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.     

在非洲爪蛙胚胎中过表达atf4影响早期神经发育及眼睛的形成

越来越多的证据表明转录激活因子4(atf4)是一个与胚胎发育相关的基因.该文研究了非洲爪蛙atf4在胚胎发育过程中的表达和功能.atf4特异性地表达在非洲爪蛙胚胎的脑部、眼睛、血岛、原肾、肝脏、胰腺以及胃和十二指肠的部分细胞.在非洲爪蛙胚胎的动物极半球过表达适量(不影响胚胎整体形态发生的剂量)的atf4,对神经上皮细胞中sox3的表达无明显影响,也不引起细胞凋亡;但是对原始神经元的标记基因以及预定形成前脑、中脑、视网膜和晶状体的前体细胞的标记基因表达都有不同程度的抑制,最终导致无晶状体小眼的表型.该研究结果首次提示对正常的早期神经发育及眼睛形成而言,atf4的活性需受到严格的调控.     

A Drosophila receptor-type tyrosine kinase (DFR1) acts as a fibroblast growth factor receptor in Xenopus embryos

Members of the fibroblast growth factor (FGF) family play important roles in various developmental processes in vertebrates. Since two genes closely related to the vertebrate FGF receptor (FGFR) genes DFR1 and DFR2/breathless have already been reported in Drosophila , the existence of a Drosophila FGF has been predicted. In the present study, we examined whether DFR1 is functionally interchangeable with a vertebrate FGFR in the Xenopus system. First, we found that the expression of DFR1 promoted Ca²⁺ efflux in response to human basic (b)FGF in Xenopus oocytes, whereas the coexpression of a dominant negative form of DFR1 (ΔDFR1) with a chick FGFR1/cek1 inhibited promotion of Ca²⁺ efflux induced by the expression of cek1 in the oocyte. Second, the expression of ΔDFR1 was observed to induce a defect in the posterior structure of the Xenopus embryo at stage 30, as observed with a dominant negative form of cek1 (Δcek1). Third, we found that the expression of ΔDFR1 inhibited the expression of FGF-regulated genes such as Xbra, Xnot , and Xshh in Xenopus embryos at stage 11, while the coexpression of DFR1 with ΔDFR1 could rescue the inhibited expression of FGF-regulated genes. These results indicate that DFR1 acts as an FGFR in Xenopus embryos and that an FGF is likely to exist in Drosophila .     

Neural inducing factors from Xenopus laevis eggs and embryos

Large foreheads can be induced by ribonucleoprotein particles from Xenopus laevis eggs and embryos. The host embryos develop only a rudimentary primary axis. A neural inducing factor from the cytosol of gastrula-neurula stages has been partially purified. The factors are associated with other proteins in larger complexes.     

Cell transformation by fibroblast growth factors can be suppressed by truncated fibroblast growth factor receptors.

Ligand-induced dimerization and transphosphorylation are thought to be important events by which receptor tyrosine kinases generate cellular signals. We have investigated the ability of signalling-defective, truncated fibroblast growth factor (FGF) receptors (FGFR-1 and FGFR-2) to block the FGF response in cells that express both types of endogenous FGF receptors. When these dominant negative receptors are expressed in NIH 3T3 cells transformed by the secreted FGF-4, the transformed properties of the cells can be reverted to various degrees, with better reversion phenotype correlating with higher levels of truncated receptor expression. Furthermore, truncated FGFR-2 is significantly more efficient at producing reversion than FGFR-1, indicating that FGF-4 preferentially utilizes the FGFR-2 signalling pathway. NIH 3T3 clones expressing these truncated receptors are more resistant to FGF-induced mitogenesis and also exhibit reduced tyrosine phosphorylation upon treatment with FGF. The block in FGF-signalling, however, can be overcome by the addition of excess growth factor. The truncated receptors have binding affinities that are four- to eightfold lower than those of wild-type receptors, as measured by Scatchard analysis. We also observed a partial specificity in the responses of truncated-receptor-expressing clones to FGF-2 or FGF-4. Our results suggest that the block to signal transduction produced by kinase-negative FGF receptors is achieved through a combination of dominant negative effects and competition for growth factor binding with functional receptors.     

