Guor Mour Her

In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio)

Mammalian liver fatty acid binding protein (L‐FABP) is a small cytosolic protein in various tissues including liver, small intestine and kidney and is thought to play a crucial role in intracellular fatty acid trafficking and metabolism. To better understand its tissue‐specific regulation during zebrafish hepatogenesis, we isolated 5′‐flanking sequences of the zebrafish L‐FABP gene and used a green fluorescent protein (GFP) transgenic strategy to generate liver‐specific transgenic zebrafish. The 2.8‐kb 5′‐flanking sequence of zebrafish L‐FABP gene was sufficient to direct GFP expression in liver primordia, first observed in 2 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. F2 inheritance rates of 42–51% in all the seven transgenic lines were consistent with the ratio of Mendelian segregation. Further, hhex and zXbp‐1 morphants displayed a visible liver defect, which suggests that it is possible to establish an in vivo system for screening genes required for liver development.

Up‐regulation of muscle‐specific transcription factors during embryonic somitogenesis of zebrafish (Danio rerio) by knock‐down of myostatin‐1

Myostatin, a secreted growth and differentiation factor (GDF‐8) belongs to transforming growth factor (TGF‐β) superfamily that plays as a negative regulator of skeletal muscle development and growth. Recently, myostatin has been isolated from fish; however, its role in muscle development and growth remains unknown. Here, we present the expression of myostatin during development and the effects of its knock‐down on various genes such as muscle regulatory transcription factors (MRFs), muscle‐specific proteins (MSP), and insulin‐like growth factors (IGFs). The myostatin expression was found to be maternal as it starts in one‐cell stage onward. The reverse transcription‐polymerase chain reaction (RT‐PCR), in situ hybridization, and Southern and Northern blots demonstrated that the myostatin expression is not only restricted to skeletal muscle, but it expressed all the tested tissues. Expression of myostatin was effected by using antisense morpholinos resulted in significant phenotypic difference in stages 18 and 20 hours postfertilization (hpf). To confirm the specificity of myostatin morpholino, furthermore, a rescue experiment was conducted. The length as well as width of somites was increased with almost no gap in between the somites. In addition, it deserves to mention that this is a first animal model that shows changes in the size of the somites. Moreover, analyses of MRFs, MSP, and IGFs in the knock‐down embryos by RT‐PCR revealed the up‐regulation of MyoD, Myogenin, and Mck transcription, whereas IGF‐2 transcription showed mild response with no effect on IGF‐1, Desmin, and Myf5. In situ hybridization showed that there was an increase in the number of somites from 3 to 4 at 13 and 22 hpf. Taken together, these data suggest that myostatin plays a major role during myogenesis, apart from inhibition of proliferation as well as differentiation. Developmental Dynamics 229:847‐856, 2004.

435‐bp liver regulatory sequence in the liver fatty acid binding protein (L‐FABP) gene is sufficient to modulate liver regional expression in transgenic zebrafish

Liver fatty acid binding protein (L‐FABP) is a small protein that is thought to play an important role in the intracellular binding and trafficking of long chain fatty acids in the liver. Expression of the gene encoding the zebrafish liver fatty acid binding protein is regulated by a 435‐bp distal region (−1944 to −1510) of the L‐FABP promoter. The 435‐bp sequence is sufficient for gene activation in the liver primordia (or bud) and continues to be active in the adult liver when positioned adjacent to the SV40 basal promoter and linked directly to green fluorescent protein. The 435‐bp sequence region has two distinct liver regulatory elements, A (−1944 to −1623) and B (−1622 to −1510), and contains multiple putative consensus binding sites. The element A sequence includes two consensus HFH and one HNF‐1α site and the element B sequence includes one consensus HNF‐3β site. Deletion of an internal 435‐bp fragment (−1944 to −1510) including the A and B elements totally ablated the liver‐specific activity of the zebrafish L‐FABP gene promoter. Deletion of either of the two elements reduces the liver activity. Mutation of the HNF‐1α site or either of the two HFH sites in the A element or the HNF‐3β site in the B element significantly altered specificity in the liver primordia of transient expression embryos. The importance of the HNF‐1α consensus binding site in the A element and the HNF‐3β consensus binding site in the B element within the 435‐bp distal region of the L‐FABP promoter region suggests that combinatorial interactions between multiple regulatory factors are responsible for the gene expression of L‐FABP in the liver. Developmental Dynamics 227:347–356, 2003.

