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Human Molecular Genetics Advance Access originally published online on May 11, 2005
Human Molecular Genetics 2005 14(13):1727-1743; doi:10.1093/hmg/ddi179
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning

Susan J. Nixon1,2,3, Jeremy Wegner4, Charles Ferguson1,2,3, Pierre-François Méry1,2,3,{dagger}, John F. Hancock1, Peter D. Currie5, Brian Key3, Monte Westerfield4 and Robert G. Parton1,2,3,*

1Institute for Molecular Bioscience, 2Centre for Microscopy and Microanalysis and 3School of Biomedical Sciences, University of Queensland, Brisbane 4072, Australia, 4Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA and 5Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia

* To whom correspondence should be addressed. Tel: +61 733656468; Fax: +61 733654422; Email: r.parton{at}imb.uq.edu.au

Received March 22, 2005; Accepted May 2, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 ({alpha} and ß), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas ß-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Caveolae, small uncoated plasma membrane pits, are a highly abundant and characteristic feature of many animal cells such as adipocytes, endothelial cells, fibroblasts and muscle (1Go–4Go). Although various roles have been ascribed to caveolae, their precise functions are still unclear. Understanding the function of caveolae is important for combating human disease because caveolae have been linked to cancer (5Go) and muscular dystrophy (6Go–8Go). The discovery and manipulation of caveolins, major integral membrane proteins associated with caveolae, have provided an avenue to study caveolae function specifically. Many non-muscle tissues express caveolin-1 and caveolin-2, whereas caveolin-3 (Cav3) appears to be the sole isoform expressed in skeletal and cardiac muscle (3Go,4Go). Caveolin-1 and Cav3 are essential for the formation of caveolae in non-muscle and muscle cells, respectively, because blocking their expression causes loss of caveolae in the corresponding tissues (9Go–12Go). Cav3 knockout mice show a complete loss of caveolae at the sarcolemma and exhibit a mild muscular dystrophy phenotype with no apparent compensation by other caveolin isoforms in caveolae formation (10Go,11Go). Cav3 overexpression in the mouse causes a more severe muscular dystrophy phenotype, with elevated levels of serum creatine kinase, down-regulation of dystrophin and ß-dystroglycan and evidence of hypertrophic, necrotic and immature or regenerating skeletal muscle fibers (13Go).

Further links between muscle disease and CAV3 have been shown by mutations in the human CAV3 gene. To date, 15 point mutations and 3 and 9 bp deletions have been associated with CAV3 in human patients with muscle diseases including limb-girdle muscular dystrophy, distal myopathy, HyperCKemia and rippling muscle disease (14Go–28Go), although three of these sequence variations are thought to be non-pathogenic polymorphisms (29Go). In some cases, identical amino acid substitutions have been associated with clinically distinct disorders suggesting modification of phenotype by additional genetic or environmental factors (19Go–23Go). Nine of these mutations, such as a well-characterized mutation in which a proline at position 104 is substituted by leucine (CAV3P104L), cause a down-regulation of CAV3 in muscle fibers and often lead to the accumulation of CAV3 in the Golgi complex rather than at the cell surface (30Go,31Go). Another mutant protein, also found in the control population, has a substitution of cysteine to tryptophan (CAV3C71W) and is found at the cell surface but was shown to disrupt H-Ras mediated Raf activation suggesting a possible defect of this mutant in the organization of lipid raft domains at the sarcolemma of muscle fibers (32Go). Caveolae have also been implicated in the pathogenesis of Duchenne muscular dystrophy (DMD). The density of caveolae increase in muscle of patients with DMD and CAV3 levels increase in dystrophin-deficient mdx mice and in DMD patients (6Go–8Go).

These studies have shown that caveolae and the precise regulation of the Cav3 protein are important for muscle health, but their roles in muscle development or maintenance are still unclear. A role for Cav3 during development has been suggested by the association of Cav3 with the extensive surface-connected network of membranes that form the transverse (T)-tubule system during myotube differentiation (33Go). Although Cav3 may facilitate T-tubule formation, it does not appear to be absolutely essential; Cav3 knockout mice form T-tubules that are abnormally organized (11Go). Recent studies suggest an important role for Amphiphysin-2 in the formation of Cav3 positive T-tubules (34Go). The role of Cav3 in myoblast fusion is also unclear; a recent study has shown that the overexpression of Cav3 perturbs myoblast fusion and Cav3 ablation enhances myoblast fusion (35Go), whereas earlier studies suggested that Cav3 ablation inhibited myoblast fusion (36Go).

In mature muscle fibers, Cav3 is predominantly located at the sarcolemma, where it associates with regularly ordered arrays of caveolae that overlap partially with the dystrophin–glycoprotein complex (DGC) (37Go). Cav3 has been shown to co-purify with components of the DGC during subcellular fractionation (38Go) and to interact with ß-dystroglycan (39Go) and dysferlin (40Go). Moreover, the distribution of dysferlin is abnormal in some cells from limb-girdle muscular dystrophy-1C, rippling muscle disease and distal myopathy patients with mutant CAV3 (14Go,17Go,18Go,41Go). Other putative interacting components include muscle phosphofructokinase (42Go) and neuronal nitric oxide synthase (43Go). Regulation of the latter by Cav3 is consistent with the finding that transgenic mice expressing the CAV3P104L mutant show decreased surface Cav3 and increased nitric oxide synthase activity (31Go).

Zebrafish provide a readily accessible model for human muscle disease (44Go). Muscle specification and differentiation follow a very well-characterized time course and allow detailed analysis with single cell resolution (45Go). Zebrafish orthologs of proteins in the human DGC have been implicated in muscle development and have been used to study human muscular dystrophy and congenital myopathy (44Go,46Go,47Go). We have used zebrafish to study the function of Cav3 during development. We show that Cav3 is expressed in the notochord indicating an unexpected role in axial patterning and novel regulation of its expression. In muscle fibers, Cav3 is initially distributed along the entire length of the sarcolemma in contrast to the ß-dystroglycan localization at fiber ends. In addition, we demonstrate the essential role of Cav3 in muscle differentiation using morpholino (MO)-induced Cav3 down-regulation, Cav3 knockdown causes severe disruption of muscle development with resultant defects in muscle function, and this can be mimicked by expression of a muscular dystrophy-associated Cav3 mutant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The sequence and genomic organization of zebrafish caveolins are conserved with mammals
We searched the expressed sequence tag (EST) and genomic databases (Entrez-PubMed and TIGR) and identified a putative zebrafish cav1 sequence. Two ESTs were identified (ESTs fb95c12, GenBank accession number AI585189 and fc37c03, GenBank accession number AI667067) with sequence homology to mammalian Caveolin-1 (Cav1). These EST sequences were used to design primers for RT–PCR of a fragment of cav1 from RNA isolated from 50 h embryos. The product was sequenced and used to design additional primers to clone a putative full-length cav1 by 5'-RACE. The 5'-RACE product was TA cloned into pGEM-T Easy vector (Promega) and sequenced (Fig. 1B). The 5'-RACE sequence revealed two isoforms of cav1 that are encoded by distinct mRNA species. The sequence and genomic organization are very similar to that of Takifugu rubripes (GenBank accession number AJ010316) and mouse Cav1 (48Go). The {alpha}-isoform is encoded from 3 exons, whereas the ß-isoform corresponds to the second and third exons and begins at the methionine residue 34 (with additional 5'-intronic sequence; Fig. 1A and B). The sequenced product of zebrafish cav1{alpha} encodes a protein with high homology to mammalian Cav1, 72% identity and 81% similarity to human CAV1; the differences are primarily at the amino terminus. Cav1ß that lacks the amino terminus is even more closely related to human CAV1ß, 77% identical and 86% similar.



