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The growing burden of liver fibrosis and lack of effective antifibrotic therapies highlight the need for identification of pathways and complementary model systems of hepatic fibrosis. A rare, monogenic disorder in which children with mutations in mannose phosphate isomerase (MPI) develop liver fibrosis led us to explore the function of MPI and mannose metabolism in liver development and adult liver diseases. Herein, analyses of transcriptomic data from three human liver cohorts demonstrate that MPI gene expression is down-regulated proportionate to fibrosis in chronic liver diseases, including nonalcoholic fatty liver disease and hepatitis B virus. Depletion of MPI in zebrafish liver in vivo and in human hepatic stellate cell (HSC) lines in culture activates fibrotic responses, indicating that loss of MPI promotes HSC activation. We further demonstrate that mannose supplementation can attenuate HSC activation, leading to reduced fibrogenic activation in zebrafish, culture-activated HSCs, and in ethanol-activated HSCs. Conclusion: These data indicate the prospect that modulation of mannose metabolism pathways could reduce HSC activation and improve hepatic fibrosis.

The intrahepatic biliary network is a highly branched three-dimensional network lined by biliary epithelial cells, but how its branching patterns are precisely established is not clear. We designed a new computer-based algorithm that quantitatively computes the structural differences of the three-dimensional networks. Utilizing the algorithm, we showed that inhibition of Cyclin-dependent kinase 5 (Cdk5) led to reduced branching in the intrahepatic biliary network in zebrafish. Further, we identified a previously unappreciated downstream kinase cascade regulated by Cdk5. Pharmacological manipulations of this downstream kinase cascade produced a crowded branching defect in the intrahepatic biliary network and influenced actin dynamics in biliary epithelial cells. We generated larvae carrying a mutation in cdk5 regulatory subunit 1a (cdk5r1a), an essential activator of Cdk5. cdk5r1a mutant larvae show similar branching defects as those observed in Cdk5 inhibitor-treated larvae. A small-molecule compound that interferes with the downstream kinase cascade rescued the mutant phenotype. These results provide new insights into branching morphogenesis of the intrahepatic biliary network.

SummaryAdipose triglyceride lipase (ATGL) and comparative gene identification 58 (CGI-58) are critical regulators of triacylglycerol (TAG) turnover. CGI-58 is thought to regulate TAG mobilization by stimulating the enzymatic activity of ATGL. However, it is not known whether this coactivation function of CGI-58 occurs in vivo. Moreover, the phenotype of human CGI-58 mutations suggests ATGL-independent functions. Through direct comparison of mice with single or double deficiency of CGI-58 and ATGL, we show here that CGI-58 knockdown causes hepatic steatosis in both the presence and absence of ATGL. CGI-58 also regulates hepatic diacylglycerol (DAG) and inflammation in an ATGL-independent manner. Interestingly, ATGL deficiency, but not CGI-58 deficiency, results in suppression of the hepatic and adipose de novo lipogenic program. Collectively, these findings show that CGI-58 regulates hepatic neutral lipid storage and inflammation in the genetic absence of ATGL, demonstrating that mechanisms driving TAG lipolysis in hepatocytes differ significantly from those in adipocytes.

Mitochondria are the site of iron utilization, wherein imported iron is incorporated into heme or iron–sulfur clusters. Previously, we showed that a cytosolic siderophore, which resembles a bacterial siderophore, facilitates mitochondrial iron import in eukaryotes, including zebrafish. An evolutionarily conserved 3-hydroxy butyrate dehydrogenase, 3-hydroxy butyrate dehydrogenase 2 (Bdh2), catalyzes a rate-limiting step in the biogenesis of the eukaryotic siderophore. We found that inactivation of bdh2 in developing zebrafish embryo results in heme deficiency and delays erythroid maturation. The basis for this erythroid maturation defect is not known. Here we show that bdh2 inactivation results in mitochondrial dysfunction and triggers their degradation by mitophagy. Thus, mitochondria are prematurely lost in bdh2-inactivated erythrocytes. Interestingly, bdh2-inactivated erythroid cells also exhibit genomic alterations as indicated by transcriptome analysis. Reestablishment of bdh2 restores mitochondrial function, prevents premature mitochondrial degradation, promotes erythroid development, and reverses altered gene expression. Thus, mitochondrial communication with the nucleus is critical for erythroid development.

Notch and fibroblast growth factor (FGF) signaling pathways have been implicated in the establishment of proper periodicity of vertebrate somites. Here, we show evidence that a Hes6-related hairy/Enhancer of split-related gene, her13.2, links FGF signaling to the Notch-regulated oscillation machinery in zebrafish. Expression of her13.2 is induced by FGF-soaked beads and decreased by an FGF signaling inhibitor. her13.2 is required for periodic repression of the Notch-regulated genes her1 and her7, and for proper somite segmentation. Furthermore, Her13.2 augments autorepression of her1 in association with Her1 protein. Therefore, FGF signaling appears to maintain the oscillation machinery by supplying a binding partner, Her13.2, for Her1.

