Zebrafish Offers New Perspective on Developmental Role of TOR Signaling

Genetic studies on the molecular basis of growth control have converged on the target of rapamycin (TOR) pathway as a key regulator. When stimulated by nutrients (i.e. amino acids) or growth factors (i.e. insulin), TOR activates protein synthesis and other anabolic pathways to promote cell growth. Our knowledge of TOR's function in vivo is still rudimentary, particularly in the setting of vertebrate development. An important question is whether TOR functions as a constitutive regulator of growth in all cell types, or as a stage- and organ-specific regulator. Recently we employed the zebrafish as a vertebrate model system to study the developmental role of TOR signaling. We found that TOR signaling was required for a discrete step prior to epithelial differentiation. The results support the view that different organs may be reliant on TOR activity to differing degrees. In the case of the zebrafish, the digestive tract exhibits the greatest sensitivity to rapamycin, which may reflect its reliance on TOR signaling for normal growth. We suggest the hypothesis that TOR signaling may regulate the size of the intestine's absorptive surface area in response to systemic nutrient demand.     

TOR-autophagy signaling in adult zebrafish models of cardiomyopathy

The target of rapamycin (TOR) kinase is part of an evolutionarily conserved signaling pathway that coordinates cell growth, survival, and autophagy. Previously, pharmacological studies using rapamycin have suggested a cardioprotective effect of TOR signaling inhibition on cardiomyopathy. We found that rapamycin exerts a conserved cardioprotective effect in two adult zebrafish models of cardiomyopathy of different etiology, and provided the first genetic evidence to support a long-term cardioprotective effect of TOR signaling inhibition. Moreover, we detected dynamic TOR-autophagy activities along different stages of cardiomyopathy. This needs to be considered when developing TOR-autophagy-based therapeutics for cardiomyopathy.     

TOR-autophagy signaling in adult zebrafish models of cardiomyopathy

The target of rapamycin (TOR) kinase is part of an evolutionarily conserved signaling pathway that coordinates cell growth, survival, and autophagy. Previously, pharmacological studies using rapamycin have suggested a cardioprotective effect of TOR signaling inhibition on cardiomyopathy. We found that rapamycin exerts a conserved cardioprotective effect in two adult zebrafish models of cardiomyopathy of different etiology, and provided the first genetic evidence to support a long-term cardioprotective effect of TOR signaling inhibition. Moreover, we detected dynamic TOR-autophagy activities along different stages of cardiomyopathy. This needs to be considered when developing TOR-autophagy-based therapeutics for cardiomyopathy.     

Rapamycin treatment causes developmental delay, pigmentation defects, and gastrointestinal malformation on Xenopus embryogenesis

Rapamycin is a drug working as an inhibitor of the TOR (target of rapamycin) signaling pathway and influences various life phenomena such as cell growth, proliferation, and life span extension in eukaryote. However, the extent to which rapamycin controls early developmental events of amphibians remains to be understood. Here we report an examination of rapamycin effects during Xenopus early development, followed by a confirmation of suppression of TOR downstream kinase S6K by rapamycin treatment. First, we found that developmental speed was declined in dose-dependent manner of rapamycin. Second, black pigment spots located at dorsal and lateral skin in tadpoles were reduced by rapamycin treatment. Moreover, in tadpole stages severe gastrointestinal malformations were observed in rapamycin-treated embryos. Taken together with these results, we conclude that treatment of the drug rapamycin causes enormous influences on early developmental period.     

PI3-kinase and TOR: PIKTORing cell growth

Regulation of growth and proliferation in higher eukaryotic cells results from an integration of nutritional, energy, and mitogenic signals. Biochemical processes underlying cell growth and proliferation are governed by the phosphatidylinositol 3-kinase (PI3K) and target of rapamycin (TOR) signaling pathways. The importance of the interplay between these two pathways is underscored by the discovery that the TOR inhibitor rapamycin is effective against tumors caused by misregulation of the PI3K pathway. We review here recent data concerning the convergence of the PI3K and TOR pathways, the role of these pathways in cell growth and proliferation, and the regulation of growth by downstream TOR targets.     

Neuronal expression of fibroblast growth factor receptors in zebrafish

Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72 h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts.     

Neuronal expression of fibroblast growth factor receptors in zebrafish

Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72 h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts.     

Microarray-based method for monitoring yeast overexpression strains reveals small-molecule targets in TOR pathway

Identification of the cellular targets of small-molecule hits in phenotypic screens is a central challenge in the development of small molecules as biological tools and potential therapeutics. To facilitate the process of small-molecule target identification, we developed a global, microarray-based method for monitoring the growth of pools of yeast strains, each overexpressing a different protein, in the presence of small molecules. Specifically, the growth of Saccharomyces cerevisiae strains harboring approximately 3,900 different overexpression plasmids was monitored in the presence of rapamycin, which inhibits the target of rapamycin (TOR) proteins. TOR was successfully identified as a candidate rapamycin target, and many additional gene products were implicated in the TOR signaling pathway. We also characterized the mechanism of LY-83583, a small-molecule suppressor of rapamycin-induced growth inhibition. These data enabled functional links to be drawn between groups of genes implicated in the TOR pathway, identified several candidate targets for LY-83583, and suggested a role for mitochondrial respiration in mediating rapamycin sensitivity.     

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.     

Semaphorin3d mediates Cx43-dependent phenotypes during fin regeneration

Gap junctions are proteinaceous channels that reside at the plasma membrane and permit the exchange of ions, metabolites, and second messengers between neighboring cells. Connexin proteins are the subunits of gap junction channels. Mutations in zebrafish cx43 cause the short fin (sof(b123)) phenotype which is characterized by short fins due to defects in length of the bony fin rays. Previous findings from our lab demonstrate that Cx43 is required for both cell proliferation and joint formation during fin regeneration. Here we demonstrate that semaphorin3d (sema3d) functions downstream of Cx43. Semas are secreted signaling molecules that have been implicated in diverse cellular functions such as axon guidance, cell migration, cell proliferation, and gene expression. We suggest that Sema3d mediates the Cx43-dependent functions on cell proliferation and joint formation. Using both in situ hybridization and quantitative RT-PCR, we validated that sema3d expression depends on Cx43 activity. Next, we found that knockdown of Sema3d recapitulates all of the sof(b123) and cx43-knockdown phenotypes, providing functional evidence that Sema3d acts downstream of Cx43. To identify the potential Sema3d receptor(s), we evaluated gene expression of neuropilins and plexins. Of these, nrp2a, plxna1, and plxna3 are expressed in the regenerating fin. Morpholino-mediated knockdown of plxna1 did not cause cx43-specific defects, suggesting that PlexinA1 does not function in this pathway. In contrast, morpholino-mediated knockdown of nrp2a caused fin overgrowth and increased cell proliferation, but did not influence joint formation. Moreover, morpholino-mediated knockdown of plxna3 caused short segments, influencing joint formation, but did not alter cell proliferation. Together, our findings reveal that Sema3d functions in a common molecular pathway with Cx43. Furthermore, functional evaluation of putative Sema3d receptors suggests that Cx43-dependent cell proliferation and joint formation utilize independent membrane-bound receptors to mediate downstream cellular phenotypes.     

Regulation of neurogenesis and epidermal growth factor receptor signaling by the insulin receptor/target of rapamycin pathway in Drosophila

Determining how growth and differentiation are coordinated is key to understanding normal development, as well as disease states such as cancer, where that control is lost. We have previously shown that growth and neuronal differentiation are coordinated by the insulin receptor/target of rapamycin (TOR) kinase (InR/TOR) pathway. Here we show that the control of growth and differentiation diverge downstream of TOR. TOR regulates growth by controlling the activity of S6 kinase (S6K) and eIF4E. Loss of s6k delays differentiation, and is epistatic to the loss of tsc2, indicating that S6K acts downstream or in parallel to TOR in differentiation as in growth. However, loss of eIF4E inhibits growth but does not affect the timing of differentiation. We also show, for the first time in Drosophila, that there is crosstalk between the InR/TOR pathway and epidermal growth factor receptor (EGFR) signaling. InR/TOR signaling regulates the expression of several EGFR pathway components including pointedP2 (pntP2). In addition, reduction of EGFR signaling levels phenocopies inhibition of the InR/TOR pathway in the regulation of differentiation. Together these data suggest that InR/TOR signaling regulates the timing of differentiation through modulation of EGFR target genes in developing photoreceptors.     

雷帕霉素靶蛋白与自噬在衰老中的交互作用

衰老是一个非常复杂的过程,与细胞和组织中累积的各种大分子（DNA、蛋白质和脂质）损伤密不可分,并且是由细胞中不同的信号通道共同调控的结果,而雷帕霉素靶标途径就是其中的一种。该途径整合了各种来自细胞内外的信号以调控细胞的生长、增殖和代谢。越来越多证据表明,雷帕霉素靶蛋白（target of rapamycin,TOR）控制着细胞和组织老化的速度,影响着整个机体衰老过程。另外TOR参与调控自噬的发生,而自噬能使生物大分子和细胞器降解并回收重复利用。多种生物模型研究发现,衰老其实是与自噬的不足有关联。本文对TOR和自噬在衰老过程中的作用和相互关系进行综述,为发展与老年疾病相关的新型治疗方法提供思路。