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  		Figure 1. Sequence, genomic organization and chromosomal localization of the caveolin family in zebrafish. (A) Genomic organization and differential splicing to yield {alpha} and ß cav1 is conserved from fish to mammals. (B–D) Nucleotide and deduced amino acid sequences of caveolin-1, -2 and -3. In all sequences, the start methionine is in bold, and the termination codon is denoted with an asterisk. Caveolin-1 isoforms are encoded by distinct mRNAs. The start codons for both isoforms are in bold, with the beta isoform start codon underlined. The predicted cav1{alpha} isoform is 181 amino acids long. The alpha isoform untranslated region is shown in normal text, whereas the beta isoform untranslated region is in italics. Dashes indicate sequence that is not found in the beta isoform. The numbering indicates nucleotide numbers and amino acid numbers for the alpha isoform. The polyadenylation signal at the 3' end, AATAAA, is underlined. While this manuscript was in preparation, cav1 was described by Smart et al. where cav1 was cloned and localized to chromosome LG25 (74Go). The predicted Cav2 is 162 amino acids long and Cav3 is 150 amino acids long (GenBank accession numbers Cav1{alpha} DQ015875 [GenBank] ; Cav1ß DQ015876 [GenBank] ; Cav2 DQ015874 [GenBank] and Cav3 DQ015873 [GenBank] ). (E) Chromosomal localization of human and mouse Cav3 along with genes found in close proximity (from http://www.ncbi.nlm.nih.gov/LocusLink/). Cav3 localizes to human chromosome (Hs) 3 and mouse chromosome (Mm) 6. Zebrafish cav3 maps to linkage group (Dr) 6, 10.65 cR from fb37d04 using the LN54 radiation hybrid panel. Sequence analysis of a contig (ctg15465 http://www.ensembl.org/Danio_rerio) reveals location of cav3 relative to rad18 and oxtr, demonstrating syntenic conservation of the region in zebrafish, human and mouse.

 
We identified putative zebrafish cav2 genomic sequence from the PubMed database (GenBank accession numbers AC087254 and AC087105) and designed primers to amplify a full-length caveolin-2 clone. The amino acid sequence revealed 50% identity and 69% similarity to human CAV2.

We identified a putative zebrafish cav3 EST using the PubMed database (cav3 fx19g04.y1). We used this cav3 EST sequence to search the Sanger Institute zebrafish genome sequence database and identified two whole-genome shotgun traces that were similar to cav3 (Z35723 [GenBank] -a3457a01.q1c and zfishG-a820c07.q1c). We used the genomic and EST sequences to design primers for RT–PCR from RNA isolated from 50 h embryos. The sequenced product encodes a protein with high homology to mammalian Cav3 with 72% identity and 84% similarity to human CAV3; the differences, as with Cav1 are primarily near the amino terminal end (Figs 1D and 2). Seventeen of 18 residues that are substituted or deleted in human diseases are conserved in the zebrafish protein (asterisks and triangles, Fig. 2); the exception is R125H that has been linked to limb-girdle muscular dystrophy-1C but was also found in two unaffected siblings suggesting that it may represent a non-disease associated polymorphism (29Go). The caveolin-scaffolding domain, a region suggested to interact with several signaling molecules (49Go,50Go), is also very well conserved with only one of 20 residues differing when compared with the human CAV3 scaffolding domain. All residues implicated in the WW-like domain of CAV3, thought to interact with ß-dystroglycan (39Go), are also conserved.



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  		Figure 2. Critical residues of the Cav3 protein are conserved among vertebrates. Alignment of human, mouse, rat, xenopus, fugu and zebrafish Cav3 (GenBank accession numbers P56539, P51637, P51638, AAH41289, sequence translated from genomic sequence CAAB01002757 and DQ015873 [GenBank] ). Identities are shaded in dark gray and similarities are shaded in light gray. Zebrafish Cav3 is 72% identical and 84% similar to human Cav3. The asterisk denotes amino acids that have been mutated or deleted in human diseases and the triangle indicates amino acids that have been mutated in human diseases but have also been found in normal populations. The caveolin-scaffolding domain, a region implicated in binding signaling molecules, is underlined.

 
We used the LN54 radiation hybridization panel to assign cav3 to linkage group 6, 10.65 cR from fb37d04 (Fig. 1E). Zebrafish cav3, like the human and mouse genes, has two exons (unpublished data). In both human and mouse, CAV3 is located in close proximity to the oxytocin receptor OXTR (4Go) and RAD18 (Fig. 1E). We found that a single contig, ctg15465 contains cav3 and two genes with high homology to RAD18 and OXTR suggesting that zebrafish cav3 lies in a region of conserved synteny (Fig. 1E). Examination of the Sanger Institute database revealed that zebrafish caveolin-1 and caveolin-2 are located on the same chromosome fragment (NA54267), as in Takifugu rubripes, indicating that the genomic organization of caveolin-1 and -2 is conserved in these two fish.

Muscle precursor cells express cav3 during early differentiation
To understand the functions of caveolins in muscle, we concentrated our analyses on Cav3, the muscle-specific caveolin family member. We detected the first cav3 expression at the 6-somite (12 h) stage in adaxial cells, a sheet of cells one cell thick adjacent to the notochord (Fig. 3A and B). We previously showed that adaxial cells are the precursors of slow skeletal muscle fibers, the first muscle fibers to differentiate (45Go). Adaxial cells extend along the anteroposterior axis shortly after formation of the somite and form the first elongated actin bundles by the 10-somite stage (14 h) and sarcomeres slightly later (51Go). Thus, the onset of cav3 expression precedes the initial appearance and organization of contractile proteins. A few hours later, by the 14-somite stage (16 h), notochord cells begin to express cav3 and expression persists until late segmentation stages (Fig. 3C–F). cav3 expression in the myotome first appears around the 14-somite stage (16 h) and persists throughout embryonic development (Fig. 3C–F, H). Starting after the second day of development, other muscles including pectoral fin (asterix, I) and facial muscles express cav3 (Fig. 3G and I). A low level of cav3 expression was detected in the developing heart at 80 h (Fig. 3G).