Overexpression of gankyrin induces liver steatosis in zebrafish (Danio rerio)

Gankyrin is a small ankyrin-repeat protein that previous research has confirmed to be overexpressed in hepatocellular carcinoma (HCC). Although relevant literature has reported on gankyrin functions in cellular proliferation and tumorigenesis, the exact role of gankyrin is poorly understood in animal model systems. This study analyzed hepatic lipid accumulation in gankyrin transgenic (GK) zebrafish. Bromodeoxyuridine (BrdU)-positive cells were predominantly increased in the liver bud of GK larvae, indicating that gankyrin functionally promoted cell proliferation at the larval stage in GK fish. However, over 90% of the viable GK adults showed an increased lipid content, leading in turn to liver steatosis. Liver histology and oil red O staining also indicated the accumulation of fatty droplets in GK fish, consistent with the specific pathological features of severe steatosis. Molecular analysis revealed that gankyrin overexpression induced hepatic steatosis and modulated the expression profiles of four hepatic microRNAs, miR-16, miR-27b, miR-122, and miR-126, and 22 genes involved in lipid metabolism. Moreover, significantly increased hepatic cell apoptosis resulted in liver damage in GK adults, leading to liver failure and death after approximately 10months. This study is the first to report gankyrin as a potential link between microRNAs and liver steatosis in zebrafish.

Increase of hepatic fat accumulation by liver specific expression of Hepatitis B virus X protein in zebrafish.

The pathogenesis of fatty liver disease remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis in zebrafish by liver specific expression of Hepatitis B virus X protein (HBx). Transgenic zebrafish lines, GBXs, which selectively express the GBx transgene (GFP-fused HBx gene) in liver, were established. GBX Liver phenotypes were evaluated by histopathology and molecular analysis of fatty acid (FA) metabolism-related genes expression. Most GBXs (66-81%) displayed obvious emaciation starting at 4 months old. Over 99% of the emaciated GBXs developed hepatic steatosis or steatohepatitis, which in turn led to liver hypoplasia. The liver histology of GBXs displayed steatosis, lobular inflammation, and balloon degeneration, similar to non-alcoholic steatohepatitis (NASH). Oil red O stain detected the accumulation of fatty droplets in GBXs. RT-PCR and Q-rt-PCR analysis revealed that GBx induced hepatic steatosis had significant increases in the expression of lipogenic genes, C/EBP-alpha, SREBP1, ChREBP and PPAR-gamma, which then activate key enzymes of the de novo FA synthesis, ACC1, FAS, SCD1, AGAPT, PAP and DGAT2. In addition, the steatohepatitic GBX liver progressed to liver degeneration and exhibited significant differential gene expression in apoptosis and stress. The GBX models exhibited both the genetic and functional factors involved in lipid accumulation and steatosis-associated liver injury. In addition, GBXs with transmissible NASH-like phenotypes provide a promising model for studying liver disease.

Epinecidin-1 peptide induces apoptosis which enhances antitumor effects in human leukemia U937 cells.

Epinecidin-1 is an antimicrobial peptide present in the grouper (Epinephelus coioides). In this study, the antitumor activity of a synthetic epinecidin-1 peptide was tested. The in vitro results showed that epinecidin-1 inhibited the proliferation of human leukemia U937 cells and increased the ADP/ATP ratio after 24h of treatment. The DNA fragmentation assay, flow cytometric assay, and caspases-3, -8, and -9 assays indicated that epinecidin-1 could induce apoptosis in U937 cells. Real-time RT-PCR results showed regular increases in tumor necrosis factor (TNF)-alpha after treatment with 4 microg/ml epinecidin-1 from 4 to 24h; interleukin (IL)-10, interferon (INF)-r, p53, IL-15, and IL-6 increased after treatment with 2 microg/ml epinecidin-1 for 4-12h. These results suggest that the epinecidn-1 inhibited U937 cells, induced apoptosis in response to cytokine production, and may have pleiotropic effects on different cells.

Functional conserved elements mediate intestinal-type fatty acid binding protein (I-FABP) expression in the gut epithelia of zebrafish larvae

Intestinal‐type fatty acid binding protein (I‐FABP) plays an important role in the intracellular binding and trafficking of long chain fatty acids in the intestine. The aim of this study, therefore, was to elucidate the regulation and spatiotemporal expression of the I‐FABP gene during zebrafish larval development. We performed in vivo reporter‐gene analysis in zebrafish by using a transient and transgenic approach. Green fluorescent protein–reporter analyses revealed that the proximal 192‐bp region of the I‐FABP promoter is sufficient to direct intestine‐specific expression during zebrafish larval development. Functional dissection of a 41‐bp region within this 192‐bp promoter revealed that one C/EBP and two GATA‐like binding sites, along with a novel 15‐bp element within it are required for I‐FABP gene expression in vivo. In addition, the six consensus sites (CCACATCAGCATGAA) in the 15‐bp element are critical for I‐FABP gene regulation in the zebrafish gut epithelia. Comparison analyses of the orthologous 15‐bp element from mammalian I‐FABP genes suggests that these mammalian elements are functionally equivalent to the zebrafish 15 element. These results provide the first in vivo evidence that these binding sites (C/EBP and GATA) and the novel 15‐bp element contribute to intestine‐specific gene expression and that they are functionally conserved across vertebrate evolution. Developmental Dynamics 230:734–742, 2004.

