In response to the lack of a transgenic line of zebrafish labeled with heart-specific fluorescence in vivo to serve as a research model, we cloned a 1.6-kb polymerase chain reaction (PCR) -product containing the upstream sequence (؊870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene, (cmlc2). A germ-line transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the green fluorescent protein (GFP) gene with ends consisting of inverted terminal repeats of an adeno-associated virus. Green fluorescence was intensively and specifically expressed in the myocardial cells located both around the heart chambers and the atrioventricular canal. Neither the epicardium nor the endocardium showed fluorescent signals. The GFP expression in the transgenic line faithfully recapitulated with the spatial and temporal expression of the endogenous cmlc2. Promoter analysis showed that the fragment consisting of nucleotides from ؊210 to 34 (؊210/34) was sufficient to drive heart-specific expression, with a ؊210/؊73 motif as a basal promoter and a ؊210/؊174 motif as an element involved in suppressing ectopic (nonheart) expression. Interestingly, a germ-line of zebrafish whose GFP appeared ectopically in all muscle types (heart, skeletal, and smooth) was generated by injecting the fragment including a single nucleotide mutation from G to A at ؊119, evidence that A at ؊119 combined with neighboring nucleotides to create a consensus sequence for binding myocyte-specific enhancer factor-2. Developmental Dynamics 228:30 -40, 2003.

To develop the first heart-specific tetracycline (Tet)-On system in zebrafish, we constructed plasmids in which the cardiac myosin light chain 2 promoter of zebrafish was used to drive the reverse Tet-controlled transactivator (rtTA) and the green fluorescent protein (GFP) reporter gene was preceded by an rtTAresponsive element. In the zebrafish fibroblast cell-line, rtTA-M2, one of rtTA's derivatives, demonstrated the highest increase in luciferase activity upon doxycycline (Dox) induction. We then generated two germ lines of transgenic zebrafish: line T03 was derived from microinjection of a plasmid containing rtTA-M2 and a plasmid containing a responsive reporter gene, whereas line T21 was derived from microinjection of a single dual plasmid. Results showed that line T21 was superior to line T03 in terms of greater GFP intensity after induction and with of minimal leakiness before induction. The photographic images of induced GFP in the heart of F2 larvae showed that the fluorescent level of GFP was dose-responsive. The level of GFP expressed in the F3 3 days postfertilization larvae that were treated with Dox for 1 hr decreased gradually after the withdrawal of the inducer; and the fluorescent signal disappeared after 5 days. The GFP induction and reduction were also tightly controlled by Dox in the F3 adult fish from line T21. This Tet-On system developed in zebrafish shows much promise for the study of the gene function in a specific tissue at the later developmental stage. Developmental Dynamics 233:1294 -1303, 2005.

Two classes of tilapia c-ski cDNA (accession nos. AJ012011, AJ012012), designated as tski1 and tski2, respectively encoded a 687 and a 714 AA protein and shared a 57% AA identity. Comparison with the Ski proteins of chickens, humans and Xenopus, tilapia TSki polypeptides shared a 60, 57, and 57% (TSki1) and 67, 63, and 61% (TSki2) AA identity, respectively. The most and the least abundant c-ski mRNAs are located in the brain and the skeletal muscle, respectively. Both tski1 and tski2 were widely expressed in the adult tissues examined, but tski2 transcripts were at higher levels except in the ovary and oocytes: tski1 transcripts were predominant in the ovary, whereas tski2 transcripts were predominant in the testes. In the oocytes, the tski1 mRNA was a maternally-inherited stockpile that subsequently was degraded, so that the expression ratio of tski1 to tski2 transcripts declined gradually as the fish developed from oocyte to 4-cm fry.

Midline convergence of organ primordia is an important mechanism that shapes the vertebrate body plan. Here, we focus on the morphogenetic movements of pronephric glomerular primordia (PGP) occurring during zebrafish embryonic kidney development. To characterize the process of PGP midline convergence, we used Wilms' tumour 1a (wt1a) as a marker to label kidney primordia, and performed quantitative analyses of the migration of the bilateral PGP. The PGP initially are approximately 350 μm apart in a wild type embryo at 10h post fertilization (hpf). The inter-PGP distance decreases exponentially between 10 and 48 hpf, while the anterior-posterior (A-P) dimension of each PGP increases linearly between 10 and 12 hpf, then decreases substantially between 12 and 24 hpf. Using mutants in the Nodal receptor cofactor one-eyed pinhead (oep) and the T-box transcription factors spadetail (spt) and no tail (ntl), we were able to define distinctive regulation underlying these sequential phases of PGP midline migration. Zygotic oep mutants (Zoep(-/-)) exhibited defects in midline convergence after 16 hpf. Spt is necessary for PGP convergence from 10 hpf, whereas ntl's effect on convergence does not begin until 24 hpf. Notably, we observed normal cardiac convergence in spt(-/-) and ntl(-/-) embryos implying that these novel roles of spt and ntl in PGP migration cannot be explained simply by generalised effects on midline convergence. These findings demonstrate that quantitative approaches to developmental migration allow the parsing of early patterning events, and in this instance suggest that the zebrafish may offer insights into midline urogenital migration anomalies in humans.