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  		Figure 3. Notochord and muscle express cav3 early in development. Developmental expression pattern of cav3 analyzed by whole-mount in situ hybridization. (A) At 12 h, adaxial cells (arrows) that are found on either side of the notochord express cav3. (B) Transverse section of the tail region of 24 h embryos indicate cav3 expression in putative slow muscle precursors, the adaxial cells. (C) Lateral view of 17 h embryo indicates expression of cav3 in the notochord, N, as confirmed in sections of 14 h embryos (D) and in a higher magnification view (E) of a 20 h embryo. (F) Lateral view of a 19 h embryo shows cav3 expression in the notochord and myotomes. (H) In 36 h embryos, cav3 is expressed throughout the somites (s). (I) At 72 h, cav3 is expressed in the myotomes, facial muscles, pectoral fin (asterisk) and at 80 h in the heart, h, (G). Note that the appearance of dark color in the eye (e) is due to pigment and not cav3 expression. (A and I) Dorsal view, anterior to left; (B and D) cross section, dorsal to top; (C, E–H) side view, anterior to the left and dorsal to the top. SC, spinal cord, Y, Yolk. Scale bars: (A, C, F, G, I) 250 µm and (B, H, D, E) 50 µm.

 
Cav3 protein is associated with T-tubules and the sarcolemma
We examined the distribution of caveolin protein using a caveolin antibody (con-cav) raised against a conserved domain of Cav3 that recognizes all caveolin isoforms (52Go). Sections of adult muscle show labeling of the sarcolemma and an intracellular pattern consistent with the organization of the T-tubules (Fig. 4A). Antibody labeling of muscle is abolished in the presence of the peptide used to generate the antibody, demonstrating that the labeling in muscle is specific for Cav3 protein (Fig. 4B). The con-cav antibody strongly labels muscle cells (Fig. 4C) as well as non-muscle cells (Fig. 5C), consistent with the recognition of multiple caveolins, as in mammalian cells. Intact muscle fibers isolated from 2-day-old embryos show highly ordered arrays of labeling on their surfaces (Fig. 4D), consistent with the labeling pattern of adult mammalian fibers (37Go).



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  		Figure 4. Cav3 and the dystrophin–glycoprotein complex only partially colocalize in developing muscle. (A–C) Antibody labeling of zebrafish muscle with con-cav antibody. (A) Section of adult muscle showing surface and internal (putative T-tubular) labeling using confocal microscopy. Labeling of sections of adult muscle with con-cav antibody in the absence (A) or presence (B) of a competing peptide immunogen. (C) Whole mount embryo at 48 h labeled with con-cav antibody; labeling can be seen at the surface of the fibers. (D–F) Isolated muscle fibers co-labeled with the con-cav antibody (red, D) and ß-dystroglycan (green, E). Caveolin labeling extends over the entire sarcolemma, whereas ß-dystroglycan labels the ends of the fibers (merge, F). Scale bars, 10 µm, except C 50 µm.

 


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  		Figure 5. Targeted down-regulation of Cav3 causes muscle abnormalities. Embryos were injected with contMO (A, C, G, I), cav3MO (B, D, F, H, J, L), cav3UTRMO (M) or not injected (E and K). (A and B) When embryos are labeled for caveolin with the con-cav antibody at 48 h, contMO-injected embryos have normal Cav3 labeling in muscle (A), but the labeling is no longer present in cav3MO-injected embryos (B). (C) Surface labeling representative of caveolin-1 is normal in cav3MO-injected embryos compared with contMO at 48 h. (E and F) At 22 h, control sibling embryos (E) are normal, with chevron shaped somites, whereas the cav3MO-injected embryos (F) show disruption to the notochord, N, and have ‘blocky’ somites. (G–J) By 48 h, cav3MO-injected embryos have curved tails (H) compared with contMO-injected embryos (G and I). Five-day-old embryos injected with cav3MO display cardiac edema (arrow, J) compared with contMO-injected (I). (K–M) At 4 days, lesions can be seen in the myotomes of both cav3MO and cav3UTRMO-injected embryos (L and M), whereas contMO had normal myotomes (K). (N–Q) At 30 h, Cav3WT-injected embryos look normal (N and O), but when mRNA encoding the Cav3P104L mutant protein is injected, the ‘mild’ embryos have disrupted somites and curved tails (P and Q). Anterior is to the left and dorsal to the top. Scale bars: (A, B, P, S, K–M) 50 µm; (C and D) 20 µm; (E and F) 125 µm; (G–J) 250 µm and (O and P) 100 µm.

 
Inhibition of Cav3 expression with MO antisense oligonucleotides disrupts muscle patterning and function
To explore Cav3 function, we injected 1–2 cell stage embryos with two independent MO antisense oligonucleotides targeted to the region flanking the start codon of Cav3 (cav3MO) or to the region –11 to –35 bp from the start codon of cav3 (cav3UTRMO). Similar results were obtained with the two MOs although the effects were consistently more severe with the cav3MO (Figs 5–9) than with the cav3UTRMO (Figs 5 and 9). We determined an optimum concentration for injection of the cav3MO and assayed Cav3 expression by labeling with the con-cav antibody. Caveolin labeling of the myotomes is completely abolished in the embryos injected with cav3MO (Fig. 5B), whereas labeling of surface epithelial cells that express Cav1 are unaffected (Fig. 5C and D). Thus, the cav3MO specifically reduces expression of the muscle-specific caveolin, Cav3.



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  		Figure 9. Down-regulation of Cav3 results in up-regulation of eng1a. (A–E) In situ hybridization of WT, contMO, cav3MO and cav3UTRMO-injected embryos at 24 h with the eng1a probe. (A and B) Embryos injected with cav3MO (B) showed enhanced expression of eng1a as compared to WT embryos (A). (C–E) Transverse sections of contMO (C) and MO injected (D and E) embryos show a higher level and larger area of expression of eng1a. Arrows indicate the border of the notochord (N). (F–K) ContMO-injected embryos at 26-somite stage demonstrate normal superficial and muscle-pioneer-like staining when labeled with a muscle-specific antibody, F59 (F and I), whereas cav3MO-injected embryos demonstrate less superficial slow muscle staining (G and H) and an increase in the number of muscle-pioneer-like cells that lie adjacent to the notochord (arrows, J and K). Anterior is to the left and dorsal is to the top; sections; dorsal is to the top. Y, yolk. Scale bars: (A and B) 250 µm and (C–K) 50 µm.

 
We then examined the specific effects of Cav3 knockdown compared with embryos injected with a control MO (contMO). cav3MO-injected embryos form blocky somites compared with wild-type chevron shaped somites (Fig. 5E and F) and at 2 days of development show greatly reduced movement. The cav3MO-injected embryos exhibit either no escape response or uncoordinated movements in response to a tactile stimulus. By mid-segmentation stages, the tails of cav3MO-injected embryos are curved and the notochord undulated (Fig. 5F, H, J). At 2 days, the cav3MO- and cav3UTRMO-injected embryos have a reduced heart rate (109±23 beats/min; n=21 embryos) compared with contMO-injected embryos (140±7 beats/min; n=15 embryos), P<0.01, although by 3 days, no significant differences are apparent (cav3MO 147±6 beats/min; n=9; contMO 150±6; n=10). At 5 days of development, the cav3MO-injected embryos have enlarged heart cavities (Fig. 5J). In cav3MO-injected embryos, lesions become obvious in the myotomes at 3 days of development. These lesions are found throughout the entire myotome indicating the degeneration of the muscle fibers but are more commonly observed in the anterior myotomes (Fig. 5L and M). These results demonstrate the essential role of Cav3 in normal skeletal and heart muscle development and maintenance.

