Repertoire of Na,K-ATPase subunit isoforms in zebrafish from gene to morphant

Na,K-ATPase is the membrane embedded protein that generates the transmembrane sodium and potassium gradients that are necessary to maintain cellular homeostasis. These gradients underlie electrical excitability in nerve and muscle, as well as the transport of numerous solutes and water across epithelia. To carry out these functions, the enzyme hydrolyzes ATP, enabling it to transport Na+ and K+ against their concentration gradients. The active enzyme consists of a catalytic a subunit and a b subunit of unspecified function. In mammalian species, four separate a and three distinct b subunits have been identified. In vitro experiments indicate that association of a and b subunits is promiscuous, raising the possibility that as many as twelve distinct Na,K-ATPase isoenzymes may be formed. Comparison of the biochemical properties between alternate a/b subunit combinations has revealed only subtle functional differences between isoenzymes. Efforts to unravel the functional properties of different Na,K-ATPase a and b subunits have been limited by the inability to purify the isoenzymes from natural sources. Also, it has proven difficult to reconcile which a/b subunit pairs actually associate in vivo within a particular cell or tissue type. This has made it difficult to correlate enzyme function with a specific a/b subunit combination. Therefore, a fundamental unresolved issue concerning Na,K-ATPase is whether the different isoenzymes are redundant or whether each subserves a unique function. To better understand the function of distinct sodium pump isoenzymes, studies were designed to characterize Na,K-ATPase genes expressed in the zebrafish, Danio rerio. Zebrafish is an excellent model system for studying vertebrate development and gene function. The ability to perform high-throughput analysis of gene expression and the capacity to analyze gene function via morpholino-based gene knockdown technology make zebrafish an attractive system in which to dissect functional differences amongst members of a multigene family. Searches of the GenBank database of expressed sequence tags revealed several cDNA clones with sequence homology to mammalian Na,K-ATPase genes. Molecular cloning revealed a cohort of fifteen distinct Na,K-ATPase subunit genes in zebrafish. Of this cohort, nine cDNAs encode a subunits and six encode b subunits. Sequence comparisons and phylogenetic analysis indicate that among the nine a subunit genes identified, six belong to the a1-like subfamily, one is an ortholog of the a2 gene, and two appear to be a3 subfamily members. Also, two zebrafish homologs were identified for each of the mammalian b1, b2 and b3 isoform genes. Using zebrafish radiation hybrid and meiotic mapping panels, linkage assignments for each a and b subunit gene were determined. Na,K-ATPase genes are dispersed in the zebrafish genome with the exception of four of the a1-like genes, which are tightly clustered on linkage group 1. Comparative mapping studies indicate that most of the zebrafish Na,K-ATPase genes localize to regions of conserved synteny (conservation of gene order over wide evolutionary distances) between zebrafish and humans. The expression pattern of Na,K-ATPase a and b subunit genes during zebrafish embryogenesis was analyzed using in situ hybridization. The most striking finding is that each of the fourteen Na,K-ATPase genes exhibits a distinct expression profile. All a and b subunit genes are expressed in the nervous system, although the pattern of expression in different regions varies dramatically. In peripheral tissues, three of a1-like genes are expressed in pronephros and mucous cells, one is expressed in heart, and one is predominant in skeletal muscle. The a2 gene is expressed in brain and heart but is most prominent in skeletal muscle, while the two a3 genes are restricted in their expression to the nervous system. Of the six b subunit genes, b1a is expressed at highest abundance in lens, pronephros, and heart, while b1b transcripts are abundant in mucous cells. The two b2-like genes are differentially expressed in nervous system. One b3 gene is expressed exclusively in brain while the other is abundantly expressed in skeletal muscle. Based on expression patterns, at least fourteen a/b subunit pairs are likely to be formed in various tissues. This high degree of isoenzyme diversity is consistent with the idea that many of the isoenzymes are likely to possess distinct functional properties. In order to test this hypothesis, expression of Na,K-ATPase b1a ( atp1b1a) and b1b (atp1b1b) in zebrafish embryos was reduced using anti-sense morpholine oligonucleotides (morpholinos). Morphants (morpholino injected embryos) bearing reduced levels of atp1b1a showed defects in cardiac morphogenesis and function while atp1b1b morphants were deficient in neural development. The atp1b1b morphant displayed overall neural degeneration at all injected levels of morpholino (1.5ng – 12ng). At higher doses, fused somites and stunted growth was observed. In contrast, atp1b1a morphants exhibited dose dependent severity of phenotype. At low doses, there was cardiac hypertrophy accompanied with pericardial edema. However, at high doses of injected morpholino, specific degeneration of ventricle occurred along with atrial hypertrophy. In some cases, there was a conduction defect, such that ventricular beats lagged behind the sinus rhythm. These data provide the first evidence for functional differences between Na,K-ATPase b subunit isoforms. The difference in morphant phenotype suggests that atp1b1a plays an integral role in fashioning the zebrafish heart while atp1b1b expression is critical for neural development. Coupled to mRNA co-injection, these morphants provide an in vivo assay for examining whether the function of either atp1b1a or atp1b1b can be substituted by another b subunit. The paradigm used to detect functional differences between isoforms can also be universally applied to the study of any gene family whose zebrafish orthologs are identified. The inhibition of gene expression using antisense-morpholino and the study of the resulting morphant phenotype is a very powerful method of reverse genetics. This experiments contained in this thesis set the precedent for the use of zebrafish genetics to answer the fundamental questions regarding isoform heterogeneity in Na,K-ATPase subunit isoforms.