Zebrafish Molecular Genetics and Genomics Laboratory
The research in the laboratory is directed towards a molecular genetic and genomic understanding of development and circadian rhythmicity using the zebrafish (Danio rerio) as a model. We are currently working on several projects as follows:
Heme regulation of exocrine zymogens. We previously determined that the porphyric phenotype of zebrafish yquem results from a missense mutation in the gene encoding uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37), the fifth enzyme in the heme biosynthesis pathway, and established zebrafish yquem as a vertebrate model for human hepatoerythropoietic porphyria (HEP) (Wang et al., 1998). In an effort to investigate the unknown aspects of UROD deficiency pathogenesis, we conducted microarray analysis of yquem/urod and wild type control zebrafish. The microarray analysis, with independent in situ hybridization confirmation, revealed down-regulation of six exocrine pancreas zymogens, i.e., carboxypeptidase A, trypsin precursor, trypsin like, chymotrypsinogen B1, chymotrypsinogen 1-like, and elastase 2 like, in yquem/urod (-/-). We then determined down-regulation of these six zymogens specifically in the exocrine pancreas of sauternes larvae, carrying a mutation in the gene encoding delta-aminolevulinate synthase (ALAS2), the first enzyme in heme biosynthesis. We also found that ptf1a, a transcription factor regulating exocrine zymogens, is down-regulated in both yquem/urod (-/-) and sau/alas2 (-/-) larvae. Further, hemin treatment rescues expression of ptf1a and these six zymogens in both yquem/urod (-/-) and sauternes/alas2 (-/-) larvae. Thus, it appears that heme deficiency down-regulates ptf1a, which in turn leads to down-regulation of exocrine zymogens. These findings provide a better understanding of heme deficiency pathogenesis and enhance our ability to diagnose and treat patients with porphyria or pancreatic diseases (Wang et al., 2007b). We are currently investigating the potential roles of PTF1A, HEME OXYGENASE1, and heme-responding proteins such as BACH2 in heme regulation of these exocrine zymogens.
Identification and characterization of ocular genes. Homozygous masterblind (mbl) mutant, exhibits reduced or absent eyes and telecephalon, the expansion of the diencephalic fates to the front of the brain. A missense mutation in the GSK3-binding domain of zebrafish AXIN1, a scaffolding protein in the Wnt signaling pathway, results in the eyeless mbl phenotype. We take advantage of the mbl/axin1 eyeless phenotype and explore its use in identification and studying eye-specific genes in zebrafish. We conducted transcriptome analysis of mbl/axin1 (-/-) and wild type control zebrafish using a 14 000-oligonucleotide microarray (Ocimum Biosolutions). Statistical analysis of the microarray data, in combination with at least two-fold differential gene expression between wild type and mbl/axin1 mutant zebrafish identified 40 down-regulated genes and 30 up-regulated genes in mbl/axin1 (-/-). We further characterized 8 down-regulated genes, including four eye-specific genes, opsin1 short-wave-sensitive1 (opn1sw1), crystallin beta a1b (cryba1b ), crystallin beta a2b (cryba2b), and crystallin gamma embryonic M2d3 (crygm2d3); two eye and brain genes, ATPase, H+ transporting, lysosomal, V0 subunit c (atp6v0c) and basic leucine zipper and W2 domains 1a (bzw1a); and two constitutive genes, heat shock protein 8 (hspa8) and ribosomal protein L7a (rpl7a). In situ hybridization experiments confirmed down-regulation of these 8 ocular formation genes in mbl (-/-) and showed their ocular and dynamic temporal expression patterns during zebrafish early development (Wang et al., 2008). We also try to identify genes that regulate early ocular specification and differentiation and are potentially Wnt/axin1 targets by examining the gastrulation stage (around 14 hpf) when the mbl phenotype first appears.