Knockdown of Cav3 function disrupts sarcomere, myofibril and intercellular patterning
To understand the effect of cav3MO injection on muscle patterning, we examined the ultrastructural changes produced by Cav3 down-regulation using EM analysis. By 2 days of development, control embryos have well-defined muscle fiber structure with highly organized bundles of myofibrils surrounded by mitochondria and intersected with the complex membrane system, mainly sarcoplasmic reticulum, of the contractile apparatus (Fig. 6A and B). In contrast, muscle cells in cav3MO-injected embryos form only small areas of organized filaments, widely dispersed throughout the cells, with no particular organization into bundles (Fig. 6C–F). Rather than the organized arrangement of mitochondria surrounding the myofibrils seen in control embryos, the cav3MO-injected embryos have numerous mitochondria scattered throughout the cells and interspersed with small areas of organized filaments. The contMO- and cav3MO-injected embryos also show striking differences in the morphology of the intercellular spaces between muscle fibers. Control embryos have close apposition of cells with intercellular spaces filled by extracellular matrix (Fig. 6A and B). In contrast, cav3MO-injected cells have obvious spaces between the muscle cells (Fig. 6C–F), possibly indicative of disrupted interactions between cells or between cells and the extracellular matrix and consistent with the lesions observed using Nomarski optics (Fig. 5L and M). Examination of nuclei in contMO- and cav3MO-injected embryos reveals no evidence of apoptosis (Fig. 6E).



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  		Figure 6. Ablation of Cav3 expression disrupts muscle ultrastructure. Two-day-old embryos were processed for electron microscopy after injection of control (A and B) or cav3MOs (C–F). (A and B) ContMO-injected embryos show the well-organized myofibrils characteristic of wild-type embryos; (B) higher magnification view of the boxed area in (A). Mitochondria, M, surround the densely packed and highly organized fibers. (C–F) In cav3MO-injected embryos, (cav3MO) well-organized myofibrils are not evident. Small patches of disorganized fibers and mitochondria are scattered throughout the cytoplasm. Intercellular spaces (asterisks) are greatly enlarged compared with contMO-injected embryos and frequently contain membranous material. Nuclei, N, in control- and cav3MO-injected embryos appear normal. Scale bars: (A) 5 µm and (B–F) 2 µm.

 
To gain further insights into the changes associated with knockdown of Cav3 expression, we prepared isolated muscle fibers from contMO-injected or cav3MO-injected 48 h embryos. Isolated muscle fibers retain their morphology in the absence of associated cells, simplifying the detailed examination of fiber structure. We waited until the fibers adhered to the culture dish and then analyzed them by light and electron microscopy. Immunofluorescence with the con-cav antibody shows that caveolin protein is distributed over the entire surface of fixed embryonic muscle fibers, consistent with labeling of adult tissue (Fig. 4D). In contrast, ß-dystroglycan (part of the DGC) is localized at the ends of fibers (Figs 4E and 7E). Thus, at this stage of development, there is limited colocalization of Cav3 with the DGC (Fig. 4F) and the DGC is specifically associated with a discrete subdomain of the cell surface, consistent with recent findings (44Go). In contrast to the control-injected fibers, fibers from cav3MO-injected embryos display negligible labeling for caveolins using the con-cav antibody (Fig. 7C and D) whereas expression of ß-dystroglycan is normal (Fig. 7E and F). Phase contrast microscopy revealed dramatic differences between the control- and Cav3-deficient fibers (Fig. 7A and B). Control fibers show prominent sarcomeric banding and are on average 1.7x longer (28±7 µm, n=79 control fibers, versus 16±4 µm, n=77 cav3MO-injected fibers) and more highly organized than fibers from the cav3MO-injected embryos. Nuclear staining revealed that 51% of Cav3 deficient fibers are mononucleate (n=383 fibers). In contrast, only 37% of control fibers are mononucleate (n=468 fibers), suggesting that Cav3 knockdown hinders myoblast fusion. Fibers from Cav3-deficient embryos are often curved or v-shaped (Fig. 8K) when compared with the generally straight fibers from control cells (Fig. 8B).



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  		Figure 7. Cav3 knockdown disrupts muscle fiber morphology. Muscle fibers were prepared from contMO-injected (contMO) (A, C, E) or cav3MO-injected (cav3MO) (B, D, F) 2-day-old embryos by collagenase digestion. (A and B) Fibers from control-injected embryos are generally long and straight with sarcomeric structures evident along the entire fiber length in contrast to the cav3MO-injected fibers that are shorter, often curved and show less evidence of sarcomeric organization (phase contrast images). (C and D) Fibers were labeled with con-cav antibody to caveolin and viewed by confocal microscopy; fibers from the control embryos show caveolin labeling over the entire surface of the fiber (C), whereas injected fibers show negligible labeling (D). (E and F) Isolated muscle fibers were labeled with anti-ß-dystroglycan antibody. This labeling indicates that ß-dystroglycan localization within the muscle fiber is not changed upon down-regulation of Cav3. (G) The S58 slow muscle antibody labels isolated slow muscle fibers, allowing the remaining muscle fibers to be classified as fast. Nuclei numbers were counted with the aid of a nuclear dye (DAPI) to determine the number of nuclei per fast muscle fiber (representative example with similar results in two independent experiments). Scale bars: (A and B) 10 µm and (C–F) 5 µm.

 


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  		Figure 8. Cav3 knockdown disrupts muscle fiber ultrastructure. Analysis of isolated muscle fibers. (A–F) contMO-injected embryos, (G–K) cav3MO-injected embryos. By electron microscopy, control fibers show classical sarcomeric organization, obvious triad structures, T, and putative caveolae (arrowheads; D and E). Surface labeling with ruthenium red during fixation confirms the presence of surface-connected T-tubules forming part of the triad structures (F) (note electron dense spots of precipitate associated with triads, T). In contrast to the organized morphology of the control fibers, the cav3MO-injected fibers are heterogeneous in shape and size (e.g. compare cells in panels G, H and K). Cells appear to be mononucleate or binucleate (nuclei, N). In some cells, the myofilaments show some sarcomeric organization (G) but most have disorganized patches of filaments, f, as seen in (K and H) and in the higher magnification views of the same cell in (I and J). Mitochondria, M, are abundant in the cav3MO-injected fibers. (K) shows a cell surface labeled with ruthenium red (F). No internal labeled structures are evident. Scale bars: (A, B, G, H, K) 5 µm; (C, I, J) 1 µm; (D and E) 0.25 µm and (F) 0.5 µm.

