类胰岛素生长因子信号途径及其对脊椎动物生长和发育的调控作用:以斑马鱼为模式动物的新进展

类胰岛素生长因子(包括IGF-I和IGF-Ⅱ)是进化上保守性很强的多肽。IGFs对脊椎动物的生长和早期发育有极其重要的调控作用。IGF的生理作用是由IGF受体中介并受几个分泌性的IGF结合蛋白调节。本文主要介绍了以斑马鱼为模式动物，用基因敲除、转基因动物和培养细胞系等现代实验方法对IGF信号途径的最新研究进展，综述了IGF配体、受体和结合蛋白的结构特点、基因表达和调节和生物学功能。此外，也对斑马鱼作为模式动物的许多优点进行了探讨。     

Fibroblast growth factor signaling in tumorigenesis

Fibroblast growth factors and their signaling receptors have been associated with multiple biological activities, including proliferation, differentiation and motility. Consequently, they have evoked interest as candidate oncogenes with the potential to initiate and/or promote tumorigenesis. This has resulted in a large literature describing the presence of these growth factors and their receptors in cancer cell lines and primary tumors of diverse origin. However, it is only recently that compelling evidence has emerged to implicate the fibroblast growth factors (Fgfs) and their receptors in the genesis of human cancers. Here, we outline the model systems that demonstrate the potential oncogenic nature of Fgf signaling and summarise recent evidence that implicates aberrant Fgf signaling as important in the natural history of some common human cancers.     

Bmp and Fgf signaling are essential for liver specification in zebrafish

Based on data from in vitro tissue explant and ex vivo cell/bead implantation experiments, Bmp and Fgf signaling have been proposed to regulate hepatic specification. However, genetic evidence for this hypothesis has been lacking. Here, we provide in vivo genetic evidence that Bmp and Fgf signaling are essential for hepatic specification. We utilized transgenic zebrafish that overexpress dominant-negative forms of Bmp or Fgf receptors following heat-shock induction. These transgenes allow one to bypass the early embryonic requirements for Bmp and Fgf signaling, and also to completely block Bmp or Fgf signaling. We found that the expression of hhex and prox1, the earliest liver markers in zebrafish, was severely reduced in the liver region when Bmp or Fgf signaling was blocked just before hepatic specification. However, hhex and prox1 expression in adjacent endodermal and mesodermal tissues appeared unaffected by these manipulations. Additional genetic studies indicate that the endoderm maintains competence for Bmp-mediated hepatogenesis over an extended window of embryonic development. Altogether, these data provide the first genetic evidence that Bmp and Fgf signaling are essential for hepatic specification, and suggest that endodermal cells remain competent to differentiate into hepatocytes for longer than anticipated.     

Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration

During appendage regeneration in urodeles and teleosts, tissue replacement is precisely regulated such that only the appropriate structures are recovered, a phenomenon referred to as positional memory. It is believed that there exists, or is quickly established after amputation, a dynamic gradient of positional information along the proximodistal (PD) axis of the appendage that assigns region-specific instructions to injured tissue. These instructions specify the amount of tissue to regenerate, as well as the rate at which regenerative growth is to occur. A striking theme among many species is that the rate of regeneration is more rapid in proximally amputated appendages compared with distal amputations. However, the underlying molecular regulation is unclear. Here, we identify position-dependent differences in the rate of growth during zebrafish caudal fin regeneration. These growth rates correlate with position-dependent differences in blastemal length, mitotic index and expression of the Fgf target genes mkp3, sef and spry4. To address whether PD differences in amounts of Fgf signaling are responsible for position-dependent blastemal function, we have generated transgenic fish in which Fgf receptor activity can be experimentally manipulated. We find that the level of Fgf signaling exhibits strict control over target gene expression, blastemal proliferation and regenerative growth rate. Our results demonstrate that Fgf signaling defines position-dependent blastemal properties and growth rates for the regenerating zebrafish appendage.     

Fibroblast growth factor 1 regulates signaling via the glycogen synthase kinase-3beta pathway. Implications for neuroprotection

We hypothesize that in neurodegenerative disorders such as Alzheimer's disease and human immunodeficiency virus encephalitis the neuroprotective activity of fibroblast growth factor 1 (FGF1) against several neurotoxic agents might involve regulation of glycogen synthase kinase-3beta (GSK3beta), a pathway important in determining cell fate. In primary rat neuronal and HT22 cells, FGF1 promoted a time-dependent inactivation of GSK3beta by phosphorylation at serine 9. Blocking FGF1 receptors with heparinase reduced this effect. The effects of FGF1 on GSK3beta were dependent on phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) because inhibitors of this pathway or infection with dominant negative Akt adenovirus blocked inactivation. Furthermore, treatment of neuronal cells with FGF1 resulted in ERK-independent Akt phosphorylation and beta-catenin translocation into the nucleus. On the other hand, infection with wild-type GSK3beta recombinant adenovirus-associated virus increased activity of GSK3beta and cell death, both of which were reduced by FGF1 treatment. Moreover, FGF1 protection against glutamate toxicity was dependent on GSK3beta inactivation by the PI3K-Akt but was independent of ERK. Taken together these results suggest that neuroprotective effects of FGF1 might involve inactivation of GSK3beta by a pathway involving activation of the PI3K-Akt cascades.     