Over-expression of fibroblast activation protein alpha increases tumor growth in xenografts of ovarian cancer cells

Fibroblast activation protein alpha （FAPα） is a 95-kDa serine protease of post-prolyl peptidase family on cell surface. FAPoL is widely expressed in tumor microenviron- ment. The wide spread association of FAPα expression with cancer suggests that it has important functions in the disease. However, the nature of FAPα＇s roles in cancer cell activity is not well-determined. It has been showed that FAPα silencing in SKOV3 cells induces ovarian tumors but significantly reduces tumor growth in a xenograft mouse model. To further determine the role of FAPoL in epithelial ovarian cancer cells, SKOV3-FAPα and HO8910-FAPα cell lines, which over-expressed FAPα stably, were con- structed and then their biological behaviors were investi- gated. It was found that FAPoL promoted ovarian cancer cell proliferation, drug resistance, invasiveness, and migra- tion in vitro. Immunochemistry assay showed that FAPα significantly facilitated tumor growth in xenograft tumor tissues. These results suggested that FAPα might directly promote tumor growth and invasiveness in ovarian cancer cells.     

Functions of fibroblast growth factors in vertebrate development

Fibroblast growth factors (FGFs) are a class of secreted polypeptide ligands which mediate diverse cellular responses during embryonic, fetal, and postnatal vertebrate development. The purposes of this review are to provide a condensed overview of FGFs and their receptors, to catalog and categorize the functions of FGFs in vertebrate development, to present recent discoveries relating to the interplay of FGFs with other secreted ligands in the control of tissue growth and patterning, and to discuss several potential directions for future research in the field.     

The role of growth factors in embryonic induction in Xenopus laevis.

Establishment of the body pattern in all animals, and especially in vertebrate embryos, depends on cell interactions. During the cleavage and blastula stages in amphibians, signal(s) from the vegetal region induce the equatorial region to become mesoderm. Two types of peptide growth factors have been shown by explant culture experiments to be active in mesoderm induction. First, there are several isoforms of fibroblast growth factor (FGF), including aFGF, bFGF, and hst/kFGF. FGF induces ventral, but not the most dorsal, levels of mesodermal tissue; bFGF and its mRNA, and an FGF receptor and its mRNA, are present in the embryo. Thus, FGF probably has a role in mesoderm induction, but is unlikely to be the sole inducing agent in vivo. Second, members of the transforming growth factor-beta (TGF-beta) family. TGF-beta 2 and TGF-beta 3 are active in induction, but the most powerful inducing factors are the distant relatives of TGF-beta named activin A and activin B, which are capable of inducing all types of mesoderm. An important question relates to the establishment of polarity during the induction of mesoderm. While all regions of the animal hemisphere of frog embryos are competent to respond to activins by mesoderm differentiation, only explants that include cells close to the equator form structures with some organization along dorsoventral and anteroposterior axes. These observations suggest that cells in the blastula animal hemisphere are already polarized to some extent, although inducers are required to make this polarity explicit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparin-copper biaffinity chromatography of fibroblast growth factors

A novel method is described to separate and identify the various forms of fibroblast growth factor (FGF) based on their differential affinities for both heparin and copper. FGFs were extracted from bovine hypothalamus and purified by batchwise adsorption to heparin-Sepharose. The partially purified FGFs were then applied to an affinity column prepared by mixing equal portions of heparin-Sepharose and copper-Sepharose. The column was rinsed consecutively with the following four reagents: (i) 2 M NaCl, (ii) 0.6 M NaCl, (iii) 0.6 M NaCl plus 10 mM imidazole, and (iv) 0.6 m NaCl. FGFs were then eluted with a linear NaCl/imidazole gradient (from 0.6 m NaCl without imidazole to 2 M NaCl plus 10 mM imidazole). Fractions eluted from the column were analyzed by sodium dodecyl sulfate-gel electrophoresis with silver staining and electrophoretic immunoblot using site-specific antibodies against basic and acidic FGF. The results demonstrate that it is possible to resolve from hypothalamus at least two basic FGF species (with Mr values of 19,000 and 18,000) and three acidic FGF species (with Mr values of 18,000, 16,400, and 15,600). These findings indicate that heparin-copper biaffinity chromatography may have wide applicability in the study of the structure and activity of FGFs.     

RNA sorting in Xenopus oocytes and embryos.