 
We confirmed and extended these results by electron microscopy. The long straight fibers prepared from contMO-injected embryos show prominent sarcomeric banding and triad junctions characteristic of mature muscle (Fig. 8A, C, F). Putative caveolae are also evident as uncoated 40–50 nm diameter invaginations of the sarcolemma (Fig. 8D and E). The general morphology and ultrastructure of the contMO-injected embryonic fibers contrast dramatically with the short and generally undifferentiated (ovoid, short and linear, or even in some cases forked, e.g. Fig. 8K) fibers from Cav3-deficient embryos. Triad junctions are generally absent (Fig. 8G–K). Ruthenium red staining to show surface-connected compartments confirms the general absence of internal labeled structures indicative of the T-tubules in the Cav3-deficient cells (Fig. 8K). Thus, Cav3 knockdown has profound effects on differentiation to form ordered myofibrils.

Cav3 inhibits multinucleate fast fiber formation
The increase in mononucleate muscle fibers could indicate a decrease in myoblast fusion, myoblast formation or a cell fate switch to mononucleate slow muscle. To examine this, we used the F59 muscle-specific antibody, which labels slow muscle early in development. We observed a slight decrease in the number of superficial slow muscle fibers in cav3MO-injected embryos compared with control embryos at the 26-somite stage, although there was an increase in slow muscle fibers adjacent to the notochord (Fig. 9K and L). This increase in muscle-pioneer-like slow muscle cells was unlikely to account for the large increase in mononucleate muscle fibers suggesting that cav3MO induced changes are not solely due to a change in muscle cell fates. To test this hypothesis directly, we isolated muscle fibers from contMO- and cav3MO-injected embryos and labeled the slow muscle with the S58 slow muscle-specific antibody to distinguish between slow and fast muscle fibers (45Go). In two independent experiments, cav3MO-injected fibers had increased numbers of mononucleate S58 negative fast fibers. Figure 7G shows results from one experiment revealing contMO-injected fibers have 28% mononucleate fast muscle fibers, whereas 52% of the cav3MO-injected fast muscle fibers have single nuclei (Fig. 7G). There is no significant difference in the fraction of S58 slow muscle cells in the isolated muscle fibers in contMO compared with cav3MO (22 versus 26%), but there is a significant difference in the number of mononucleate fast muscle fibers. These results are consistent with an inhibition of differentiation or fusion of muscle fibers rather than a fate switch to slow muscle.

Expression of a muscular dystrophy-associated mutant, Cav3P104L, produces a dominant phenotype more severe than Cav3 knockdown
A CAV3 mutation, CAV3P104L, has been identified in human patients with limb girdle muscular dystrophy (LGMD-1C) (20Go) and rippling muscle disease (22Go). The mutant protein localizes to the Golgi complex causing intracellular retention of endogenous wild-type CAV3 in a dominant fashion. We examined whether expression of the equivalent mutation in the zebrafish protein perturbs muscle development. We transcribed mRNAs from the zebrafish Cav3P104L or Cav3WT cDNAs and injected them into 1–4 cell stage embryos together with mRNA encoding GFP. Cav3P104L expression had dramatic effects on embryonic development and viability (Table 1). In contrast, we observed no effect of Cav3WT on gross morphology or muscle differentiation (Fig. 5N and O) even when expressed at several fold higher levels (Table 1). Embryos injected with Cav3P104L at 125 pg/embryo (n=29) were classified further as ‘very severe’ undifferentiated spherical form (21%), ‘severe’ embryos curled up but tail still distinguishable (24%), ‘mild’ curled tails, blocky somites and undulating notochord (10%, Fig. 5P and Q) or normal (41% Cav3P104L, 100% of Cav3WT n=16). The ‘mild’ phenotype showed similarities to the cav3MO-injected embryos and this was investigated further by examining heart rate and myotube fusion in this group of embryos. Cav3P104L embryos had significantly reduced heart rates compared with Cav3WT-injected embryos and non-injected controls (unpublished data). Muscle fibers isolated from embryos injected at 125 pg/embryo, at 48 h with a ‘mild’ phenotype, also had a reduced number of nuclei per muscle fiber, with 41% of isolated fibers mononucleate (n=114 fibers) compared with 16% in the Cav3WT injected fibers (n=143 fibers) and 14% in the non-injected controls (n=78 fibers). Isolated fibers demonstrated similar phenotypes as those isolated from cav3MO-injected embryos, including short, forked and curled fibers (unpublished data). These results are consistent with the interpretation that the Cav3P104L mutant protein, like Cav3 knockdown (Fig. 7), reduces myoblast fusion and has a dominant effect on muscle development that is dosage dependent. However, the strong effects of P104L observed in the most severely affected embryos suggest the possible interference with other caveolins in non-muscle cell types. In conclusion, reduction of Cav3 expression and expression of a Cav3 dystrophy-associated mutant perturb muscle development in a similar manner.


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  		Table 1. Survival rate of Cav3WT and Cav3P104L-injected embryos
 
Knockdown of Cav3 causes up-regulation of a pioneer-slow muscle marker, eng1a
To gain further insight into the changes in slow muscle development we observe with Cav3 knockdown, we examined the effect of Cav3 knockdown on the Hedgehog signaling pathway. Disruption of Hedgehog signaling leads to U-shaped somites, which is also characteristic of Cav3 knockdown (Fig. 5F). To investigate possible changes in Hedgehog signaling, we examined the expression of sonic hedgehog (shh) and its receptor, patched1. No change in mRNA distribution or levels is detected in cav3MO-injected embryos (unpublished data). However, examination of the downstream target, engrailed1a, by in situ hybridization shows a dramatic increase in the number of eng1a positive muscle-pioneer-like cells using two independent cav3 MOs (Fig. 9B, D, E). This increase in eng1a correlates with an increase in the number of slow muscle cells adjacent to the notochord (Fig. 9J and K). This suggests that cav3 knockdown causes an increase in the number of muscle-pioneer cells with subsequent reduction in the number of slow muscle cells at the periphery. Thus, these results suggest an unexpected role for cav3 in the specification of muscle-pioneer cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
This study represents the first description of the caveolin family in the zebrafish and the detailed functional characterization of one member of that family, Cav3. We show a role for Cav3 in muscle development and a striking effect of a muscular dystrophy-associated mutation in Cav3 on muscle development. Finally, we provide evidence for a link between Cav3 and downstream components of the hedgehog signaling pathway.

Caveolin-1 and Caveolin-2 show high evolutionary conservation in sequence and chromosomal organization
Analysis of the sequences of caveolins demonstrates a high level of evolutionary conservation. At the protein level, zebrafish Cav1 shows 72% identity and 81% similarity with human caveolin-1{alpha}, whereas zebrafish Cav2 is 50% identical and 69% similar. Database mining reveals that zebrafish caveolin-1 and caveolin-2 are found in close proximity to each other on the same chromosome, indicating conservation in the chromosomal localization in zebrafish compared with mammals. Isolation of zebrafish cav1 also reveals distinct mRNA species for the {alpha} and ß isoforms. A recent study of mouse developing lung has shown differential expression of {alpha} and ß isoforms at the transcriptional level (53Go); Cav1{alpha} is expressed constantly during lung development but Cav1ß is not expressed until 17.5 dpc, when alveolar type I cells are differentiating. The existence of two isoforms in fish indicates important evolutionarily-conserved functional roles.

