Holtzman et al., 2007 - Endocardium is necessary for cardiomyocyte movement during heart tube assembly. Development (Cambridge, England)   134(13):2379-2386 Full text @ Development

Fig. 1 Tracking wild-type cardiomyocytes reveals two morphologically distinct phases of cell behavior during cardiac fusion. (A) Schematic of cardiac fusion in the zebrafish embryo. Dorsal views; locations of cardiomyocyte populations shown in green; location of endocardium at the 20-somite stage indicated with an E. (B-D) Selected images from a time-lapse of cardiac fusion in a typical wild-type embryo expressing Tg(cmlc2:egfp) (see Movie 1 in the supplementary material), exhibiting cardiac morphology at the (B) 16-somite, (C) 18-somite and (D) 20-somite stages. Dorsal views, anterior to the top. (E-I) Paths traveled by individual cardiomyocytes during (E,F) the initial 50 minutes, (G,H) the final 80 minutes and (I) the final 20 minutes of fusion. Tracks displayed represent a subset of the tracked cells in this time-lapse. Each arrow extends from a cell's starting position to its ending position. Red arrows, medial movement; yellow arrows, angular movement; asterisks, cells exhibiting no net displacement over the elapsed time. Cardiac fusion begins with a phase of coherent medial movement (E,F), followed by a transition to angular movement in anterior and posterior regions (G,H). Scale bar: in I, 20 μm for B-I. (J-M) Quantitative analysis of the direction of cardiomyocyte movement. (J,L) Radial bar graphs depict degree of cardiomyocyte displacement during the first (J) and second (L) phases. For all tracked wild-type cardiomyocytes, net displacement along the anterior-posterior axis was calculated relative to a directly medial path (0° displacement). Grouping the data in 10° increments, the length of each radial bar reflects the percentage of cells exhibiting each degree of displacement. Red bars are classified as medial movement, and yellow bars are classified as angular movement. Significantly more cells exhibit angular movement during the second phase than during the first phase (52% versus 17%; t-test, P<0.01). (K,M) Location of cardiomyocytes moving angularly during first (K) and second (M) phases. Colors overlaid on egfp expression reflect anterior (purple), central (green) and posterior (blue) regions. Most of the cells exhibiting angular movement are located in anterior or posterior regions. In these regions, the percentage of cells moving angularly during the second phase is significantly greater than that observed during the first phase (*, posterior: 86% versus 20%; **, anterior: 77% versus 17%; t-test, P<0.01 for both regions). See Materials and methods for details of time-lapse analysis and Table 1 for additional data.

PHENOTYPE:
Fish:
Observed In:
Stage: 20-25 somites

Fig. 2 No angular movement in the absence of endocardium. (A,B) Selected images from a time-lapse of cardiac fusion in a typical clo mutant zebrafish embryo expressing Tg(cmlc2:egfp) (see Movie 2 in the supplementary material), exhibiting cardiac morphology at the (A) 17-somite and (B) 20-somite stages. Dorsal views, anterior to the top. (C-E) Paths traveled during the (C,D) entire 90 minutes and (E) final 20 minutes of fusion (arrows and asterisks as described for Fig. 1). Tracks displayed represent a subset of the tracked cells in this time-lapse. In clo mutants, cardiomyocytes exhibit medial movement throughout the duration of cardiac fusion. Scale bar: in E, 20 μm for A-E. (F) Radial bar graph (see Fig. 1J,L) depicting degree of displacement for all tracked clo mutant cardiomyocytes. Few cells exhibit angular movement, resembling the pattern of cell behavior observed during the first phase of wild-type fusion (see Fig. 1J). (G,H) Location of cardiomyocytes moving angularly in clo mutants. In anterior (purple) and posterior (blue) regions (), the percentage of cells moving angularly is significantly reduced (t-test, P<0.01) compared with that observed during the second phase of wild-type fusion (see Fig. 1M). See Table 1 for additional data. (I,J) In situ hybridization depicts cmlc2 expression in wild-type (I) and clo mutant (J) embryos at 28 hpf. Dorsal views, anterior to the top; both images shown at the same magnification. Elongation of the heart tube is delayed and aberrant in clo mutant embryos.

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Term:
Stage: Prim-5
PHENOTYPE:
Fish:
Observed In:
Stage: Prim-5

Fig. 3 Angular cell movement occurs in the absence of initial medial movement. (A-D) Selected images from a time-lapse of cardiac fusion in a typical mil mutant zebrafish embryo expressing Tg(cmlc2:egfp) (see Movie 3 in the supplementary material), exhibiting cardiac morphology at the (A) 19-somite and (B) 23-somite stages. Dorsal views, anterior to the top. (C,D) Paths traveled by cardiomyocytes in the right lateral heart field during the entire time-lapse (arrows and asterisks as described for Fig. 1). Tracks displayed represent a subset of the tracked cells in this time-lapse. Although initial medial movement is lost, mil mutant cardiomyocytes exhibit a phase of angular movement. Images in C,D are double the magnification of those in A,B. Scale bar: 20 μm. (E) Radial bar graph (see Fig. 1J,L) depicting degree of displacement for all tracked mil mutant cardiomyocytes. Most cells exhibit angular movement. (F,G) Location of cardiomyocytes moving angularly in mil mutants. In mil mutants, as in wild-type embryos, angular movement is regionally restricted. See Table 1 for additional data. (H,I) In situ hybridization for flk1 (kdr - ZFIN) expression in wild-type (H) and mil mutant (I) embryos at the 21-somite stage. Dorsal views, anterior to the top; both images shown at the same magnification. Arrows indicate clusters of presumed endocardial precursors. (J,K) Two-color fluorescent in situ hybridization for expression of cmlc2 (red) and fli1a (green) in wild-type (J) and mil mutant (K) embryos at the 21-somite stage. Dorsal views, anterior to the top; only the right lateral heart field is shown in K; both images shown at the same magnification. In both wild-type and mil mutant embryos, the presumed endocardial precursors are clustered adjacent to the central cardiomyocytes.

EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Term:
Stage: 20-25 somites
PHENOTYPE:
Fish:
Observed In:
Stage: 20-25 somites

Fig. 4 Endocardium directs cardiomyocyte movement, even in the absence of initial medial movement. (A-D) Selected images from a time-lapse of cardiac fusion in a mil;clo double-mutant zebrafish embryo expressing Tg(cmlc2:egfp) (see Movie 4 in the supplementary material), exhibiting cardiac morphology at the (A) 19-somite and (B) 23-somite stages. Dorsal views, anterior to the top. (C,D) Paths traveled by cardiomyocytes in the right lateral heart field during the entire time-lapse (asterisks as described in Fig. 1). Images in C,D are three times the magnification of those in A,B. Scale bar: 20 μm. Example shown is representative of four heart fields analyzed. Cardiomyocytes in mil;clo double mutants exhibit no net directed movement.

Acknowledgments:
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