[Show abstract][Hide abstract] ABSTRACT: The WNK1 (WNK lysine deficient protein kinase 1) protein is a serine/threonine protein kinase with emerging roles in cancer. WNK1 causes hypertension and hyperkalemia when overexpressed and cardiovascular defects when ablated in mice. In this study, the role of Wnk1 in angiogenesis was explored using the zebrafish model. There are two zebrafish wnk1 isoforms, wnk1a and wnk1b, and both contain all the functional domains found in the human WNK1 protein. Both isoforms are expressed in the embryo at the initiation of angiogenesis and in the posterior cardinal vein (PCV), similar to fms-related tyrosine kinase 4 (flt4). Using morpholino antisense oligonucleotides against wnk1a and wnk1b, we observed that wnk1 morphants have defects in angiogenesis in the head and trunk, similar to flk1/vegfr2 morphants. Furthermore, both wnk1a and wnk1b mRNA can partially rescue the defects in vascular formation caused by flk1/vegfr2 knockdown. Mutation of the kinase domain or the Akt/PI3K phosphorylation site within wnk1 destroys this rescue capability. The rescue experiments provide evidence that wnk1 is a downstream target for Vegfr2 (vascular endothelial growth factor receptor-2) and Akt/PI3K signaling and thereby affects angiogenesis in zebrafish embryos. Furthermore, we found that knockdown of vascular endothelial growth factor receptor-2 (flk1/vegfr2) or vascular endothelial growth factor receptor-3 (flt4/vegfr3) results in a decrease in wnk1a expression, as assessed by in
situ hybridization and q-RT-PCR analysis. Thus, the Vegf/Vegfr signaling pathway controls angiogenesis in zebrafish via Akt kinase-mediated phosphorylation and activation of Wnk1 as well as transcriptional regulation of wnk1 expression.
[Show abstract][Hide abstract] ABSTRACT: Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx) and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC) formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of src in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and src transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential in vivo platforms for drug screening for therapies for human liver cancer.
[Show abstract][Hide abstract] ABSTRACT: There are four cell lineages derived from intestinal stem cells that are located at the crypt and villus in the mammalian intestine the non-secretory absorptive enterocytes, and the secretory cells, which include mucous-secreting goblet cells, regulatory peptide-secreting enteroendocrine cells and antimicrobial peptide-secreting Paneth cells. Although fibroblast growth factor (Fgf) signaling is important for cell proliferation and differentiation in various tissues, its role in intestinal differentiation is less well understood.
We used a loss of function approach to investigate the importance of Fgf signaling in intestinal cell differentiation in zebrafish; abnormal differentiation of goblet cells was observed when Fgf signaling was inhibited using SU5402 or in the Tg(hsp70ldnfgfr1-EGFP) transgenic line. We identified Fgfr2c as an important receptor for cell differentiation. The number of goblet cells and enteroendocrine cells was reduced in fgfr2c morphants. In addition to secretory cells, enterocyte differentiation was also disrupted in fgfr2c morphants. Furthermore, proliferating cells were increased in the morphants. Interestingly, the loss of fgfr2c expression repressed secretory cell differentiation and increased cell proliferation in the mibta52b mutant that had defective Notch signaling.
In conclusion, we found that Fgfr2c signaling derived from mesenchymal cells is important for regulating the differentiation of zebrafish intestine epithelial cells by promoting cell cycle exit. The results of Fgfr2c knockdown in mibta52b mutants indicated that Fgfr2c signaling is required for intestinal cell differentiation. These findings provide new evidences that Fgf signaling is required for the differentiation of intestinal cells in the zebrafish developing gut.
[Show abstract][Hide abstract] ABSTRACT: The expression of fgfr2 and fgfr4 in adult zebrafish intestine. Section in situ hybridization was used to analyze the expression of fgfr2 and fgfr4 genes. (A)
fgfr2 was detected in the lamina propria, and (B)
fgfr4 was expressed mainly in the epithelial layer of the intestine. Scale bar = 50 µm.
