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First published online 22 October 2003
doi: 10.1242/dev.00838

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Mutation of weak atrium/atrial myosin heavy chain disrupts atrial function and influences ventricular morphogenesis in zebrafish

¹ Developmental Genetics Program and Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA
² Department of Biochemistry and Biophysics and Programs in Developmental Biology, Genetics, and Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA

View larger version (141K): [in a new window]   Fig. 1. Cardiac morphology in wea mutant embryos. (A-D) Lateral views of live embryos at 48 hpf, anterior to the left. (A,B) Atrial plane of focus. Compared with the wild-type (wt) atrium (A, arrow), the wea mutant atrium (B, arrow) appears dilated. (C,D) Ventricular plane of focus. Compared with the wild-type ventricle (C, arrow), the wea mutant ventricle (D, arrow) appears slightly compact. Mutant embryos shown are weam58 homozygotes. The weam58 and weask7 mutant phenotypes are identical in all characterized aspects; data in all figures are from weam58 mutants.

View larger version (70K): [in a new window]   Fig. 2. Myocardial ultrastructure is disrupted in the wea mutant atrium and intact in the wea mutant ventricle. (A-D) Longitudinal sections of myocardiocytes at 48 hpf viewed by transmission electron microscopy. (A,B) Atrial cells in wild-type embryos contain myofibrillar arrays (A), but myofibrils are rarely found in wea mutant atrial cells (B). Occasionally, wea mutant atrial cells contain a few disorganized myofilaments (B, arrow). (C,D) Ventricular cells in wild-type (C) and wea mutant embryos (D) contain normal myofibrillar arrays.

View larger version (82K): [in a new window]   Fig. 3. The wea mutant atrium lacks atrial myosin heavy chain. (A-D) Lateral views at 48 hpf, anterior to the left. (A,B) Whole-mount immunofluorescence with MF20 (TRITC) and S46 (FITC). In wild-type embryos (A), double exposure indicates MF20 staining in the ventricle (red), and overlap of MF20 and S46 staining in the atrium (yellow). Ventricle (V) and atrium (A) are indicated. Inset shows MF20 staining only; immunoreactivity in both chambers is clear. In wea mutant embryos (B), double exposure indicates that MF20 stains the ventricle (red), but neither MF20 nor S46 stain the atrium. Inset, showing MF20 alone, reinforces that MF20 stains only the ventricle in wea mutants. (C,D) Whole-mount immunofluorescence with the anti-tropomyosin antibody CH1 (FITC). CH1 stains both chambers in wild-type (C) and wea mutant embryos (D). Note that the wea mutant ventricle appears smaller than the wild-type ventricle.

View larger version (92K): [in a new window]   Fig. 4. amhc is expressed only in atrial myocardium. (A-L) Whole-mount in situ hybridization compares expression of cmlc2 (A,E,I), vmhc (B,F,J) and amhc (C,D,G,H,K,L). (A-D) Dorsal views at the 21-somite stage, anterior to the top. In wild-type embryos (A-C), cmlc2 expression (A) is observed throughout the cardiac cone. Presumed ventricular precursors express vmhc (B) and are found in the central portion of the cardiac cone. At this stage, presumed atrial precursors are beginning to express amhc (C) and are found in the outer portion of the cardiac cone. (E-H) Dorsal views through the head at 24 hpf, anterior to the bottom. In wild-type embryos (E-G), cmlc2 (E) is expressed throughout the heart tube, vmhc (F) is expressed in the ventricular precursors, and amhc (G) is expressed in the atrial precursors. (I-L) Frontal views at 48 hpf, head to the top. In wild-type embryos, cmlc2 (I) is expressed throughout the heart, vmhc (J) is expressed in the ventricle, and amhc (K) is expressed in the atrium. wea mutant embryos express amhc in atrial precursors initially (D), but do not maintain amhc expression (H,L). At all stages examined, ranging from the 19-somite stage through adulthood, there appears to be little, if any, overlap between vmhc and amhc expression. We have not observed amhc expression anywhere outside of the atrium.