Cytoplasmic localization of mRNA molecules has emerged as a powerful mechanism for generating spatially restricted gene expression. This process is an important contributor to cell polarity in both somatic cells and oocytes, and can provide the basis for patterning during embryonic development. In vertebrates, this phenomenon is perhaps best documented in the frog, Xenopus laevis, where polarity along the animal-vegetal axis coincides with the localization of numerous mRNA molecules. Research over the last several years has made exciting progress toward understanding the molecular mechanisms underlying cytoplasmic mRNA localization.     

Regulation of rat proximal tubule epithelial cell growth by fibroblast growth factors, insulin-like growth factor-1 and transforming growth factor-beta, and analysis of fibroblast growth factors in rat kidney.

Growth factors may play an important role in regulating the growth of the proximal tubule epithelium. To determine which growth factors could be involved, we have investigated the mitogenicity of various purified factors in rat kidney proximal tubule epithelial (RPTE) cells cultured in defined medium. Fibroblast growth factors, aFGF (acidic FGF) and bFGF (basic FGF), stimulate DNA synthesis in a dose-dependent manner, with ED50 values of 4.5 and 3.2 ng/ml, respectively; their effects are not additive. With cholera toxin in the medium, both aFGF and bFGF can replace insulin or epidermal growth factor (EGF) to attain the maximum level of cell growth, but they cannot replace cholera toxin. Cholera toxin specifically potentiates the effects of FGFs on DNA synthesis. At high cell density, both insulin and insulin-like growth factor 1 (IGF-1) induce DNA synthesis more effectively than EGF, FGFs and cholera toxin. The high concentration (0.2-1.0 microgram/ml) of insulin required for cell growth can be replaced by a low concentration of IGF-1 (10-20 ng/ml), indicating that insulin probably acts through a low affinity interaction with the IGF-1 receptor. Transforming growth factor-beta 1 (TGF-beta 1) inhibits DNA synthesis induced by individual factors and combinations of factors in a concentration-dependent manner. Northern blot analysis shows that mRNA for TGF-beta 1, IGF-1, and aFGF, but not bFGF are present in rat kidney. Western blot analysis and bioassay data confirmed that the majority of FGF-like protein in rat kidney is aFGF. The data suggest that in addition to EGF, IGFs, and TGF-beta, FGFs may also be important kidney-derived regulators of proximal tubule epithelial cell growth in vivo and in vitro.     

Hormone-like fibroblast growth factors and metabolic regulation

The family of fibroblast growth factors (FGFs) consisting now of 22 members is generally considered to control a wide range of biological functions such as development, differentiation and survival. However, research during the past decade provided substantial evidence that a so called “hormone-like” subgroup of FGFs, comprised of FGF19, FGF21 and FGF23, is involved in the regulation of diverse metabolic pathways to control glucose, lipid, bile acid, phosphate and vitamin D metabolism. The unique properties of these FGFs include predominant production of the factors in selective tissues, their abundance in the blood due to the lack of extracellular heparin-mediated sequestration, and highly specific tissue-targeted action via engagement of their respective co-receptors. The important metabolic context of FGF19, FGF21, and FGF23 actions has revealed important novel roles for FGFs and provided significant means to explore an opportunity for therapeutic targeting of these factors and their corresponding pathways.     

Regeneration of the tail bud in Xenopus embryos.

In an attempt to solve some aspect of the long-standing controversy about the regenerative ability of appendages in vertebrate embryos, the tail bud of Xenopus laevis embryos has beenamputated at stage sranging from St. 26 to St. 32 and its ability to regenerate duringa culture period of 2-3 days has been studied. At amputation stages 26-28,the tail bud consisted only undifferentialted mesoderm and ectoderm, but at stage 32 it had afully differentiated neural tube, a vaculotaed notochord and segmented somites. A total of 137amputations at differnt stages gace consistent results: a tail formed in all the operated larvacand it had normal, well-developed axial tissues in most cases. The relatively few cases with abnormal tail struture were stunted, oedematour larvae with defects in the trunk region as well. It is concluded from these experiments that cells near the original tail budare able to differentiate into tialbud tissues and to replace the amputated regoin, even at these late embryoic stages. The implications of these findings for comparative studies on regeneration in vertebrates are discussed.