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First published online 6 October 2004
doi: 10.1242/dev.01410

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accordion, a zebrafish behavioral mutant, has a muscle relaxation defect due to a mutation in the ATPase Ca²⁺ pump SERCA1

¹ Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-0720, USA
² Mental Health Research Institute, University of Michigan, Ann Arbor, MI 48109-0720, USA
³ Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058, USA

View larger version (23K): [in a new window]   Fig. 1. acc embryos exhibit an aberrant response to touch. Embryos (24 hpf) were stimulated by touching them with forceps. (A) Touch induced a wild-type embryo to coil twice within 1 second. Relaxation of the trunk after the second coil took less than 0.5 seconds. (B) Touch induced an acc embryo to contract both sides in an apparent simultaneous fashion to cause the trunk to bend dorsally followed by a slow relaxation (>4 seconds).

View larger version (74K): [in a new window]   Fig. 2. Morphological defects observed at 48 hpf in acc embryos are secondary to the abnormal behavior. Tricaine is applied to acc embryos from 24 to 48 hpf to immobilize the embryos. Wild-type sibling and a tricaine-treated acc embryo (C) display a straight trunk while an acc mutant (B) displays a bent trunk. DIC images show that the notochord in a wild-type sibling (D) and tricaine-treated acc embryo (F) are undamaged, while the notochord of an acc mutant is breaking apart (E). The normal pattern of slow twitch muscle fibers labeled with MAb F59 is seen in wild-type sibling (G) and tricaine-treated acc mutant (I), but the slow twitch fibers are disarrayed in acc mutants (H).

View larger version (29K): [in a new window]   Fig. 3. The CNS and the NMJ are normal in acc. (A) Schematic summary of the experimental procedure. Embryos (48 hpf) are pinned on a dish through the notochord. The skin is peeled off to allow access to the muscle cells. Muscle voltage responses are evoked by mechanosensory stimulation delivered by a puff of water and are measured with a patch electrode. Wild-type siblings (B) and acc (D) display similar rhythmic depolarizations (fictive swimming), whereas strychnine-treated wild-type siblings (C) respond with a non-rhythmic, shorter depolarization.

View larger version (31K): [in a new window]   Fig. 4. Decay in Ca2+ transient is slower in acc muscles during relaxation. (A) Schematic summary of the experimental procedures. Calcium Green 1 dextran is injected into 1- to 4-cell stage embryos. At 24 hpf embryos are pinned on a dish and mosaically fluorescent muscle cells are observed by line-scanning with a confocal microscope during spontaneous coiling. (B) There is no difference in the timing of the increase in fluorescence during Ca2+ transients between wild-type siblings (black) and acc (red) embryos, but the decay of fluorescence is much slower in mutant embryos. Small and large arrowheads indicate the peak and half decay of fluorescence, respectively. (C) Quantification of the difference in time to half decay from peak fluorescence. Ca2+ decay is slower in acc muscles than in wild-type siblings.

View larger version (83K): [in a new window]   Fig. 5. acc embryos have mutations in atp2a1/SERCA1. (A) Physical mapping to the LN54 radiation hybrid panel shows that atp2a1 is close to netrin 1. (B) Missense mutations are found in SERCA1 of all three acc alleles. SERCA1 contains 10 transmembrane domains (1-10, purple), phosphorylation domains (blue) and a nucleotide binding domain (green). The white crosses indicate the positions of the mutation in the acc allele. The nucleotide and amino acid changes are shown on the right. (C) Alignment of vertebrate SERCA1 proteins showing that all mutations are found in the completely conserved residues. Shaded residues indicate conserved amino acids. Triangles (red) indicate the position of the mutation in the acc allele.

View larger version (84K): [in a new window]   Fig. 6. SERCA1 mRNA is specifically expressed by muscles. Expression is observed only in muscles at 15 hpf (A) and 29 hpf (B). Axial section of 29 hpf embryos clearly shows exclusive expression in muscles (C). Expression is not observed in the spinal cord. SC, spinal cord; NC, notochord; YT, yolk tube.

View larger version (57K): [in a new window]   Fig. 7. Mutant rescue and antisense phenocopy confirm that atp2a1 is the acc gene. All panels show video frames 1 second after touch. (A-D) Wild-type SERCA1 mRNA rescues the acc phenotype. Uninjected (A) and acctq206 mRNA-injected (C) acc embryos respond with the aberrant dorsal bend following touch, while acc embryos injected with wild-type SERCA1 mRNA respond normally to touch (B). This embryo coiled twice and relaxed 1 second after touching. Injection of SERCA1 mRNA carrying a cognate Brody disease mutation does not rescue the acc phenotype (D). (E-H) Antisense atp2a1/SERCA1 phenocopies the acc phenotype. Antisense MO-injected wild-type embryos respond to touch with the dorsal bend (F), while control MO-injected embryos respond with coils and are relaxed 1 second after touching (E). Co-injection of wild-type SERCA1 mRNA with antisense MO blocks the induction of the mutant phenotype and shows the normal coils and relaxation to touch (G), whereas co-injection with mutant mRNA does not (H).

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