Changes in FGF and FGF receptor expression in low-frequency-stimulated rat muscles and rat satellite cell cultures

This study compares effects of chronic electrical stimulation on the expression levels of FGF-1, FGF-2 and their receptors (FGFRI, FGFR4) in rat tibialis anterior (TA) muscle of hypothyroid rat, as well as in satellite cell cultures derived from normal rat TA and soleus (SOL) muscles. In 5-day (5-d)-stimulated hypothyroid TA muscle, FGF-1 and FGF-2 mRNA levels were threefold elevated over control. FGFR1 and FGFR4 mRNAs were twofold and 1.5-fold elevated, respectively. In longer stimulated muscles, FGF-1 and FGFR4 mRNAs returned to basal levels, whereas FGF-2 mRNA remained elevated. FGFR1 mRNA decreased to control levels in 10-d stimulated muscles, but increased again after 20 days of stimulation. SOL- and TA-derived satellite cell cultures were stimulated for 5 days. At this time point, changes in myosin heavy chain isoforms were detectable consisting of increases in MHCI mRNA and decreases in MHCIIb and MHCIId mRNA. The comparison between 5-d-stimulated hypothyroid TA muscle and 5-d-stimulated TA- and SOL-derived satellite cell cultures revealed differences in the expression of FGF-1 and FGF-2, but similar expression levels of FGFR1 and FGFR4. Even though FGF-1 and FGF-2 mRNAs were elevated in the satellite cell cultures, their increases were less pronounced than in the stimulated hypothyroid muscle. Taking into consideration that skeletal muscle contains muscle fibres and various non-muscle tissues, e.g. blood vessels, these results suggest that the latter contribute to the observed increases in FGF-1 and FGF-2 expression in stimulated muscle.     

Expression of cholecystokinin type A receptor gene correlates with DNA demethylation during postnatal development of rat pancreas.

Cholecystokinin stimulates pancreatic amylase secretion, gallbladder contraction, and pancreatic growth, etc. by binding with high affinity to a cholecystokinin type A receptor (CCKAR). To better understand the expression of CCKAR mRNA in terms of tissue specificity and postnatal development, we determined the methylation status of BssHII sites (5'-B sites) in the rat CCKAR gene promoter. The 5'-B sites in adult pancreas expressing CCKAR mRNA were much less extensively methylated than those in fetal pancreas not expressing the mRNA. In brain, liver, and kidney of adult rats not expressing CCKAR mRNA, the 5'-B sites were methylated. In pancreas, the demethylation level of the sites increased at 21 days after birth. Concomitant with the DNA demethylation level in the 5'-B sites, the mRNA level rose rapidly in 21 days. These results demonstrate that methylation and expression of the CCKAR gene reveal a good inverse correlation.     

Expression and glycosylation studies of human FGF receptor 4

Fibroblast growth factor receptor subtype 4 (FGFR4) has been shown to have special activation properties and just one splicing form, unlike the other FGFRs. FGFR4 overexpression is correlated with breast cancer and therefore FGFR4 is a target for drug design. Our aim is to overexpress high amounts of homogeneous FGFR4 extracellular domain (FGFR4(ed)) for structural studies. We show that baculovirus-insect cell-expressed FGFR4(ed) is glycosylated on three (N88, N234, and N266) of the six possible N-glycosylation sites but is not O-glycosylated. The deglycosylated triple mutant was expressed and had binding properties similar to those of glycosylated FGFR4(ed), but was still heterogeneous. Large amounts of FGFR4(ed) have been produced into inclusion bodies in Escherichia coli and refolded at least partly correctly but the refolded E. coli-produced FGFR4(ed) still aggregates.     

Spatial and temporal expression of heparan sulfate in mouse development regulates FGF and FGF receptor assembly

Heparan sulfate (HS) interacts with diverse growth factors, including Wnt, Hh, BMP, VEGF, EGF, and FGF family members, and is a necessary component for their signaling. These proteins regulate multiple cellular processes that are critical during development. However, a major question is whether developmental changes occur in HS that regulate the activity of these factors. Using a ligand and carbohydrate engagement assay, and focusing on FGF1 and FGF8b interactions with FGF receptor (FR)2c and FR3c, this paper reveals global changes in HS expression in mouse embryos during development that regulate FGF and FR complex assembly. Furthermore, distinct HS requirements are identified for both complex formation and signaling for each FGF and FR pair. Overall, these results suggest that changes in HS act as critical temporal regulators of growth factor and morphogen signaling during embryogenesis.     

