miR-122 targets an anti-apoptotic gene, Bcl-w, in human hepatocellular carcinoma cell lines.

miR-122, a hepato-specific microRNA (miRNA), is frequently down-regulated in human hepatocellular carcinoma (HCC). In an effort to identify novel miR-122 targets, we performed an in silico analysis and detected a putative binding site in the 3'-untranslated region (3'-UTR) of Bcl-w, an anti-apoptotic Bcl-2 family member. In the HCC-derived cell lines, Hep3B and HepG2, we confirmed that miR-122 modulates Bcl-w expression by directly targeting binding site within the 3'-UTR. The cellular mRNA and protein levels of Bcl-w were repressed by elevated levels of miR-122, which subsequently led to reduction of cell viability and activation of caspase-3. Thus, Bcl-w is a direct target of miR-122 that functions as an endogenous apoptosis regulator in these HCC-derived cell lines.

Overexpression of Myostatin2 in zebrafish reduces the expression of dystrophin associated protein complex (DAPC) which leads to muscle dystrophy.

Myostatin, a member of the TGF-beta superfamily, is a potent negative regulator of skeletal muscle and growth. Previously, we reported Mstn1 from zebrafish and studied its influence on muscle development. In this study, we identified another form of Myostatin protein which is referred to as Mstn2. The size of Mstn2 cDNA is 1342 bp with 109 and 132 bp of 5' and 3'-untranslated regions (UTRs), respectively. The coding region is 1101 bp encoding 367 amino acids. The identity between zebrafish Mstn1 and 2 is 66%. The phylogenetic tree revealed that the Mstn2 is an ancestral form of Mstn1. To study the functional aspects, we overexpressed mstn2 and noticed that embryos became less active and the juveniles with bent and curved phenotypes when compared to the control. The RT-PCR and in situ hybridization showed concurrent reduction of dystrophin associated protein complex (DAPC). In cryosection and in situ hybridization, we observed the disintegration of somites, lack of transverse myoseptum and loss of muscle integrity due to the failure of muscle attachment in mstn2 overexpressed embryos. Immunohistochemistry and western blot showed that there was a reduction of dystrophin, dystroglycan and sarcoglycan at translational level in overexpressed embryos. Taken together, these results indicate the suitability of zebrafish as an excellent animal model and our data provide the first in vivo evidence of muscle attachment failure by the overexpression of mstn2 and it leads to muscle loss which results in muscle dystrophy that may contribute to Duchenne syndrome and other muscle related diseases.

Steroid hormones (17beta-estradiol and hydrocortisone) upregulate hepatocyte nuclear factor (HNF)-3beta and insulin-like growth factors I and II expression in the gonads of tilapia (Oreochromis mossambicus) in vitro.

Hepatocyte nuclear factors (HNF-1alpha, -1beta and -3beta) and insulin-like growth factors (IGF-I and -II), which are involved in liver-specific gene expression, metabolism, development and cell growth, have been found in the gonads of tilapia (Oreochromis mossambicus). However, the functions of these factors and how they interact within the gonads of bony fish are not understood. In the present study, we provided experimental evidence that the expression of HNF-3beta in the gonads of tilapia, but not HNF-1alpha and -1beta, was affected in vitro by 17beta-estradiol and hydrocortisone. Immunohistochemical staining confirmed that tilapia HNF-3beta was mainly found in the nuclei of hepatocytes, the follicular granulosa cells of the ovaries, and the interstitial cells of the testes of adult tilapia. Further data were gathered at various steroid concentrations (0.1, 1, 10, 100, and 1000 nM) over various culture intervals (6, 12, 18, 24, 30, and 36 h) and subjected to semi-quantitative RT-PCR analysis. The expression of downstream genes (IGF-I and -II) followed the same temporal patterns as HNF-3beta, albeit at decreased levels for 30 and 36 h culture intervals. Both hormones upregulated HNF-3beta mRNA expression at concentrations of 0.1-10 nM, and reached optimal physiological concentrations for induction of IGFs at 1-10 nM. The identity of the PCR fragments was concurrently verified by sequencing and PCR-Southern hybridization. We inferred that HNF-3beta and IGFs may play a regulatory role in tilapia gonads during oocyte maturation and spermatogenesis.

