 3 (4-fold), and type I collagen (4-fold). Furthermore, rhFGF-2 increased dural cell expression of osteopontin (2-fold), a "late" marker of osteoblastic differentiation. Interestingly, dural cell alkaline phosphatase activity, an "earlier" marker of osteoblast differentiation, was significantly decreased by treatment with rhFGF-2 compared with control cultures at 24 hours (0.005 +/- 0.001 versus 0.01 +/- 0.003, p < or = 0.01) and 48 hours (0.004 +/- 0.0009 versus 0.01 +/- 0.0009). Together these data provide insight into the autocrine and paracrine effects of FGF-2 on the biology of the dura mater.