Expression of the FGF receptor 2 gene (fgfr2) during embryogenesis in the zebrafish Danio rerio

We isolated a full-length cDNA clone for the zebrafish homologue of fibroblast growth factor receptor (FGFR) 2. The deduced protein sequence is typical of vertebrate FGFRs in that it has three Ig-like domains in the extracellular region. The expression of fgfr2 is initiated during epiboly in the paraxial mesoderm. During early somitogenesis, fgfr2 expression was noted in the anterior neural plate as well as in newly formed somites. Whereas fgfr2 expression in somites is transient, it increases in the central nervous system (CNS), i.e. in the ventral telencephalon, anterior diencephalon, midbrain, and respective rhombomeres of the hindbrain, from the mid-somitogenesis stage. The dorsal telencephalon and the region around the midbrain-hindbrain boundary are devoid of fgfr2 expression. Essentially the same expression pattern is observed until 48 h post-fertilization in the CNS, although rhombomeric expression in the hindbrain is progressively confined to narrower stripes. After somitogenesis, fgfr2 expression was also observed in the lens, hypochord, endoderm, and fin mesenchyme. We compared the expression of the four fgfr genes (fgfr1-4) in the CNS of zebrafish embryos and show that fgfr1 is the only fgfr gene that is expressed in the dorsal telencephalon and isthmic region from which expression of fgfr2-4 is absent.     

Expression of the FGF receptor 2 gene (fgfr2) during embryogenesis in the zebrafish Danio rerio

We isolated a full-length cDNA clone for the zebrafish homologue of fibroblast growth factor receptor (FGFR) 2. The deduced protein sequence is typical of vertebrate FGFRs in that it has three Ig-like domains in the extracellular region. The expression of fgfr2 is initiated during epiboly in the paraxial mesoderm. During early somitogenesis, fgfr2 expression was noted in the anterior neural plate as well as in newly formed somites. Whereas fgfr2 expression in somites is transient, it increases in the central nervous system (CNS), i.e. in the ventral telencephalon, anterior diencephalon, midbrain, and respective rhombomeres of the hindbrain, from the mid-somitogenesis stage. The dorsal telencephalon and the region around the midbrain-hindbrain boundary are devoid of fgfr2 expression. Essentially the same expression pattern is observed until 48 h post-fertilization in the CNS, although rhombomeric expression in the hindbrain is progressively confined to narrower stripes. After somitogenesis, fgfr2 expression was also observed in the lens, hypochord, endoderm, and fin mesenchyme. We compared the expression of the four fgfr genes (fgfr1-4) in the CNS of zebrafish embryos and show that fgfr1 is the only fgfr gene that is expressed in the dorsal telencephalon and isthmic region from which expression of fgfr2-4 is absent.     

Expression of EGF receptor and FGF receptor isoforms during neuroepithelial stem cell differentiation

To characterize the role of epidermal growth factor (EGF) and fibroblast growth factor (FGF) in regulating neuroepithelial stem cells differentiation, we have examined the expression of FGF, EGF, and their receptors by neuroepithelial (NEP) cells and their derivatives. Our results indicate that undifferentiated NEP cells express a subset of FGF receptor (FGFR) isoforms, but do not express platelet-derived growth factor receptors (PDGFRs) or epidermal growth factor receptor (EGFR). The FGFR pattern of expression by differentiated neuron and glial cells differs from that found on NEP stem cells. FGFR-4 is uniquely expressed on NEP cells, while FGFR-1 is expressed by both NEP cells and neurons, and FGFR-2 is down-regulated during neuronal differentiation. FGFRs present on astrocytes and oligodendrocytes also represent a subset of those present on NEP cells. Expression of FGF and EGF by NEP cells and their progeny was also examined. NEP cells synthesize detectable levels of both FGF-1 and FGF-2, and EGF. FGF-1 and FGF-2 synthesis is likely to be biologically relevant, as cells grown at high density do not require exogenous FGF for their survival and cells grown in the presence of neutralizing antibodies to FGF show a reduction in cell survival and division. Thus, neuroepithelial cells synthesize and respond to FGF, but not to EGF, and are therefore distinct from other neural stem cells (neurospheres). The unique pattern of expression of FGF isoforms may serve to distinguish NEP cells from their more differentiated progeny.     