Two distinct teleost hepatocyte nuclear factor 1 genes, hnf1alpha/tcf1 and hnf1beta/tcf2, abundantly expressed in liver, pancreas, gut and kidney of zebrafish.

Two distinct forms of zebrafish hepatocyte nuclear factor 1 (hnf1) were identified and referred to as hnf1alpha/tcf1 and hnf1beta/tcf2. Both hnf1 genes were shown to be expressed abundantly in liver, pancreas, gut and kidney. Zebrafish HNF1alpha and HNF1beta proteins contain all HNF1 signature domains including the dimerization domain, POU-like domain and atypical homeodomain. Sequence and phylogenetic analysis reveals that zebrafish hnf1alpha is closer to tetrapodian hnf1alpha than to tetrapodian hnf1beta and zebrafish hnf1beta is highly conserved with tetrapodian hnf1beta. Existences of hnf1alpha and hnf1beta in teleost zebrafish, tilapia and fugu suggest that hnf1 gene duplication might occur before the divergence of teleost and tetrapod ancestors. Zebrafish hnf1alpha and hnf1beta genes were mapped to linkage group LG8 and LG15 in T51 panel by RH mapping and are composed of 10 and 9 exons, respectively. Zebrafish hnf1beta gene with at least 11 genes in LG15 was identified to maintain the conserved synteny with those of human in chromosome 17 and those of mouse in chromosome 11. Our results indicate that distinct hnf1alpha and hnf1beta genes in teleosts had been evolved from the hnf1 ancestor gene of chordate.

Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus).

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.

Thioacetamide induced liver damage in zebrafish embryo as a disease model for steatohepatitis.

Steatohepatitis has recently been increasing as a cofactor influencing the progression of fibrosis, cirrhosis, adenoma and carcinoma in liver; however, the mechanisms by which it contributes to liver injury remain uncertain. We induced steatohepatitis in zebrafish embryos using thioacetamide (TAA). TUNEL assay revealed significant increasing of apoptosis in liver after 5 days post fertilization and the increasing of apoptosis was observed to be associated with the up-regulation of apoptotic genes such as, bad, bax, P-38a, caspase-3 and 8, and JNK-1. Histological sections by oil red O stain showed the accumulation of fatty droplets which causes the pushing of the nucleus towards one side. Up-regulation of steatosis markers such as, ACC, adiponectin, PTL, CEBP- alpha and beta, SREBP-1 was also observed. Furthermore, the elevation of glutathione peroxidase in TAA treated embryos indicated that TAA induces lipid peroxidation which leads to causes liver damage. Zebrafish has already been considered as a good human disease model and in this context; TAA-treated zebrafish may serve as a good animal model to study the molecular pathogenesis of steatohepatitis. Moreover, non-availability of specific drugs to prevent steatohepatitis, this animal model may serve as a powerful preclinical platform to study the therapeutic strategies and for evaluating chemoprevention strategies for this disease.

Phosphatidylserine receptor is required for the engulfment of dead apoptotic cells and for normal embryonic development in zebrafish.

During development, the role of the phosphatidylserine receptor (PSR) in the removal of apoptotic cells that have died is poorly understood. We have investigated this role of PSR in developing zebrafish. Programmed cell death began during the shield stage, with dead cells being engulfed by a neighboring cell that showed a normal-looking nucleus and the nuclear condensation multi-micronuclei of an apoptotic cell. The zebrafish PSR engulfing receptor was cloned (zfpsr), and its nucleotide sequence was compared with corresponding sequences in Drosophila melanogaster (76% identity), human (74%), mouse (72%) and Caenorhabditis elegans (60%). The PSR receptor contained a jmjC domain (residues 143-206) that is a member of the cupin metalloenzyme superfamily, but in this case serves an as yet unknown function(s). psr knockdown by a PSR morpholino oligonucleotide led to accumulation of a large number of dead apoptotic cells in whole early embryo. These cells interfered with embryonic cell migration. In addition, normal development of the somite, brain, heart and notochord was sequentially disrupted up to 24 hours post-fertilization. Development could be rescued in defective embryos by injecting psr mRNA. These results are consistent with a PSR-dependent system in zebrafish embryos that engulfs apoptotic cells mediated by PSR-phagocytes during development, with the system assuming an important role in the normal development of tissues such as the brain, heart, notochord and somite.

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-beta) 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.