An ENU screen for zebrafish skeletal muscle mutants led to identification of a dystrophic mutant, patchytail
In an attempt to identify new genes in congenital skeletal muscle diseases, an ENU F2 mutagenesis screen for homozygous recessive mutations was performed in zebrafish (36
). Zebrafish embryos with skeletal muscle abnormalities were identified using a birefringence assay that involves examination of axial skeletal muscles of live zebrafish embryos using polarized filter microscopy (37
). Wild-type embryos from 3 days post fertilization (dpf) onwards display highly birefringent skeletal muscles due to the ordered array of myofilaments (Fig. A and B). Skeletal muscle mutants identified with this screen, which displayed reduced or patchy birefringence compared with wild-type embryos, were selected for subsequent studies. One of the mutants, designated patchytail
, displayed patchy birefringence indicative of areas of muscle degeneration in somites of homozygotes. During earlier development, the formation of muscle fibers in patchytail
was indistinguishable from wild-type embryos. The first signs of muscle degeneration in patchytail
were observed at 3–4 dpf and were identified as dark areas in somites under polarized light when compared with wild-type (Fig. B and D). In patchytail
fish, muscle degeneration is initially restricted to a very few posterior somites at this stage (4 dpf). This is a much milder phenotype in comparison with other dystrophic fish models, such as sapje
(dystrophin deficient) (Fig. E and F) or candyfloss
’ or laminin α2 deficient) fish (Fig. G and H) where generalized muscle degeneration was seen in most somites by 2–3 dpf (Table ) (34
). Muscle degeneration in patchytail
progressed rapidly after 4 dpf, and most of the somites exhibited muscle degeneration by 7 dpf (Fig. I and J). By this stage, skeletal muscle disorganization was seen randomly distributed in all somites in the trunk. Muscle degeneration in patchytail
appeared to be restricted to skeletal muscles. There was no evidence for any morphological abnormalities of the heart, cardiac enlargement or edema of the cardiac sac through 7 dpf. Furthermore, cardiac function appeared to be unaffected as observed by similar heart rates in mutant in comparison with wild-type embryos. A large number of mutant embryos were raised (~300), and all homozygotes died by 8–10 dpf; however, in the heterozygous state, these fish were fully viable, fertile and apparently unaffected.
Figure 1. Patchytail mutant fish show loss in birefringence and muscle degeneration similar to a dystrophic phenotype. (A and B) Wild-type fish show bright birefringence indicative of highly organized muscles at 4 dpf. (C and D) Patchytail fish appear grossly normal (more ...)
A comparison of known zebrafish models of dystrophies
Patchytail fish show impaired locomotor activity
Mutant patchytail embryos exhibited a defect in motor activity starting early in development. In developing zebrafish, muscle activity results in hatching from their chorion ~2–2.5 dpf. Typically 95 ± 3% (P < 0.005) of wild-type embryos were hatched from their chorions by 2.5 dpf. In contrast, only 85 ± 4% (P < 0.005) of mutant embryos hatched by 2.5 dpf, suggesting a mild muscle weakness during early development.
By 7 dpf, mutant fish exhibited significantly slower swimming than wild-type controls. The swimming phenotype was examined by video microscopy with a touch-evoked escape behavior assay. Typically, wild-type 7 dpf larvae respond to tactile stimuli with a rapid and vigorous escape contraction, followed by swimming, which often resulted in larvae rapidly swimming out of the field of view (Fig. A–D). In contrast, patchytail
embryos displayed weak escape contractions, followed by reduced swimming that often failed to propel the larvae more than a few lengths (Fig. E–J and Supplementary Material, Movies 1 and 2
) (wild-type at 7 dpf: 6.2 ± 0.4 cm/0.1 s, n
= 10; mutant at 7 dpf: 0.75 ± 0.08 cm/0.1 s, n
= 10; Student's t
< 0.001). A highly diminished touch-evoked escape behavior at 7 dpf is indicative of impaired motor function and overall skeletal muscle weakness. Together, these data suggest only mild normal muscle dysfunction during early patchytail
development followed by progressive muscle degeneration by 7 dpf suggestive of a dystrophic phenotype.