[Show abstract][Hide abstract] ABSTRACT: The expression of fgfrs and the secretory cell differentiation of fgfr2b morphants. (A)
fgfr1a, fgfr2, fgfr3 and fgfr4 gene were analyzed in 5 dpf zebrafish gut tissue by RT-PCR. (C) WT embryos and (D)
fgfr2b morphants were double labeled using 2F11 antibody and WGA. The magnified image shows (C’–D’) enteroendocrine cells and (C”–D”) goblet cells. DAPI was used for nuclear counter staining (blue). (B) The bar charts show the percentages of 2F11 and WGA positive cells. Error bars indicate SD. Scale bar = 50 µm.
[Show abstract][Hide abstract] ABSTRACT: In mammals, fibroblast growth factor (FGF) signaling controls liver specification and regulates the metabolism of lipids, cholesterol, and bile acids. FGF signaling also promotes hepatocyte proliferation, and helps detoxify hepatotoxin during liver regeneration after partial hepatectomy. However, the function of Fgf in zebrafish liver is not yet well understood, specifically for postnatal homeostasis. The current study analyzed the expression of fgf receptors (fgfrs) in the liver of zebrafish. We then investigated the function of Fgf signaling in the zebrafish liver by expressing a dominant-negative Fgf receptor in hepatocytes (lfabp:dnfgfr1-egfp, lf:dnfr). Histological analysis showed that our genetic intervention resulted in a small liver size with defected medial expansion of developing livers in transgenic (Tg) larvae. Morphologically, the liver lobe of lf:dnfr adult fish was shorter than that of control. Ballooning degeneration of hepatocytes was observed in fish as young as 3 months. Further examination revealed the development of hepatic steatosis and cholestasis. In adult Tg fish, we unexpectedly observed increased liver-to-body-weight ratios, with higher percentages of proliferating hepatocytes. Considering all these findings, we concluded that as in mammals, in adult zebrafish the metabolism of lipid and bile acids in the liver are regulated by Fgf signaling. Disruption of the Fgf signal-mediated metabolism might indirectly affect hepatocyte proliferation.
No preview · Article · Jul 2012 · Transgenic Research
[Show abstract][Hide abstract] ABSTRACT: The effects of fgfr2c MO specific on DFCs. The normal expression pattern of foxA3 in liver (l) and pancreas (p) was shown in DFCfgfr2c MO and DFCfgfr2c-5 mm MO morphants (A and C). Abnormal pattern of visceral organs was also observed in these embryos (B and D). The development of heart was examined using cmlc2 probe (E∼H). Normal (E and G) and abnormal heart looping (F and H) can be observed in both morphants. The bar charts showed the percentage of embryos with different expression distribution of foxA3 or cmlc2 in both morphants (I). Panel A to D were dorsal view and panel E to H were ventral-anterior view. A: atrium, V: ventricle.
[Show abstract][Hide abstract] ABSTRACT: The percentage of abnormal heart looping in fgfr2c and fgf ligand morphants. In order to test the synergistic effect of Fgfr2c and Fgf ligands (Fgf4, Fgf8 and Fgf24), different combinations of low dosage fgfr2c MO and fgf MOs were injected into Line 544. Double morphants of fgfr2c and fgf4 (2, 1 or 0.5 ng/embryo for fgfr2c MO; 34, 22.5 or 12 ng/embryo for fgf4 MO) did not have synergistic effect on the abnormal heart looping, including L-loop and no loop pattern (A). Co-injection with fgfr2c MO (0.5 ng/embryo) and fgf24 MO (1.25 ng/embryo) also did not greatly increase the abnormal percentage (B). In contrast to above results, a synergistic effect was detected in fgfr2c-fgf8 double morphants (0.5 ng/embryo for fgfr2c MO and 1 ng/embryo for fgf8 MO; C).