Vertebrate endoderm development has recently become the focus of intense investigation. We have identified a novel sox gene, 226D7, which is important in zebrafish endoderm development. 226D7 was isolated by an in situ hybridization screening for genes expressed in the yolk syncytial layer (YSL) at the blastula stage. 226D7 is expressed mainly in the YSL at this stage and, during gastrulation, its expression is also detected in the forerunner cells and endodermal precursor cells. The expression of 226D7 is positively regulated by Nodal signaling. The knockdown of 226D7 using morpholino antisense oligonucleotides results in a lack of sox17-expressing endodermal precursor cells during gastrulation, and, consequently, lacks endodermal derivatives such as gut tissue. The effect is strictly restricted to the endodermal lineage, while the mesoderm is normally formed, a phenotype that is nearly identical to that of the casanova mutant (Dev. Biol. 215 (1999) 343). We further demonstrate that overexpression of 226D7 increases the number of sox17-expressing endodermal progenitor cells without upregulating the expression of the Nodal genes, cyclops and squint. Region-specific knockdown and overexpression of 226D7 by injection into the YSL suggest that 226D7 in the YSL is not involved in endoderm formation and 226D7 in the endoderm progenitor cells is important for endoderm development. Taken together, our data demonstrate that 226D7 is a downstream target of Nodal signal and a critical transcriptional regulator of early endoderm formation.

Nonalcoholic fatty liver disease is the most common liver disease in both adults and children. The earliest stage of this disease is hepatic steatosis, in which triglycerides are deposited as cytoplasmic lipid droplets in hepatocytes. Through a forward genetic approach in zebrafish, we found that guanosine monophosphate (GMP) synthetase mutant larvae develop hepatic steatosis. We further demonstrate that activity of the small GTPase Rac1 and Rac1-mediated production of reactive oxygen species (ROS) are down-regulated in GMP synthetase mutant larvae. Inhibition of Rac1 activity or ROS production in wild-type larvae by small molecule inhibitors was sufficient to induce hepatic steatosis. More conclusively, treating larvae with hydrogen peroxide, a diffusible ROS that has been implicated as a signaling molecule, alleviated hepatic steatosis in both GMP synthetase mutant and Rac1 inhibitor-treated larvae, indicating that homeostatic production of ROS is required to prevent hepatic steatosis. We further found that ROS positively regulate the expression of the triglyceride hydrolase gene, which is responsible for the mobilization of stored triglycerides in hepatocytes. Consistently, inhibition of triglyceride hydrolase activity in wild-type larvae by a small molecule inhibitor was sufficient to induce hepatic steatosis.De novo GMP synthesis influences the activation of the small GTPase Rac1, which controls hepatic lipid dynamics through ROS-mediated regulation of triglyceride hydrolase expression in hepatocytes.

BTG/tob family proteins are thought to be a potential tumor suppressor due to their anti-proliferative activity. We cloned zebrafish btg-b, a member of the BTG1/2 subfamily, using in situ hybridization screening. The tissue-specific expression of btg-b is first observed in the organizer region at the early gastrula stage. Later in development, the forebrain, the hindbrain, the polster and the paraxial mesoderm transiently express btg-b. Recently, mouse Btg1 and Btg2 have been shown to be a cofactor for Hoxb9. Double in situ hybridization with zebrafish btg-b and hoxb9a indicates that the expression domains of these two genes overlap in the posterior paraxial mesoderm.

SummaryEmerging evidence indicates that paracrine signals from endothelial cells play a role in tissue differentiation and organ formation [1–3]. Here, we identify a novel role for endothelial cells in modulating hepatocyte polarization during liver organogenesis. We find that in zebrafish, the apical domain of the hepatocytes predicts the location of the intrahepatic biliary network. The refinement of hepatocyte polarization coincides with the invasion of endothelial cells into the liver, and these endothelial cells migrate along the maturing basal surface of the hepatocytes. Using genetic, pharmacological, and transplantation experiments, we provide evidence that endothelial cells influence the polarization of the adjacent hepatocytes. This influence of endothelial cells on hepatocytes is mediated at least in part by the cell-surface protein Heart of glass and contributes to the establishment of coordinately aligned hepatocyte apical membranes and evenly spaced intrahepatic conduits.

The roles of extra-embryonic tissues in early vertebrate body patterning have been extensively studied, yet we know little about their function during later developmental events. Here, we analyze the function of the zebrafish extra-embryonic yolk syncytial layer (YSL) specific transcription factor,Mtx1, and find that it plays an essential role in myocardial migration. Downregulating the function of Mtx1 in the YSL leads to cardia bifida, a phenotype in which the myocardial cells fail to migrate to the midline. Mtx1 in the extra-embryonic YSL appears to regulate the embryonic expression of fibronectin, a gene previously implicated in myocardial migration. We further show dosage-sensitive genetic interactions between mtx1 and fibronectin. Based on these data, we propose that the extra-embryonic YSL regulates myocardial migration, at least in part by influencing fibronectin expression and subsequent assembly of the extracellular matrix in embryonic tissues.

The medaka is becoming an attractive model organism for the study of vertebrate early development and organogenesis and large-scale mutagenesis projects that are aimed at creating developmentally defective mutants are now being conducted by several groups in Japan. To strengthen the study of medaka developmental genetics, we have conducted a large-scale isolation of ESTs from medaka embryos and developed tools that facilitate mutant analysis. In this study, we have characterized a total of 132,082 sequences from both ends of cloned insert cDNAs from libraries generated at different stages of medaka embryo development. Clustering analysis with 3-prime sequences finally identified a total of 12,429 clusters. As a pilot analysis, 924 clusters were subjected to in situ hybridization to determine the spatial localization of their transcripts. Using EST sequence data generated in the present study, a 60-mer oligonucleotide microarray with 8,091 unigenes (Medaka Microarray 8K) was constructed and tested for its usefulness in expression profiling. Furthermore, we have developed a rapid and reliable mutant mapping system using a set of mapped EST markers (M-marker 2003) that covers the entire medaka genome. These resources will accelerate medaka mutant analyses and make an important contribution to the medaka genome project.

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1, 2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila[3, 4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5–8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6–8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9, 10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11–13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation.