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.

Developing organs acquire a specific three-dimensional form that ensures their normal function. Cardiac function, for example, depends upon properly shaped chambers that emerge from a primitive heart tube. The cellular mechanisms that control chamber shape are not yet understood. Here, we demonstrate that chamber morphology develops via changes in cell morphology, and we determine key regulatory influences on this process. Focusing on the development of the ventricular chamber in zebrafish, we show that cardiomyocyte cell shape changes underlie the formation of characteristic chamber curvatures. In particular, cardiomyocyte elongation occurs within a confined area that forms the ventricular outer curvature. Because cardiac contractility and blood flow begin before chambers emerge, cardiac function has the potential to influence chamber curvature formation. Employing zebrafish mutants with functional deficiencies, we find that blood flow and contractility independently regulate cell shape changes in the emerging ventricle. Reduction of circulation limits the extent of cardiomyocyte elongation; in contrast, disruption of sarcomere formation releases limitations on cardiomyocyte dimensions. Thus, the acquisition of normal cardiomyocyte morphology requires a balance between extrinsic and intrinsic physical forces. Together, these data establish regionally confined cell shape change as a cellular mechanism for chamber emergence and as a link in the relationship between form and function during organ morphogenesis.

The embryonic vertebrate heart begins pumping blood long before the development of discernable chambers and valves. At these early stages, the heart tube has been described as a peristaltic pump. Recent advances in confocal laser scanning microscopy and four-dimensional visualization have warranted another look at early cardiac structure and function. We examined the movement of cells in the embryonic zebrafish heart tube and the flow of blood through the heart and obtained results that contradict peristalsis as a pumping mechanism in the embryonic heart. We propose a more likely explanation of early cardiac dynamics in which the pumping action results from suction due to elastic wave propagation in the heart tube.

SUMMARYThe secondary heart field is a conserved developmental domain in avian and mammalian embryos that contributes myocardium and smooth muscle to the definitive cardiac arterial pole. This field is part of the overall heart field and its myocardial component has been fate mapped from the epiblast to the heart in both mammals and birds. In this study we show that the population that gives rise to the arterial pole of the zebrafish can be traced from the epiblast, is a discrete part of the mesodermal heart field, and contributes myocardium after initial heart tube formation, giving rise to both smooth muscle and myocardium. We also show that Isl1, a transcription factor associated with undifferentiated cells in the secondary heart field in other species, is active in this field. Furthermore, Bmp signaling promotes myocardial differentiation from the arterial pole progenitor population, whereas inhibiting Smad1/5/8 phosphorylation leads to reduced myocardial differentiation with subsequent increased smooth muscle differentiation. Molecular pathways required for secondary heart field development are conserved in teleosts, as we demonstrate that the transcription factor Tbx1 and the Sonic hedgehog pathway are necessary for normal development of the zebrafish arterial pole.

Acute lymphoblastic leukemia (ALL) is a clonal disease that evolves through the accrual of genetic rearrangements and͞or mutations within the dominant clone. The TEL-AML1 (ETV6-RUNX1) fusion in precursor-B (pre-B) ALL is the most common genetic rearrangement in childhood cancer; however, the cellular origin and the molecular pathogenesis of TEL-AML1-induced leukemia have not been identified. To study the origin of TEL-AML1-induced ALL, we generated transgenic zebrafish expressing TEL-AML1 either ubiquitously or in lymphoid progenitors. TEL-AML1 expression in all lineages, but not lymphoid-restricted expression, led to progenitor cell expansion that evolved into oligoclonal B-lineage ALL in 3% of the transgenic zebrafish. This leukemia was transplantable to conditioned wildtype recipients. We demonstrate that TEL-AML1 induces a B cell differentiation arrest, and that leukemia development is associated with loss of TEL expression and elevated Bcl2͞Bax ratio. The TEL-AML1 transgenic zebrafish models human pre-B ALL, identifies the molecular pathways associated with leukemia development, and serves as the foundation for subsequent genetic screens to identify modifiers and leukemia therapeutic targets.stem cell ͉ translocation ͉ childhood cancer ͉ genetics T he TEL-AML1 fusion generated by the t(12, 21)(p13;q22) chromosomal translocation is present in 25% of childhood pre-B acute lymphoblastic leukemia (ALL), making it the most common genetic rearrangement in childhood cancer (1-3). The translocation fuses the first five exons of the Ets transcription factor TEL (also known as ETV6) in-frame to nearly the entire AML1 gene (also known as RUNX1). Retrospective studies in twins with pre-B ALL, as well as Guthrie cards studies from 567 normal newborns (4), reveal that the TEL-AML1 fusion occurs in utero, with a protracted time course for leukemia development (5, 6).Murine studies involving TEL-AML1 suggest that this fusion protein confers a low transforming ability. Transgenic mice expressing TEL-AML1 from the Ig heavy chain promoter (E) did not develop any hematological disorder (7). Mice transplanted with bone marrow cells transduced with retroviral vectors expressing TEL-AML1 developed a preleukemic state without occult leukemia (8-10). The incidence of leukemia in such mice increased only in the presence of cooperating mutations (11).The cell initially transformed by TEL-AML1 remains to be elucidated; however, in ALL patients, the TEL-AML1 fusion event precedes differentiation of lymphoid progenitors to pre-B cells (12). This finding confines the origin of pre-B ALL to a B-lineage restricted progenitor(s) (4) or a multipotent hematopoietic stem cell (HSC) with preferential B-lymphoid clonal expansion (13).We used the zebrafish to study TEL-AML1 leukemogenesis for several reasons. First, the zebrafish has well conserved genetic processes controlling hematopoesis (14, 15). Second, zebrafish develop tumors that are histologically similar to human tumors (16)(17)(18)(19)(20). The lymphoid expression of mouse c-Myc led to...

Embryonic heart formation requires the union of bilateral populations of cardiomyocytes and their reorganization into a simple tube. Little is known about the morphogenetic mechanisms that coordinate assembly of the heart tube and determine its dimensions. Using time-lapse confocal microscopy to track individual cardiomyocyte movements in the zebrafish embryo, we identify two morphologically and genetically separable phases of cell movement that coordinate heart tube assembly. First, all cardiomyocytes undergo coherent medial movement. Next, peripherally located cardiomyocytes change their direction of movement, angling toward the endocardial precursors and thereby establishing the initial circumference of the nascent heart tube. These two phases of cardiomyocyte behavior are independently regulated. Furthermore, we find that myocardial-endocardial interactions influence the second phase by regulating the induction, direction and duration of cardiomyocyte movement. Thus, the endocardium plays a crucial early role in cardiac morphogenesis, organizing cardiomyocytes into a configuration appropriate for heart tube assembly. Together, our data reveal a dynamic cellular mechanism by which tissue interactions establish organ architecture.

Na,K-ATPase is an essential gene maintaining electrochemical gradients across the plasma membrane. Although previous studies have intensively focused on the role of Na,K-ATPase in regulating cardiac function in the adults, little is known about the requirement for Na,KATPase during embryonic heart development. Here, we report the identification of a zebrafish mutant, heart and mind, which exhibits multiple cardiac defects, including the primitive heart tube extension abnormality, aberrant cardiomyocyte differentiation, and reduced heart rate and contractility. Molecular cloning reveals that the heart and mind lesion resides in the α1B1 isoform of Na,K-ATPase. Blocking Na,K-ATPase α1B1 activity by pharmacological means or by morpholino antisense oligonucleotides phenocopies the patterning and functional defects of heart and mind mutant hearts, suggesting crucial roles for Na,KATPase α1B1 in embryonic zebrafish hearts. In addition to α1B1, the Na,K-ATPase α2 isoform is required for embryonic cardiac patterning. Although the α1B1 and α2 isoforms share high degrees of similarities in their coding sequences, they have distinct roles in patterning zebrafish hearts. The phenotypes of heart and mind mutants can be rescued by supplementing α1B1, but not α2, mRNA to the mutant embryos, demonstrating that α1B1 and α2 are not functionally equivalent. Furthermore, instead of interfering with primitive heart tube formation or cardiac chamber differentiation, blocking the translation of Na,KATPase α2 isoform leads to cardiac laterality defects. Supplemental figure available online

We isolated a 1,438 bp cDNA fragment that encoded Myf-5 myogenic factor of zebrafish. The deduced amino acid contained 237 residues, including the basic helix-loop-helix domain that is conserved in all known Myf-5. The zebrafish myf-5 transcripts were first detectable at 7.5 hpf, increased substantially until 16 hpf, and then declined gradually to an undetectable level by 26 hpf. During somitogenesis, zebrafish myf-5 transcripts were distributed mainly in the somites and segmental plates. Prominent signals occurred transiently in adaxial cells in two parallel rows but did not extend beyond the positive-signal somites. Various lengths of upstream region of zebrafish myf-5 fused with EGFP gene were used to carry out transgenic analysis. Results showed that a small, 82 bp (nucleotide positions from -82 to -1), regulatory cassette is sufficient to control the somite- and stage-specific expression of zebrafish myf-5 during early development.