Zebrafish Cav3 structure and function are highly conserved compared with human
Zebrafish and mammalian Cav3 also show high homology (72% identity to human CAV3) and amino acids in CAV3 that are altered in human diseases are conserved in the zebrafish (Fig. 1). A putative Cav3 ortholog has previously been described in another non-mammalian species, Xenopus laevis, although that sequence shows relatively low homology to mammalian CAV3 (54Go) when compared with the sequence described here. In fact, examination of the Xenopus database reveals a fourth caveolin gene, with higher homology to the Cav3 gene in mammals and zebrafish and it is likely that this protein (AAH41289 [GenBank] is the closest Cav3 ortholog in Xenopus. The genomic organization and chromosomal localization of zebrafish cav3 are also well conserved with mammals; the Oxytocin receptor and Rad18 genes lie adjacent to Cav3 in fish, mice and humans.

Mutation of the zebrafish cav3 gene equivalent to a human muscular dystrophy-associated CAV3 mutant, caused extremely severe changes in zebrafish muscle development. CAV3P104L accumulates in the Golgi complex of mammalian cells and down-regulates localization of CAV3 at the cell surface (30Go). Our results show that, as in humans with the CAV3P104L mutation (20Go), the expression of the zebrafish Cav3P104L mutant produces a similar dominant phenotype. The severity of the effect of expression of this mutant in the zebrafish demonstrates the significance of this model system for dissecting the effect of this and other CAV3 mutants on muscle function in vivo.

Cav3 functions early in developing muscle and notochord
We show for the first time in any system that cav3 is expressed early in development within the notochord and that knockdown of Cav3 expression results in notochord defects. This implicates Cav3 in notochord function and also suggests the involvement of additional regulatory factors in Cav3 expression; in mammalian skeletal muscle Cav3 expression is regulated by myogenin (55Go), which is not expressed in the notochord (56Go). Our analysis of cav3 expression in adaxial cells shows that cav3 is present prior to the formation of elongated actin filaments and sarcomeres, consistent with it playing a role in myofibril organization. This interpretation is further supported by the loss of myofibril organization we observe after Cav3 knockdown.

As expected, cav3 is abundantly expressed in skeletal muscle in the fish, as in mammals. Lower levels of expression are also found in the heart. Expression of cav3 in adaxial cells, prior to myofibril formation, differs from observations in cultured mammalian cells in which Cav3 expression was observed at the time of myotube formation, with caveolin-1 expressed in myoblasts prior to fusion (4Go). The difference may reflect differences between muscle cell lines and in vivo differentiation of muscle cells.

Caveolae are an abundant feature of many cell types in the zebrafish embryo. At the cellular level, we show that in 2-day-old embryos, abundant caveolae are present along the length of the embryonic muscle sarcolemma, and this correlates with the distribution of Cav3 as observed by light microscopy. The distribution of Cav3 at this developmental stage does not overlap significantly with that of the DGC protein, ß-dystroglycan, a putative Cav3-interacting protein. Whether the interaction of Cav3 with ß-dystroglycan occurs later in development or involves only a relatively minor fraction of the two proteins remains to be examined. Interestingly, previous studies indicated that Cav3 and dystrophin bind to the same region on ß-dystroglycan suggesting that they are mutually exclusive binding partners and that Cav3 may competitively regulate the interaction of these two proteins at the sarcolemma (39Go).

Functional characterization of Cav3
The specific function(s) of caveolae in muscle are presently unknown. Reduction of Cav3 expression by antisense MO injection caused embryos to have slowed or completely uncoordinated movement. Examination of gross morphology revealed dramatic tail curvature in the cav3MO-treated embryos. Whether this is solely due to effects on muscle differentiation or, in addition, reflects an effect on the notochord (57Go) that we have shown also expresses Cav3 and has abundant caveolae (unpublished data), awaits further investigation. Cav3MO-injected embryos demonstrate an enlarged pericardium, and at early stages, a decreased heart rate. In mouse, Cav3 is expressed in the heart at embryonic day 10 (55Go). Overexpression of Cav3 results in severe cardiac tissue degeneration, fibrosis and reduced cardiac functions (58Go), whereas ablation of Cav3 causes a progressive cardiomyopathic phenotype characterized by myocyte hypertrophy (59Go).

Previous experiments in mammals have produced somewhat contradictory conclusions about the function of Cav3 in myoblast development. Cav3 has been implicated in the fusion of mammalian myoblasts based on studies of cultured C2C12 cell lines in which Cav3 expression was reduced by antisense treatments (36Go). More recent studies of transformed muscle cell lines derived from either transgenic mice overexpressing Cav3 or Cav3 null mice suggested that Cav3 overexpression reduces and Cav3 loss enhances fusion of myoblasts to form myotubes (35Go). In contrast, however, Cav3 knockout mice exhibit only a mild dystrophic phenotype suggesting that myoblast fusion in vivo is apparently relatively normal (10Go,11Go).

Our results provide new insights into Cav3 function. In contrast to some of the Cav3 overexpression studies in mouse (13Go,35Go), we see no gross effect of Cav3WT mRNA injection on the whole organism, including no differences in heart rates compared with uninjected controls. Isolated muscle fibers from Cav3WT-injected embryos had the same number of nuclei as compared to uninjected controls indicating similar levels of myoblast fusion. On the other hand, knockdown of Cav3 function resulted in severe skeletal muscle defects. Developing muscle fibers had disorganized fibrils and large intercellular spaces. These results demonstrate an essential role for Cav3 in muscle development or maintenance. Muscle fibers isolated from the cav3MO-injected embryos have disorganized myofibrils, are shorter in length, and typically have only one nucleus when compared with 3–4 nuclei on average in control embryonic muscle fibers. This increase in mononucleate muscle fibers is not due to a fate switch to slow muscle as demonstrated with the S58 slow muscle antibody. The cav3MO-injected fibers also develop aberrant branched and ovoid shapes in contrast to the uniform long straight fibers isolated from control embryos. Together these observations suggest that Cav3 deficiency leads to a reduction in myoblast fusion and that the fusion events may not be spatially coordinated.

In addition to, or causally related to these effects on myotube formation, Cav3 deficiency produced severe defects in the organization of the contractile apparatus and membrane systems in immature myotubes. Control fibers at 2 days of development already show the well-organized contractile apparatus and membrane systems of adult muscle; myofibrils, surrounded by mitochondria have well-organized sarcomeres. Triad junctions, indicative of sarcoplasmic reticulum-T-tubule coupling, are already evident. In contrast, Cav3-deficient fibers display mainly disorganized filament bundles interspersed by numerous mitochondria and poorly developed membrane systems. Although Cav3 has been implicated in T-tubule formation (33Go) further work will be required to establish whether the effects described here directly result from lack of Cav3 or from aberrant or retarded differentiation.