Expression of Fibroblast growth factor 19 (Fgf19) during chicken embryogenesis and eye development, compared with Fgf15 expression in the mouse

The normal development of eyes relies on proper signaling through Fibroblast growth factor (FGF) receptors, but the source and identity of cognate ligands have remained largely unknown. We have found that Fgf19 is expressed in the developing chicken retina. In situ hybridization discloses dynamic expression patterns for Fgf19 in the optic vesicle, lens primordia and retinal horizontal cells. Overall expression pattern of Fgf19 during chicken embryogenesis was also examined: Fgf19 is expressed in the regions associated with cranial placodes induction, boundary regions of rhombomeres, somites, specific groups of neural cells in midbrain, hindbrain, and those derived from epibranchial placodes, and the apical ectodermal ridge of limb buds. Expression pattern of the Fgf19-orthologous gene Fgf15 was further examined in the mouse developing eye. Fgf15 is expressed in the optic vesicle, a subset of progenitor cells of neural retina, and emerging ganglion and amacrine cells during retinogenesis.     

Fgf signaling is required for zebrafish tooth development

We have investigated fibroblast growth factor (FGF) signaling during the development of the zebrafish pharyngeal dentition with the goal of uncovering novel roles for FGFs in tooth development as well as phylogenetic and topographic diversity in the tooth developmental pathway. We found that the tooth-related expression of several zebrafish genes is similar to that of their mouse orthologs, including both epithelial and mesenchymal markers. Additionally, significant differences in gene expression between zebrafish and mouse teeth are indicated by the apparent lack of fgf8 and pax9 expression in zebrafish tooth germs. FGF receptor inhibition with SU5402 at 32 h blocked dental epithelial morphogenesis and tooth mineralization. While the pharyngeal epithelium remained intact as judged by normal pitx2 expression, not only was the mesenchymal expression of lhx6 and lhx7 eliminated as expected from mouse studies, but the epithelial expression of dlx2a, dlx2b, fgf3, and fgf4 was as well. This latter result provides novel evidence that the dental epithelium is a target of FGF signaling. However, the failure of SU5402 to block localized expression of pitx2 suggests that the earliest steps of tooth initiation are FGF-independent. Investigations of specific FGF ligands with morpholino antisense oligonucleotides revealed only a mild tooth shape phenotype following fgf4 knockdown, while fgf8 inhibition revealed only a subtle down-regulation of dental dlx2b expression with no apparent effect on tooth morphology. Our results suggest redundant FGF signals target the dental epithelium and together are required for dental morphogenesis. Further work will be required to elucidate the nature of these signals, particularly with respect to their origins and whether they act through the mesenchyme.     

Fibroblast growth factor 2 regulates dopaminergic neuron development in vivo

Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson's disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development.     

Dhx15 regulates zebrafish intestinal development through the Wnt signaling pathway

DEAH-box helicase 15 (DHX15) is ATP-dependent RNA helicase which is known for its role in RNA metabolism. Recent studies reported DHX15 involves in the intestinal immunity. However, the role of DHX15 (or RNA helicase) in intestinal development is poorly understood. Here, we revealed an unidentified role for dhx15 in regulating zebrafish intestinal development. We found the profound intestinal defects in dhx15 knockout zebrafish. Decreased proliferation and increased apoptosis of the intestine cells were observed when dhx15 were deleted. Further RNA genome wide analysis and qRT-PCR analysis showed the Wnt signaling pathway is down-regulated in the dhx15 knockout zebrafish. Thus, we concluded that dhx15 regulates zebrafish intestinal development through the Wnt signaling pathway. Here, we provided new insights into the role of dhx15 in intestinal development beyond its well-characterized role in intestinal immunity.     

Fibroblast growth factor receptor 1 signaling in the osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation

Mutations in fibroblast growth factor receptors (Fgfrs) 1-3 cause skeletal disease syndromes in humans. Although these Fgfrs are expressed at various stages of chondrocyte and osteoblast development, their function in specific skeletal cell types is poorly understood. Using conditional inactivation of Fgfr1 in osteo-chondrocyte progenitor cells and in differentiated osteoblasts, we provide evidence that FGFR1 signaling is important for different stages of osteoblast maturation. Examination of osteogenic markers showed that inactivation of FGFR1 in osteo-chondro-progenitor cells delayed osteoblast differentiation, but that inactivation of FGFR1 in differentiated osteoblasts accelerated differentiation. In vitro osteoblast cultures recapitulated the in vivo effect of FGFR1 on stage-specific osteoblast maturation. In immature osteoblasts, FGFR1 deficiency increased proliferation and delayed differentiation and matrix mineralization, whereas in differentiated osteoblasts, FGFR1 deficiency enhanced mineralization. Furthermore, FGFR1 deficiency in differentiated osteoblasts resulted in increased expression of Fgfr3, a molecule that regulates the activity of differentiated osteoblasts. Mice lacking Fgfr1, either in progenitor cells or in differentiated osteoblasts, showed increased bone mass as adults. These data demonstrate that signaling through FGFR1 in osteoblasts is necessary to maintain the balance between bone formation and remodeling through a direct effect on osteoblast maturation.     