Imbalance in liver homeostasis leading to hyperplasia by overexpressing either one of the Bcl-2-related genes, zfBLP1 and zfMcl-1a

Apoptosis is an essential part of normal embryonic development in vertebrates, and it is involved in sculpturing organs and controlling cell populations. In previous studies, we identified two novel proteins, zfBLP1 and zfMcl‐1a, which are similar to those of the Bcl‐2 family as a group of evolutionarily conserved proteins that regulate cellular anti‐apoptosis. To evaluate the effect of dysregulated hepatocyte apoptosis during zebrafish hepatogenesis, we demonstrate the transgenic overexpression of either zfBLP1 or zfMcl‐1a in zebrafish larval liver. Results showed that 18%–43% of larvae overexpressed zfBLP1 and that 16%–37% of larvae overexpressed zfMc1‐1a in the liver leading to liver hyperplasia in 5‐day postfertilization (dpf) zebrafish larvae. Histologically, zebrafish larvae exhibiting liver hyperplasia displayed a normal type of hepatocyte and the same cell numbers in their two liver buds compared with only one liver bud of wild‐type larvae. Of interest, the expression of cyclin genes (A2, B, D1, and E), hepatocyte nuclear factor genes (HNF‐1α, β, ‐3β, and 4α), and oncogenic markers (P53, c‐myc, β‐catenin, N‐ras, and gankyrin) were up‐regulated, while the expression of C/EBP‐α was down‐regulated in a zfMcl‐1a–mediated anti‐apoptotic process of the liver. Increased cell death and proliferation was found in both hepatic cells of zebrafish larvae overexpressing either zfBLP1 or zfMcl‐1a. However, those zebrafish larvae with liver hyperplasia only lived approximately 10 days. (This finding may have been due to liver abnormalities that led to failure of liver function.) In conclusion, transgenic overexpression of zfBLP1 or zfMcl‐1a in zebrafish larvae interrupts regulation of the homeostatic balance between cell proliferation and programmed cell death during hepatogenesis and leads to liver hyperplasia. Developmental Dynamics 235:515–523, 2006.

Betanodavirus induces oxidative stress-mediated cell death that prevented by anti-oxidants and zfcatalase in fish cells.

The role of oxidative stress in the pathogenesis of RNA nervous necrosis virus infection is still unknown. Red-spotted grouper nervous necrosis virus (RGNNV) induced free radical species (ROS) production at 12–24 h post-infection (pi; early replication stage) in fish GF-1 cells, and then at middle replication stage (24–48 h pi), this ROS signal may upregulate some expressions of the anti-oxidant enzymes Cu/Zn SOD and catalase, and eventually expression of the transcription factor Nrf2. Furthermore, both antioxidants diphenyliodonium and N-acetylcysteine or overexpression of zebrafish catalase in GF-1 cells also reduced ROS production and protected cells for enhancing host survival rate due to RGNNV infection. Furthermore, localization of ROS production using esterase activity and Mitotracker staining assays found that the ROS generated can affect mitochondrial morphology changes and causes ΔΨ loss, both of which can be reversed by antioxidant treatment. Taken together, our data suggest that RGNNV induced oxidative stress response for playing dual role that can initiate the host oxidative stress defense system to upregulate expression of antioxidant enzymes and induces cell death via disrupting the mitochondrial morphology and inducing ΔΨ loss, which can be reversed by anti-oxidants and zfcatalase, which provide new insight into betanodavirus-induced ROS-mediated pathogenesis.

Ubiquitous transcription factor YY1 promotes zebrafish liver steatosis and lipotoxicity by inhibiting CHOP-10 expression.

The ubiquitous transcription factor Yin Yang 1 (YY1) is known to have diverse and complex cellular functions. Although relevant literature has reported that YY1 expression can induce the down-regulation of C/EBP homologous protein 10 (CHOP-10) and then allow the transactivation of certain transcription factors required for lipogenesis, similar properties of YY1 are poorly understood in animal model systems. In this study, we demonstrate hepatic lipid accumulation in YY1 transgenic zebrafish (GY). Oil-red staining cells were predominantly increased in the livers of both GY larvae and adults, indicating that YY1 functionally promoted lipid accumulation in GY livers. Molecular analysis revealed that YY1 over-expression contributed to the accumulation of hepatic triglycerides (TGs) by inhibiting CHOP-10 expression in the juvenile GY and 3 other fish cell lines; the decreased CHOP-10 expression then induced the transactivation of C/EBP-α and PPAR-γ expression. CHOP-10 morpholino (MO)-injected and rosiglitazone-treated G-liver larvae showed liver steatosis by transactivating PPAR-γ. PPAR-γ MO-injected, and GW9662- and astaxanthin-treated GY larvae showed no liver steatosis by inhibiting PPAR-γ. Moreover, a fatty acid (FA) accumulation and a TG decrease were found in the liver of aged GY, leading to the induction of FA-oxidizing systems that increased hepatic oxidative stress and liver damage. This study is the first to examine YY1 as a potential stimulator for GY liver steatosis and lipotoxicity.