Cav3 knockdown expands engrailed expression
We found that Cav3 knockdown unexpectedly resulted in increased Engrailed expression and an increase in the number of muscle pioneer cells. Because slow muscle, and muscle pioneers in particular, depends on Hedgehog signaling (60Go–62Go), this increase in Engrailed suggested that Cav3 might function in the Hedgehog signaling pathway. To explore this possibility, we examined expression of Hedgehog signaling pathway proteins. We observed no change in the expression of Shh or its target, Ptc1, after cav3MO injection; however, the number of muscle pioneers increased and eng1a expression was up-regulated dramatically. We previously showed that over-expression of sonic or tiggy-winkle hedgehog caused a dramatic induction of muscle pioneer cells along with a large increase in the number of slow muscle cells at the expense of fast muscle fibers (62Go). This increase in the number of muscle pioneer cells is inhibited by the TGF-ß gene family member, Dorsalin-1, which antagonizes the effect of Hedgehog without inhibiting slow muscle cell differentiation (62Go). Loss of Sonic Hedgehog signaling from the notochord in mutants with notochord defects results in a loss of slow muscle, but these mutants can be rescued by exogenous Sonic Hedgehog (shh) mRNA (63Go–65Go). How the slow muscle differentiates into non-pioneer and pioneer slow muscle cells are not well defined. The increase in muscle pioneer cells in embryos that lack Cav3 indicates that there may be a link between Cav3 and the Hedgehog signaling pathway, or alternatively that Cav3 acts directly in cell fate specification of muscle pioneer cells. Although no interaction between Cav3 and any hedgehog signaling proteins have been found, there has been a report suggesting the association of Patched (Ptc) with Cav1 in tissue culture cells (66Go). Whether Cav1 is involved in the transport of Ptc to the cell surface or in sequestering Ptc to particular domains on the cell surface is unknown (67Go). If caveolin is involved in the delivery of Ptc to the cell surface, the role of Ptc in suppressing smoothened could be hindered allowing activation of the Hedgehog signaling pathway. Alternatively it has been proposed that caveolin could promote the interaction of Hedgehog with Ptc in lipid raft domains, promoting smoothened activation (66Go). Interestingly, Engrailed has also been linked to caveolae. A small fraction of mammalian Engrailed has been shown to be segregated into lipid raft fractions in both tissue culture cells and neuronal tissues and to associate with caveolae in cultured cells (68Go). Further analysis of Ptc trafficking, the Hedgehog protein, and downstream targets in Cav3 null cells should be particularly interesting.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Embryo collection
Zebrafish embryos were raised, removed from their chorions and fixed in 4% paraformaldehyde as described previously (69Go). Animals were staged according to standard criteria (70Go) as hours post-fertilization (h). Fixed embryos were stored at –20°C in methanol until required.

Cloning of the caveolin cDNAs
Total RNA was isolated from 50 h embryos using Trizol reagent (Gibco, Carlsbad, CA, USA) according to instructions. Primers were made from EST and genomic sequence of cav1, cav2 or cav3 (Entrez-PubMed and Sanger Institute) to amplify partial or full-length cDNA clones. The primers used were Cav1Forward: 5'-GAAGGTGGACTTTGAGGACG-3' and Cav1Reverse: 5'-GAGAGTCAACCCTTCACTTCTG-3'; Cav2Forward 5'-CGCCCTTGCAACTCCAAAACTTAA-3' and Cav2Reverse 5'-GCGCCACCCATAACAAAGTTGACAT-3'; Cav3Forward1 5'-CCCCACTTTTTCTTCACTCGCTCAC-3' and Cav3Reverse 5'-AGCTTCACTCTCCGTCGTCTTCCAG-3'. RNA was transcribed using Superscript II (Gibco) according to instructions. This cDNA was used as a template for PCR using the previous primers. A product of ~500 bp for cav1, 690 pb for cav2 and 750 bp for cav3 was cloned into pGEM-T Easy (Promega, Madison, WI, USA) and sequenced using ABI PRISM BigDye Terminator v2 or v3.1 (Applied Biosystems, Foster City, CA, USA) in the Australian Genome Research Facility, University of Queensland (Brisbane). Sequences were aligned using MacVector 7.1.1.

5'-RACE
Cav1Reverse and Cav1Reverse2: 5'-CCGATCCCTCTGGCTCTTCTCTTT-3' primers were made to the cav1 fragment as described earlier. 5'-RACE was carried out using FirstChoice-RLM RACE kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions using RNA from 50 h embryos. The PCR product was visualized on an agarose gel, the band was cloned using pGEM-T Easy (Promega) and sequenced.

Genomic mapping of cav3
The cav3 gene was assigned to a linkage group using the LN54 radiation hybrid panel (71Go). Forward2 5'-GTTACACCACCTTCACCGTCTCCAA-3' and reverse primer (mentioned previously) to exon 2 of the cav3 gene were used for PCR. Each reaction contained 1x Thermopol buffer (New England Biolabs, Beverly, MA, USA), 0.2 µM each of forward and reverse primer, 0.25 mM dNTPs, 1 U of Taq DNA polymerase and 100 ng of hybrid-cell DNA. The three control reactions had 100 ng zebrafish DNA, 100 ng mouse DNA or no DNA. PCR conditions were initial denaturation for 2 min at 94°C followed by 35 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 40 s followed by final extension at 72°C for 10 min. The 25 µl reactions were run on a 1.5% agarose gel and the panel was scored on the basis of the absence or presence of the expected 461 bp band. The resulting RH vector was analyzed according to directions at http://mgchd1.nichd.nih.gov:8000/zfrh/beta.cgi.

mRNA in situ hybridization of whole mount embryos
Digoxigenin-labeled probes were synthesized by in vitro transcription using SP6 or T7 polymerase (NEB) and digoxigenin-11-UTP RNA labeling mix (Roche Diagnostics, Mannheim, Germany). mRNA in situ hybridizations were carried out essentially as previously described with minor alterations (69Go,72Go). Embryos were mounted in glycerol and observed with Zeiss Axiophot 2 microscope, Leica Dissecting microscope or Olympus dissecting microscope and images were captured digitally. Figures were generated using Adobe Photoshop 7; modifications were applied to whole images only.

Sectioning
Embryos were labeled by mRNA in situ hybridization and then mounted in 0.1% agar, 1% agarose and 5% sucrose or paraffin for serial sectioning. The blocks were trimmed, sectioned, observed using a Zeiss Axiophot 2 or an Olympus BH2 microscope and images were captured digitally. Figures were generated using Adobe Photoshop 7; modifications were applied to whole images only.