Ligand-dependent activation of breathless FGF receptor gene in Drosophila developing trachea

Spatially and temporally regulated activity of Branchless/Breathless signaling is essential for trachea development in Drosophila. Early ubiquitous breathless (btl) expression is controlled by binding of Trachealess/Tango heterodimers to the btl minimum enhancer. Branchless/Breathless signaling includes a Sprouty-dependent negative feedback loop. We show that late btl expression is a target of Branchless/Breathless signaling and hence, Branchless/Breathless signaling contains a positive feedback loop, which may guarantee a continuous supply of fresh receptors to membranes of growing tracheal branch cells. Branchless/Breathless signaling activates MAP-kinase, which in turn, activates late btl expression and destabilizes Anterior-open, a repressor for late btl expression. Biochemical and genetic analysis indicated that the minimum btl enhancer includes binding sites of Anterior-open.     

FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development

FGF receptor 2 isoform IIIb (FGFR2b), originally discovered as a receptor for FGF7, is known to be an important receptor in vertebrate morphogenesis, because FGFR2b null mice exhibit agenesis or dysgenesis of various organs, which undergo budding and branching morphogenesis. Since FGF7 null mice do not exhibit marked defects in organogenesis, it has been considered that other FGF(s) than FGF7 might function as a major ligand for FGFR2b during organogenesis. One of the candidate ligands is FGF10, because FGF10 binds to FGFR2b with high affinity and the formation of the limb and lung is arrested in FGF10 null mice as found in FGFR2b-deficient mice. Previous analyses of FGF10 null mice revealed that FGF10 is required for limb and lung development. To elucidate the role of FGF10 in wide-range organogenesis, we further analyzed the phenotypes of the FGF10 knockout mice. We found diverse phenotypes closely related to those for FGFR2b-deficient mice, which includes the absence of thyroid, pituitary, and salivary glands, while minor defects were observed in the formation of teeth, kidneys, hair follicles, and digestive organs. These results suggest that FGF10 acts as a major ligand for FGFR2b in mouse multi-organ development.     

Expression and functions of FGF ligands during early otic development

Classical studies have postulated the action of an endomesodermal signal initiating inner ear induction, subsequently followed by a neural tube-derived signal to complete the process of otic placode formation in the surface ectoderm. Members of the Fibroblast growth factor (FGF) gene family have been implicated in these processes. In this review, expression analysis and recent experimental evidence for candidate inner ear FGF ligands during inner ear induction is discussed. Careful examination of the spatiotemporal expression patterns of different FGFs during inner ear induction reveals that the sequential appearance of FGF members in the endoderm and/or mesoderm is followed by expression in the posterior hindbrain in all vertebrate species analysed to date. Experimental manipulations have demonstrated the sufficiency and/or necessity of some FGFs during different steps of inner ear induction in vitro and in vivo. Combining the advantages of the molecular tools and approaches available in different experimental systems such as zebrafish, chicken or mouse will eventually lead to a complete understanding of how FGFs control inner ear induction in vertebrates.     

阿片受体样受体ORL1基因在大鼠脑内的表达

我们采用地主辛标记的寡聚核苷酸探针和原位杂产技术研究了新发现的阿片受体样受体ＯＲＬ１基因在正常大鼠脑仙的表达和分布。发现ＯＲＬ１基因在大鼠脑内广泛表达。尤其在大脑皮层、丘脑、下丘脑、杏仁核、海马、隔核、缰核、导水管周围灰质、中缝核群及蓝斑等区域。提示ＯＲＬ１除参与痛觉调制外,还可能参与脑内多项生理功能的调控。     

FGF receptor availability regulates skeletal myogenesis.

Fibroblast growth factors (FGFs) and their receptors are critical participants in embryonic development, including the genesis of skeletal, cardiac, and smooth muscle. FGF signaling is mediated through interactions between multiple FGF ligands and transmembrane tyrosine kinase receptors, resulting in activation of a number of signal transduction pathways. Skeletal myocytes express FGF ligands and receptors in a coordinated fashion, suggesting that these molecules participate in autocrine signaling in the myocyte. Endogenously produced FGF has been shown to inhibit myogenesis, but the role of FGF receptor availability in directing myocyte proliferation and differentiation has not been established. To determine the contribution of receptor availability to the regulation of myogenesis, receptor availability was either increased by expressing a full-length FGF receptor-1 or decreased by expressing a truncated FGF receptor-1 in cultured skeletal myocytes. Constitutive expression of a full-length FGF receptor-1 increased myocyte proliferation and delayed differentiation. Conversely, a reduction in functional FGF receptor signaling by expression of a truncated FGF receptor-1 decreased proliferation and enhanced differentiation of myocytes. These data demonstrate that FGF receptor availability plays a critical regulatory role in skeletal myogenesis.     

Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development.

