Low oxygen levels (hypoxia) play a role in clinical conditions such as stroke, chronic ischemia, and cancer. To better understand these diseases, it is crucial to study the responses of vertebrates to hypoxia. Among vertebrates, some teleosts have developed the ability to adapt to extremely low oxygen levels. We have studied long-term adaptive responses to hypoxia in adult zebrafish. We used zebrafish that survived severe hypoxic conditions for 3 wk and showed adaptive behavioral and phenotypic changes. We used cDNA microarrays to investigate hypoxia-induced changes in expression of 15,532 genes in the respiratory organs (the gills). We have identified 367 differentially expressed genes of which 117 showed hypoxia-induced and 250 hypoxia-reduced expressions. Metabolic depression was indicated by repression of genes in the TCA cycle in the electron transport chain and of genes involved in protein biosynthesis. We observed enhanced expression of the monocarboxylate transporter and of the oxygen transporter myoglobin. The hypoxia-induced group further included the genes for Niemann-Pick C disease and for Wolman disease [lysosomal acid lipase (LAL)]. Both diseases lead to a similar intra- and extracellular accumulation of cholesterol and glycolipids. The Niemann-Pick C protein binds to cholesterol from internal lysosomal membranes and is involved in cholesterol trafficking. LAL is responsible for lysosomal cholesterol degradation. Our data suggest a novel adaptive mechanism to hypoxia, the induction of genes for lysosomal lipid trafficking and degradation. Studying physiological responses to hypoxia in species tolerant for extremely low oxygen levels can help identify novel regulatory genes, which may have important clinical implications.

The transcription factor peroxisome proliferator-activated receptor α (PPARα) is an important regulator of hepatic lipid metabolism. While PPARα is known to activate transcription of numerous genes, no comprehensive picture of PPARα binding to endogenous genes has yet been reported. To fill this gap, we performed Chromatin immunoprecipitation (ChIP)-chip in combination with transcriptional profiling on HepG2 human hepatoma cells treated with the PPARα agonist GW7647. We found that GW7647 increased PPARα binding to 4220 binding regions. GW7647-induced binding regions showed a bias around the transcription start site and most contained a predicted PPAR binding motif. Several genes known to be regulated by PPARα, such as ACOX1, SULT2A1, ACADL, CD36, IGFBP1 and G0S2, showed GW7647-induced PPARα binding to their promoter. A GW7647-induced PPARα-binding region was also assigned to SREBP-targets HMGCS1, HMGCR, FDFT1, SC4MOL, and LPIN1, expression of which was induced by GW7647, suggesting cross-talk between PPARα and SREBP signaling. Our data furthermore demonstrate interaction between PPARα and STAT transcription factors in PPARα-mediated transcriptional repression, and suggest interaction between PPARα and TBP, and PPARα and C/EBPα in PPARα-mediated transcriptional activation. Overall, our analysis leads to important new insights into the mechanisms and impact of transcriptional regulation by PPARα in human liver and highlight the importance of cross-talk with other transcription factors.

Mammalian CYPHER (Oracle, KIA0613), a member of the PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36), has been associated with cardiac and muscular myopathies. Targeted deletion of Cypher in mice is neonatal lethal possibly caused by myopathies. To further investigate the role of cypher in development, we have cloned the zebrafish orthologue. We present here the gene, domain structure, and expression pattern of zebrafish cypher during development. Cypher was not present as a maternal mRNA and was absent during early development. Cypher mRNA was first detected at the 3-somite stage in adaxial somites, and as somites matured, cypher expression gradually enveloped the whole somite. Later, cypher expression was also found in the heart, in head and jaw musculature, and in the brain. We further identified 13 alternative spliced forms of cypher from zebrafish heart and skeletal muscle tissue, among them a very short form containing the PDZ domain but lacking the ZM (ZASP-like) motif and the LIM domains. Targeted gene knock-down experiments using cypher antisense morpholinos led to severe defects, including truncation of the embryo, deformation of somites, dilatation of the pericardium, and thinning of the ventricular wall. The phenotype could be rescued by a cypher form, which contains the PDZ domain and the ZM motif, but lacks all three LIM domains. These findings indicate that a PDZ domain protein is important for normal somite formation and in normal heart development. Treatment of zebrafish embryos with cyclopamine, which disrupts hedgehog signaling, abolished cypher expression in 9 somite and 15-somite stage embryos. Taken together, our data suggest that cypher may play a role downstream of sonic hedgehog, in a late stage of somite development, when slow muscle fibers differentiate and migrate from the adaxial cells.

Wnt signaling is essential during animal development and also plays important roles in pathological conditions. Two mayor pathways have been described: the β-catenin-dependent canonical (or classical) pathway and the β-catenin-independent non-canonical Wnt pathway. Recent binding studies suggest links between the small PDZ protein TIP-1 (Tax-1 interacting protein) to components of both Wnt pathways. We have cloned and characterized the zebrafish tip-1 gene. Whole mount in situ hybridization and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that zebrafish tip-1 is present as a maternal RNA and is ubiquitously expressed during early development. After 24 h of development, tip-1 expression was high in the central nervous system (CNS) whereas only weak expression was detected in the caudal regions of the zebrafish embryo. Tip-1 knockdown using antisense morpholino oligonucleotides, as well as ectopic tip-1 expression, led to elongation defects in zebrafish embryos and larvae. Both knockdown and overexpression of tip-1 resulted in a widened goosecoid (gsc) expression domain in shield stage embryos, led to an abbreviated prechordal plate, and to reduced convergent extension movements during gastrulation. We constructed a green fluorescence protein (GFP)/TIP-1 fusion protein which, when expressed in cultured fibroblasts (ZF4-cells), induced filopodia growth. Our observations indicate a role for TIP-1 in gastrulation movements and in filopodia growth induction.