[Show abstract][Hide abstract] ABSTRACT: The cas expression pattern in fgfr2c morphants. Embryos at 90% epiboly were stained with cas probe for labeling DFCs. The morphology of normal DFC cluster in wild type was shown in panel A (79%, n = 140). The mild and severe disorganization of DFC pattern could also be detected. However, the percentages of abnormal pattern were increased in fgfr2c morphants (B and C, 56.9%, n = 195).
[Show abstract][Hide abstract] ABSTRACT: Many organs in vertebrates are left-right asymmetrical located. For example, liver is at the right side and stomach is at the left side in human. Fibroblast growth factor (Fgf) signaling is important for left-right asymmetry. To investigate the roles of Fgfr2 signaling in zebrafish left-right asymmetry, we used splicing blocking morpholinos to specifically block the splicing of fgfr2b and fgfr2c variants, respectively. We found that the relative position of the liver and the pancreas were disrupted in fgfr2c morphants. Furthermore, the left-right asymmetry of the heart became random. Expression pattern of the laterality controlling genes, spaw and pitx2c, also became random in the morphants. Furthermore, lefty1 was not expressed in the posterior notochord, indicating that the molecular midline barrier had been disrupted. It was also not expressed in the brain diencephalon. Kupffer's vesicle (KV) size became smaller in fgfr2c morphants. Furthermore, KV cilia were shorter in fgfr2c morphants. We conclude that the fgfr2c isoform plays an important role in the left-right asymmetry during zebrafish development.
[Show abstract][Hide abstract] ABSTRACT: Expression pattern of fgfr2. The expression of fgfr2 was detected in marginal YSL (A∼C, arrow, 95%∼100% epiboly) and in the area near KV (arrowhead in D′, 5 somite-stage). Boxed area shown in panel D is enlarged in panel D′.
[Show abstract][Hide abstract] ABSTRACT: The effects of fgfr2b and fgfr2c specific MOs. (A and B): Blue arrows were primer sites for RT-PCR to detect the splicing products. Red thick lines were MO target sites. Injection of fgfr2b (4 ng per embryo) or fgfr2c (1, 2 or 4 ng per embryo) MOs caused partial deletion of exon 8 (b) and exon 9 (c), respectively, that had been confirmed by sequencing. The original splice donor sites were blocked and cryptic splice donor sites in exon8 and exon9 were activated (indicated by bottom red lines) by the corresponding MOs. The partial cDNA sequences were shown (exon7, 8, 10 and 11 for fgfr2b and exon 7, 9 10 and 11 for fgfr2c). Underline indicated the primer sequence. The deleted regions were highlighted.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that fibroblast growth factors (FGFs) are involved in the process of liver injury repair. Liver regeneration after partial hepatectomy (PH) is impaired in transgenic mice expressing dominant-negative FGFR2b in hepatocytes. Although FGF7, a ligand specifically bound to FGFR2b, is expressed by activated hepatic stellate cells (HSCs) in fibrotic livers, the expressions and functions of FGF7 and FGFR2b after PH remain unexplored. Therefore, this study sought to examine the potential role of FGF7 signaling during liver regeneration.
We examined the expression of FGF7 and FGFR2b in normal and regenerating livers. Effects of FGF7 on hepatocytes were examined in vitro using primary hepatocyte culture with FGF7 recombinant protein and in vivo by hydrodynamic-based gene transfer method.
We found that FGF7 expression was increased according to the activation status of HSCs after PH. The receptor, FGFR2b, was also increased in hepatocytes during liver regeneration. In vitro treatment with FGF7 protein activated ERK1/2 and promoted proliferation of hepatocytes isolated from regenerating livers. In vivo overexpression of exogenous FGF7 could notably promote hepatic proliferation and activate MAPKs after PH.
This study suggests a role for activated HSC-expressed FGF7 in stimulating FGF signaling pathways in hepatocytes and regulating liver regeneration.
No preview · Article · Jun 2011 · Cellular Physiology and Biochemistry