The Drosophila sugarless and sulfateless genes encode enzymes required for the biosynthesis of heparan sulfate glycosaminoglycans. Biochemical studies have shown that heparan sulfate glycosaminoglycans are involved in signaling by fibroblast growth factor receptors, but evidence for such a requirement in an intact organism has not been available. We now demonstrate that sugarless and sulfateless mutant embryos have phenotypes similar to those lacking the functions of two Drosophila fibroblast growth factor receptors, Heartless and Breathless. Moreover, both Heartless- and Breathless-dependent MAPK activation is significantly reduced in embryos which fail to synthesize heparan sulfate glycosaminoglycans. Consistent with an involvement of Sulfateless and Sugarless in fibroblast growth factor receptor signaling, a constitutively activated form of Heartless partially rescues sugarless and sulfateless mutants, and dosage-sensitive interactions occur between heartless and the heparan sulfate glycosaminoglycan biosynthetic enzyme genes. We also find that overexpression of Branchless, the Breathless ligand, can partially overcome the requirement of Sugarless and Sulfateless for Breathless activity. These results provide the first genetic evidence that heparan sulfate glycosaminoglycans are essential for fibroblast growth factor receptor signaling in a well defined developmental context, and support a model in which heparan sulfate glycosaminoglycans facilitate fibroblast growth factor ligand and/or ligand-receptor oligomerization.     

Expression of the cystic fibrosis gene in human development.

The specialised epithelia lining the respiratory tract, pancreatic ducts, male genital ducts and sweat gland ducts are defective in the severe inherited disease, cystic fibrosis (CF). We have looked at the expression of the CF gene in human fetal tissues to throw light on the development of function in specialised ductal epithelia and to determine the age of onset of the CF disease process. The CF gene is already seen to be transcribed in mid-trimester fetal lung, pancreas and male genital ducts. Hence, by this developmental stage, and before they are fully differentiated, these epithelia have the capability to perform important transport functions. Epithelial cell cultures derived from fetal pancreas and male genital ducts maintain expression of the CF gene in vitro and so form good models for analysing CF gene function and differentiation of these specialised epithelia.     

Expression of glucocorticoid receptor and glucose transporter-1 during placental development in the diabetic rat

In various tissues, glucocorticoids (GCs) are known to downregulate glucose transport systems; however, their effects on glucose transporters (GLUTs) in the placenta of a diabetic rat are unknown. Glucocorticoid hormone action within the cell is regulated by the glucocorticoid receptor (GR). Thus, this study was designed to investigate the relationship between GR and glucose transporter expression in the placenta of the diabetic rat. Our immunohistochemical results indicated that GR and glucose transporter protein 1 (GLUT 1) are expressed ubiquitously in the trophoblast and endothelial cells of the labyrinthine zone, where maternal fetal transport takes place in the rat placenta. Expression of GR in the junctional zone of the rat placenta was detected in giant cells, and in some spongiotrophoblast cells, but not in the glycogen cells. GLUT 1 was present, especially in glycogen cells during early pregnancy, and in the spongiotrophoblast cells of the junctional zone during late pregnancy. Amounts of GR and GLUT 1 protein were increased towards the end of gestation both in the control and the diabetic placenta. However, at days 17 and 19 of gestation, only the placental GR protein was significantly increased in the streptozotocin-induced diabetic rats compared to control rats. Diabetes led to a significant decrease in placental weight at gestation day 15. In contrast, at gestational days 17 and 21, the weights of the diabetic placenta were significantly increased as compared with the controls. Moreover, diabetes induced fetus intrauterine growth retardation at gestational days 13, 17 and 21. In conclusion, the localization pattern of GR and GLUT 1 proteins in the same cell types led us to believe that there might be a relationship between GR and GLUT 1 expressions at the cellular level. GLUT 1 does not play a pivotal role in diabetic pregnancies. However, placental growth abnormalities during diabetic pregnancy may be related to the amount of GR.     

Structural basis for FGF receptor dimerization and activation.

The crystal structure of FGF2 bound to a naturally occurring variant of FGF receptor 1 (FGFR1) consisting of immunoglobulin-like domains 2 (D2) and 3 (D3) has been determined at 2.8 A resolution. Two FGF2:FGFR1 complexes form a 2-fold symmetric dimer. Within each complex, FGF2 interacts extensively with D2 and D3 as well as with the linker between the two domains. The dimer is stabilized by interactions between FGF2 and D2 of the adjoining complex and by a direct interaction between D2 of each receptor. A positively charged canyon formed by a cluster of exposed basic residues likely represents the heparin-binding site. A general model for FGF- and heparin-induced FGFR dimerization is inferred from the crystal structure, unifying a wealth of biochemical data.