View larger version (32K): [in a new window]   Fig. 5. weam58 and weask7 are strong loss-of-function alleles of amhc. (A) The weam58 mutation is a deletion of a single T at position 4024 of the amhc orf, which creates a frame-shift (arrow points to the position of the missing T). (B) The weask7 mutation is a T to A substitution at position 4577 of the amhc orf, which creates a premature stop codon (arrow points to the mutation). (C) Both mutant amhc cDNAs are predicted to encode truncated Amhc proteins. The wild-type Amhc protein contains 1937 amino acids, the predicted weam58 Amhc protein would contain 66 missense amino acids (gray bar) and would terminate after amino acid 1407, and the predicted weask7 Amhc protein would terminate after amino acid 1524. (D-F) Lateral views at 48 hpf, anterior to the left, of whole-mount immunofluorescence with MF20 (TRITC) and S46 (FITC). Ventricle (V) and atrium (A) are indicated. Phenotype of wild-type (D) and wea mutant embryos (E) is as described in Fig. 3. Embryos injected with an anti-amhc morpholino (F) exhibit a phenotype indistinguishable from that of wea mutant embryos (E). 138/138 morpholino-injected embryos phenocopy the wea mutation, and 120/120 embryos injected with a control morpholino appear wild type. Note that the wea mutant ventricle (E) and the morpholino-injected ventricle (F) both appear smaller than the wild-type ventricle (D).

View larger version (46K): [in a new window]   Fig. 6. Defects in ventricular morphology emerge by 48 hpf in wea mutants. (A-F) Lateral views, anterior to the left, of whole-mount immunofluorescence with the anti-tropomyosin antibody CH1 (FITC). Ventricle (V) and atrium (A) are indicated. (A-C) At 36 hpf, the wild-type ventricle (A) and the wea mutant ventricle (B,C) have similar morphology. The wea mutant atrium is slightly dilated at this stage. (D-F) At 48 hpf, the wea mutant ventricle (E,F) is noticeably smaller than the wild-type ventricle (D). The morphology of the wea mutant ventricle varies between individuals (E,F), as does the degree of dilation of the wea mutant atrium. Distortion of cardiac looping is also apparent in wea mutants at this stage.

View larger version (166K): [in a new window]   Fig. 7. Ventricular morphology responds to atrial dysfunction in wea mutants. (A-D) Longitudinal sections through the heart at 72 hpf, stained with Hematoxylin and Eosin, anterior to the top. (A,B) Sections through the wild-type (A, arrow) and wea mutant atrium (B, arrow) demonstrate similar thickness of the atrial wall. The wea mutant atrium is dilated in comparison with the wild-type atrium and contains less blood. (C,D) Comparison of sections through the wild-type (C, arrow) and wea mutant ventricle (D, arrow) demonstrates that the ventricular wall is thicker, and that the ventricular lumen is narrower, in wea mutants. The morphology of the wea mutant ventricle varies between individuals (D); insets show two additional examples of ventricular sections from other wea mutant embryos. Variability is also apparent within individual embryos, as the wea mutant ventricular wall does not exhibit a uniform thickness. (D) Thickening of the wea mutant ventricular wall is apparent by 48 hpf, and increases between 48 and 72 hpf (data not shown). All sections shown are the central section from serial sectioning through the respective chamber; results are representative of the examination of more than 15 embryos of each genotype.

View larger version (136K): [in a new window]   Fig. 8. Myocardial gene expression responds to atrial dysfunction in wea mutants. (A-D) Lateral views at 96 hpf, anterior to the left. Whole-mount in situ hybridization compares expression of anf (A,B) and cmlc2 (C,D). Ventricle (V) and atrium (A) are indicated. Embryos were treated with 0.003% phenylthiourea from 24-96 hpf to inhibit pigmentation (Elsalini and Rohr, 2003). Embryos were sorted by phenotype prior to fixing, and tails were marked to distinguish between wild-type and wea mutant embryos. Mutant and wild-type siblings were then kept together throughout the in situ protocol, and were stained for the same length of time. (A,B) Comparison of wild-type (A) and wea mutant embryos (B) indicates substantial upregulation of anf expression throughout the wea mutant heart. (C,D) Likewise, the wea mutant heart exhibits upregulation of cmlc2 expression at this stage. Intensity of anf and cmlc2 expression is increased on a per cell basis; this is especially clear for the atrium, which is the same thickness in wild-type and wea mutant embryos.

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