ABSTRACT
Oxygen availability has been a major force in shaping the physiological evolution of animals. Under reduced oxygen availability (hypoxia) major changes in gene expression are mediated by hypoxia-inducible factors (HIF alphas). Tetrapods have three hif alpha genes, whereas zebrafish (Danio rerio) and other cyprinids have six due to a teleost lineage-specific genome duplication. We studied the transcriptional divergence of the six teleost-specific hif alphas by inspecting the tissue-specific transcription patterns in adult zebrafish and by monitoring the early developmental transcription of normoxia- and hypoxia-grown zebrafish embryos. Overall we observed the highest hif alpha mRNA levels in tissues that are important for hypoxic survival, including the brain, gill and heart. Of the paralogs that have not previously received attention (hif alpha-1A, hif alpha-2B and hif alpha-3B) especially the hif alpha-2B transcription levels suggest functional relevance. The hif alpha-1A/B paralogs that have considerable coding sequence divergence displayed more overall transcriptional divergence than the hif alpha-2A/B paralog pair. The hif alpha-2A/B paralogs that are similarly conserved in coding sequence had a divergent transcription pattern during early development. When zebrafish grown in modest hypoxia were compared to normoxia grown fish, only hif alpha-3A transcription was significantly altered. These results suggest that, in zebrafish, the evolutionary retention of each hif alpha paralog pair has involved unique patterns of coding sequence divergence, adult tissue-specific transcriptional divergence or developmental transcriptional divergence.
Subject(s)
Gene Duplication , Gene Expression Regulation , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Transcription, Genetic , Animals , Brain/metabolism , Evolution, Molecular , Eye/metabolism , Female , Genes, Duplicate , Gills/metabolism , Hypoxia , Male , Myocardium/metabolism , Oxygen/metabolism , Phylogeny , Polymerase Chain Reaction , RNA, Messenger/metabolism , Tissue Distribution , ZebrafishABSTRACT
Depolarization of cultured mouse cerebellar granule cells with potassium or kainate results in developmentally arrested state that includes down-regulation of GABA(A) receptor alpha1, alpha6 and beta2 subunit expression. These subunits are normally strongly expressed in cerebellar granule cells from second postnatal week throughout the adulthood. In the present study we demonstrate that selective activation of AMPA subtype of glutamate receptors down-regulates alpha1 and alpha6 subunit mRNA expression. Removal of AMPA agonist from culture medium restores expression of these subunits indicating reversibility of the down-regulation. In serum-free culture medium AMPA receptor activation did not down-regulate alpha1 or alpha6 subunit expression. Furthermore, the down-regulation was strongly attenuated when the cells were cultured in the presence of dialysed fetal calf serum. The results indicate that down-regulation of GABA(A) receptor alpha1 and alpha6 subunits by AMPA receptor activation is dependent on the presence of low molecular weight compounds present in fetal calf serum. In order to study mouse cerebellar granule cell maturation and/or regulation of GABA(A) receptor subunit expression in culture, the experiments should be performed in the absence of fetal calf serum.
Subject(s)
Cerebellum/metabolism , Neurons/metabolism , Receptors, AMPA/physiology , Receptors, GABA-A/biosynthesis , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Cells, Cultured , Cerebellum/cytology , Coloring Agents , Culture Media, Serum-Free , Down-Regulation , GABA Agonists/pharmacology , GABA Antagonists/pharmacology , GABA-A Receptor Antagonists , Kainic Acid/pharmacology , Mice , Neurons/drug effects , Protein Binding , Pyrimidinones/pharmacology , Quinoxalines/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, AMPA/antagonists & inhibitors , Receptors, AMPA/genetics , Receptors, GABA-A/genetics , Receptors, Kainic Acid/antagonists & inhibitors , Reverse Transcriptase Polymerase Chain Reaction , Tetrazolium Salts , Thiazoles , GluK2 Kainate ReceptorABSTRACT
We have studied the effects of AMPA/kainate receptor agonists on GABA(A) receptor subunit mRNA expression in vitro in cultured rat cerebellar granule cells (CGCs). Kainate (KA) (100 microM) and high K(+) (25 mM) dramatically up-regulated delta subunit mRNA expression to 500-700% of that in control cells grown in low K(+) (5 mM). KA or high K(+) had no effect on the expression of the other major GABA(A) receptor subunits alpha1, alpha6, beta2, beta3 or gamma2. Up-regulation of delta mRNA was also detected with the AMPA receptor-selective agonist CPW-399 and to a lesser extent with the KA receptor-selective agonist ATPA. AMPA/kainate receptor-selective antagonist DNQX completely inhibited KA-, CPW-399- and ATPA-induced delta mRNA up-regulation indicating that the effects were mediated via AMPA and KA receptor activation. NMDA receptor-selective antagonist MK-801 inhibited 76% of the KA- and 57% of the CPW-399-induced delta up-regulation suggesting that KA and CPW-399 treatments may induce glutamate release resulting in NMDA receptor activation, and subsequently to delta mRNA up-regulation. In CGCs, delta subunit is a component of extrasynaptic alpha6betadelta receptors that mediate tonic inhibition. Up-regulation of delta during prolonged glutamate receptor activation or cell membrane depolarization may be a mechanism to increase tonic inhibition to counteract excessive excitation.