Comparative analysis of circadian clock genes in teleost fish genomes. The release of the genome sequences of the Japanese pufferfish (Takifugu rubripes), the spotted green pufferfish (Tetraodon nigroviridis), the Japanese medaka (Oryzias latipes), the three spine stickleback (Gasterosteus aculeatus) and the zebrafish (Danio rerio) allow us to conduct comparative analysis of circadian clock genes in the teleost fish genomes. How many circadian clock genes are there in teleost fish genomes? How many circadian clock genes arose from ancient genome duplication? How had these duplicated genes been preserved? Using known circadian clock genes from zebrafish as well as other organisms, we searched these five teleost genomes and uncovered a number of families of duplicated circadian clock genes. Phylogenetic reconstruction showed that zebrafish has two period1 genes, period1a and period1b. Syntenic analysis then indicated zebrafish period1a/period1b are a duplicated pair originated from the ancient genome-wide duplication (Wang, 2008). Temporal and spatial subfunctionalization of period1 could be responsible for preservation of period1a/period1b in the zebrafish genome, because the duplicate peaks at different times of the day and exhibits distinct expression domains as shown by in situ hybridization.
Ontogeny of zebrafish circadian clocks. Zebrafish eggs are fertilized externally and the resulting embryos are immediately exposed to the external environment. If early embryos have circadian rhythms, they should rhythmically express key circadian clock genes. We have tested this hypothesis by determining the oscillations of a number of circadian clock genes in zebrafish embryos and larvae using in situ hybridization and quantitative real-time PCR. In situ hybridization was conducted using embryos and larvae from the second day to the fifth day. We also examined gene expression in constant darkness, constant light, and an 8:8 h light/dark cycle. For instance, period1a and period1b are rhythmically expressed in a robust fashion during zebrafish early development.
Pineal regulation of circadian rhythmicity. The brain, eyes and pineal gland have been implicated as important circadian oscillators. From an EST analysis of zebrafish brain and eyes, we found that ndrg1b (N-myc downstream regulated gene 1b) exhibits rhythmic pineal expression: ndgr1b is expressed only at ZT12 and ZT16. The same rhythmic pineal expression pattern persists under DD and LL conditions. We are currently investigating molecular genetic mechanisms underlying ndrg1b rhythmic pineal expression.
Selected Publications:
H. Wang. 2008. Comparative genomic analysis of teleost fish bmal genes. Submitted.
H. Wang. 2008. Comparative analysis of teleost fish genomes reveals preservation of different ancient clock duplicates in different fishes. Submitted.
H. Wang. 2008. Comparative analysis of period genes in teleost fish genomes. Journal of Molecular Evolution In press.
H. Wang, J. W. Kesinger, Q. Zhou, J. D. Wren, G. Martin, S. Turner, Y. Tang, M. B. Frank and M. Centola. 2008. Identification and characterization of zebrafish ocular formation genes. Genome 51:222-235.
H. Wang, Q. Zhou, J. Kesinger, C. Norris and Cammi Valdez. 2007b. Heme regulates exocrine peptidase precursor genes in zebrafish. Experimental Biology and Medicine 232:1170-1180.
H. Wang, E. M. Lee, S. Sperber, S. Lin, M. Ekker and Q. Long. 2007a. Isolation and expression of zebrafish zinc-finger transcription factor gene tsh1. Gene Expression Patterns 7:318-322.
B. A. Roe, C. Lau, S. Oommen, J. Li, A. Hua, H. S. Lai, S. Kenton, J. White and H. Wang. 2003. Comparative analysis of human chromosome 22q11.1-q12.3 with syntenic regions in the chimpanzee, baboon, bovine, mouse, pufferfish, and zebrafish genomes. Cold Spring Harbor Symposia on Quantitative Biology 68:265-274.
A. E. Tucker, I. I. Salles, D. E. Voth, W. Ortiz-Leduc, H. Wang, I. Dozmorov, M. Centola and J. D. Ballard. 2003. Decreased glycogen synthase kinase 3-beta levels and related physiological changes in Bacillus anthracis lethal toxin-treated macrophages. Cellular Microbiology 5:523-532.
R. M. Young, S. Marty, Y. Nakano, H. Wang, D. Yamamoto, S. Lin, and M. L. Allende. 2002. Zebrafish yolk-specific not really started (nrs) gene is a vertebrate homolog of the Drosophila spinster gene and is essential for embryogenesis. Developmental Dynamics 223:298-305.
H. Wang, Q. Long, S. D. Marty, S. Sassa, and S. Lin. 1998. A zebrafish model for hepatoerythropoietic porphyria. Nature Genetics 20:239-243.
Q. Long, A. Meng, H. Wang, J. R. Jessen, M. J. Farrell, and S. Lin. 1997. GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene. Development 124:4105-4111.