MO injection
cav3MO antisense oligonucleotide was designed for sequence flanking the ATG of zebrafish Cav3, the second MO, cav3UTRMO complements the sequence at position –11 to –35. MOs of the following sequence cav3MO 5'-CGTTAGTGTTGTACTGGTCCGCCAT-3', cav3UTRMO 5'-TCTGGCCCAAGAGCTGTCAAAAAGT-3' and contMO 5'-CCTCTTACCTCAGTTACAATTTATA-3' were obtained from Gene Tools, LLC (Philomath, OR) and diluted for injection in 1x Danieau solution (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM HEPES and pH 7.6) with phenol red as an indicator. cav3MO (3–4.5 ng), cav3UTRMO (4.5–6 ng) or contMO (4.5–6 ng) was injected into the one cell of zebrafish embryos between the 1 and 2 cell stage.

Analysis of live embryos
Embryos were immobilized in embryo medium containing tricaine (0.2% 3-aminobenzoic acid ethyl ester) and images were captured using an Olympus dissecting microscope and Advance Spot software. Heart beats were counted over a 10 s period at room temperature (RT) and converted to heart rate/minute.

Synthesis of caveolin mutant, Cav3P104L, and in vitro transcription of DNA constructs for microinjection
Site directed mutagenesis of zebrafish Cav3 was carried out essentially according to the QuickChange site directed mutagenesis kit protocol from Stratagene (Cedar Creek, TX). Two complementary primers were used to mutate the proline at position 104 to a leucine in Cav3. The primers used were 5'-CCTTCTGTCACATCTGGGCCGTGATGCTTTGCATTAAAAGC-3' and 5'-GCTTTTAATGCAAAGCATCACGGCCCAGATGTGACAGAAGG-3'; these primers allowed detection by the removal of a NdeI site. The resultant Cav3P104L clones were sequenced as described earlier and cloned into pCS2+ vector with EcoRI and BamHI. The DNA plasmids were linearized with NotI, purified and precipitated. The RNA for microinjection into embryos was synthesized from linearized plasmid DNA using the Ambion (Austin, TX) mMACHINE SP6 in vitro transcription kit according to the manufacturer's instructions.

Microinjection of mRNA
Approximately 10 min after natural spawning of adults, the embryos were collected and placed in embryo medium for microinjection of mRNA (125–500 pg/embryo) from Cav3P104L (125–175 pg/embryo) or Cav3WT (wild-type Cav3) together with GFP mRNA. Injections were made into embryos between the one and four cell stages. A group of uninjected control embryos was taken from the same clutch. To ensure that there was complete penetration of the injected RNA throughout the developing embryo, the injected embryos were screened for fluorescence of GFP after shield stage. Embryos that did not fluoresce, or showed regionalized, non-uniform fluorescence were removed. Embryos were immobilized and images captured as described earlier.

Antibody labeling of whole mount embryos
Embryos were fixed and stored in methanol at –20°C. They were brought into PBST (PBS with 0.1% Tween) by progressively decreasing the percentage of methanol. Embryos were blocked with PBSS (1% DMSO, 1x PBS, 1% BSA, 0.2% saponin and 1% Horse serum), then incubated overnight with a pan-caveolin antibody (anti-con-cav) (52Go). The embryos were then washed in PBSS 8x15 min. Embryos were incubated in secondary antibody (anti-rabbit CY3, Jackson ImmunoResearch Laboratories Inc., Westgrove, PA, USA) for 4 h then washed in PBSS (3x15 min). Embryos were visualized using a laser-scanning confocal microscope and BioRAD Lasersharp 2000 software. Embryos labeled with F59 that specifically recognizes slow muscle during early developmental stages (45Go) were labeled and imaged according to Currie and Ingham (61Go). F59 antibody was a gift from F. Stockdale (Stanford University). Images were captured using a Zeiss LSM 510 and Zeiss LSM software. Embryos were mounted in a cavity slide in 80% glycerol in phosphate-buffered saline.

Isolation of muscle fibers
Muscle fibers were isolated from 2-day-old embryos and incubated in collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ, USA) at 3.125 mg/ml in CO2-independent medium (Gibco) at 37°C for 1.5 h. Muscle fibers were triturated and transferred to a microfuge tube. The fibers were spun at 380g in a benchtop centrifuge for 1 min, the supernatant was removed and the fibers were resuspended in CO2-independent medium until they settled on the dish or coverslip. The fibers were washed in CO2-independent medium then treated or fixed as required. Images of unfixed fibers were captured using an Olympus IX70 inverted microscope with Spot digital camera and Spot 3.2.6 software. Fiber length was measured using Adobe Photoshop 5.0.

Antibody labeling of muscle fibers and adult muscle sections
Isolated muscle fibers were fixed in 4% paraformaldehyde permeabilized in 0.5% Triton X-100, quenched in 50 mM NH4Cl and blocked in 0.2% BSA/0.2% FSG in PBS. Muscle fibers labeled with the S58 antibody were fixed in methanol at –20°C for 10 min followed by blocking in 0.2% BSA/0.2% Fish Skin Geletin in PBS. Fibers and sections were labeled with anti-con-cav (52Go), ß-dystroglycan antibody (43DAG/8D5, Novocastra Laboratories Ltd., Newcastle upon Tyne, UK) or the S58 monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa) overnight at 4°C or 30 min at RT for sections. The samples were then washed 4x in PBS and incubated in secondary antibody (anti-mouse alexa-488, Molecular Probes, Eugene, OR, USA or anti-rabbit CY3, Jackson ImmunoResearch Laboratories Inc.) for 1 h (fibers) or 30 min (sections). Peptide inhibition of the muscle labeling with anti-con-cav was carried out by incubating the con-cav peptide at 10 µg/ml with the antibody for 30 min at RT prior to labeling. DAPI was used to label nuclei. Staining was visualized using a laser-scanning confocal microscope and BioRAD Lasersharp 2000 software or an Olympus AX-70 fluorescence microscope to count muscle fiber numbers.

Electron microscopy
Embryos were fixed in 2.5% glutaraldehyde in PBS for 2 h at RT. They were then embedded in epon according to standard methods. Transverse sections were obtained from the region immediately posterior to the pronephric duct. Isolated muscle fibers on 3 cm dishes were fixed in situ as mentioned earlier and, after processing, sections were prepared parallel to the culture dish surface. In some experiments, the cell surface was labeled with ruthenium red as described previously (73Go). Results shown are from contMO- and cav3MO-injected embryos injected at the same time and are representative of at least three separate sets of injections.


   ACKNOWLEDGEMENTS
 
We are grateful to A. Carter, B. Carrigan, A. Carozzi and P.K. Loi for technical assistance. We would also like to thank C. Wicking and S. Martin for comments on the manuscript. This work was supported by grants from the National Health and Medical Research Council of Australia, The National Heart Foundation of Australia and the National Institutes of Health (AR45575 and HD22486). The Institute for Molecular Bioscience is a Special Research Centre of the Australian Research Council. The S58 monoclonal antibody was developed by Stockdale, FE (Stanford, CA, USA) was obtained from the Developmental Studies Hybridoma Band developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: Inserm U469, Endocrinologie Moléculaire: Signalisation Cellulaire et Pathologie, CCIPE, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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