Presenilin 1 regulates epidermal growth factor receptor turnover and signaling in the endosomal-lysosomal pathway

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.     

Fibroblast growth factor signaling in embryonic and cancer stem cells

Cancer stem cells are cancer cells that originate from the transformation of normal stem cells. The most important property of any stem cell is the ability to self-renew. Through this property, there are striking parallels between normal stem cells and cancer stem cells. Both cell types share various markers of “stemness”. In particular, normal stem cells and cancer stem cells utilize similar molecular mechanisms to drive self-renewal, and similar signaling pathways may induce their differentiation.The fibroblast growth factor 2 (FGF-2) pathway is one of the most significant regulators of human embryonic stem cell (hESC) self-renewal and cancer cell tumorigenesis. Here we summarize recent data on the effects of FGF-2 and its receptors on hESCs and leukemic stem/progenitor cells. Also, we discuss the similarities of these findings with stem cell renewal and differentiation phenotypes.     

A neuropeptide signaling pathway regulates synaptic growth in Drosophila

Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. Here, we demonstrate that cholecystokinin-like receptor (CCKLR) and drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR or dsk produced severe NMJ undergrowth, whereas overexpression of CCKLR caused overgrowth. Presynaptic expression of CCKLR was necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduced synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. Our results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms.     

Fibroblast growth factor homologous factors are intracellular signaling proteins

Fibroblast growth factors (FGFs) mediate cell growth, differentiation, migration, and morphogenesis by binding to the extracellular domain of cell surface receptors, triggering receptor tyrosine phosphorylation and signal transduction [1-5]. FGF homologous factors (FHFs) were discovered within vertebrate DNA sequence databases by virtue of their sequence similarity to FGFs [3, 6, 7], but the mechanism of FHF action has not been reported. We show here that FHF-1 is associated with the MAP kinase (MAPK) scaffold protein Islet-Brain-2 (IB2) [8] in the brain and in specific cell lines. FHF/IB2 interaction is highly specific, as FHFs do not bind to the related scaffold protein IB1(JIP-1b) [9, 10], nor can FGF-1 bind to IB2. We further show that FHFs enable IB2 to recruit a specific MAPK in transfected cells, and our data suggest that the scaffolds IB1 and IB2 have different MAPK specificities. Hence, FHFs are intracellular components of a tissue-specific protein kinase signaling module.     

Carbonic anhydrase 5 regulates acid-base homeostasis in zebrafish

The regulation of the acid-base balance in cells is essential for proper cellular homeostasis. Disturbed acid-base balance directly affects cellular physiology, which often results in various pathological conditions. In every living organism, the protein family of carbonic anhydrases regulate a broad variety of homeostatic processes. Here we describe the identification, mapping and cloning of a zebrafish carbonic anhydrase 5 (ca5) mutation, collapse of fins (cof), which causes initially a collapse of the medial fins followed by necrosis and rapid degeneration of the embryo. These phenotypical characteristics can be mimicked in wild-type embryos by acetazolamide treatment, suggesting that CA5 activity in zebrafish is essential for a proper development. In addition we show that CA5 regulates acid-base balance during embryonic development, since lowering the pH can compensate for the loss of CA5 activity. Identification of selective modulators of CA5 activity could have a major impact on the development of new therapeutics involved in the treatment of a variety of disorders.     

Insulin-like growth factor signaling regulates zebrafish embryonic growth and development by promoting cell survival and cell cycle progression

Although much is known about the global effects of insulin-like growth factor 1 receptor (IGF1R)-mediated signaling on fetal growth and the clinical manifestations resulting from IGF/IGF1R deficiencies, we have an incomplete understanding of the cellular actions of this essential pathway during vertebrate embryogenesis. In this study, we inhibited IGF1R signaling during zebrafish embryogenesis using antisense morpholino oligonucleotides or a dominant-negative IGF1R fusion protein. IGF1R inhibition resulted in reduced embryonic growth, arrested development and increased lethality. IGF1R-deficient embryos had significant defects in the retina, inner ear, motoneurons and heart. No patterning abnormalities, however, were found in the brain or other embryonic tissues. At the cellular level, IGF1R inhibition increased caspase 3 activity and induced neuronal apoptosis. Coinjection of antiapoptotic bcl2-like mRNA attenuated the elevated apoptosis and rescued the retinal and motoneuron defects, but not the developmental arrest. Subsequent cell cycle analysis indicated an increased percentage of cells in G1 and a decreased percentage in S phase in IGF1R-deficient embryos independent of apoptosis. These results provide novel insight into the cellular basis of IGF1R function and show that IGF1R signaling does not function as an anteriorizing signal but regulates embryonic growth and development by promoting cell survival and cell cycle progression.