Figure 2. Patchytail mutant embryo exhibit slow swimming in response to touch. (A–D) Mechanosensory stimulation induced a wild-type embryo (5 dpf) to swim away rapidly. (E–J) Touch induced the patchytail mutant embryo (5 dpf) to swim, but only slowly. (more ...)
Mapping the patchytail locus identifies a mutation in dag1
To identify the mutation underlying the patchytail dystrophic phenotype, genetic mapping was performed. Pooled DNA of 40 patchytail and 40 control siblings of 5 dpf embryos, respectively, were tested with 288 simple sequence length polymorphism markers resulting in linkage of the mutation to chromosome 22. Using standard bulk segregant analysis, a first-pass map position for the patchytail locus was established to markers z6850 (29.1 cM), z9084 (56 cM), z28677 (59.3 cM). Using a fine mapping strategy, this region was further refined to flanking markers z21243 (43.3 cM) and z9084 (55.9 cM), approximating a genomic region of 13 cM (Fig. A). As the phenotype observed in patchytail fish suggests a dystrophic process in skeletal muscles, an analysis of this chromosomal area for skeletal muscle-specific genes resulted in identification of the dystroglycan gene (dag1) as a candidate. Sequencing of dag1 revealed a point mutation c.1700 T>A in exon 3 that segregated with the mutant phenotype (Fig. B).
Figure 3. The patchytail phenotype is a result of mutation in the zebrafish ortholog of the DAG1 gene. (A) An initial bulk segregant analysis implicated marker z228677 on LG22 as being linked to patchytail phenotype. Further fine mapping resolved flanking markers (more ...)
Mutation in dag1 results in decreased RNA and absence of dystroglycan protein
The point mutation identified in dag1-mutated fish (c.1700T>A) results in a missense amino acid change of valine (V) to aspartic acid (D) (p.V567D). This change is present in the C-terminal domain of α-dystroglycan that is required for DGC assembly by interacting with the C-terminal domain of β-dystroglycan (Fig. C). Computational predictions for the effect of this missense change on protein were evaluated using the Polyphen-2, SIFT and PMut programs. Polyphen-2 predicted this mutation to be ‘probably damaging’ (score 0.969, sensitivity, 0.70, specificity 0.94). Mutation at 567 from V to D was predicted to be not tolerated and affecting protein function with a score of 0.00 by SIFT. Amino acids with probabilities <0.05 are predicted to be deleterious by SIFT. Finally, the pMut program predicted V567D change to be pathological (NN output 0.9055, reliability 8).
The effect of this point mutation in patchytail fish was experimentally evaluated at the RNA and protein levels. RT–PCR showed decreased gel band intensity of dag1 transcripts in patchytail mutant embryos in comparison with the wild-type controls, suggesting that dag1 mRNA levels were likely to be reduced in mutant fish (Fig. D). To assess the effect of mutation on α- and β-dystroglycan proteins, western blotting was performed on wild-type as well as patchytail zebrafish embryos. Dystroglycan protein is post-translationally cleaved into α- and β-dystroglycan and expression of both of these polypeptides were examined. Western blot analysis revealed protein products of expected sizes in wild-type embryos. In comparison with the positive controls, no detectable α- or β-dystroglycan expression was seen in mutant embryos (Fig. E). Therefore, these studies show that the p.V567D change in dag1 is associated with reduced levels of dag1 mRNA and absence of both α- or β-dystroglycan proteins.
To further validate that the dystrophic phenotype observed in patchytail fish is a result of dystroglycan deficiency, rescue studies were performed. This involved the rescue of mutant phenotype by overexpression of zebrafish dag1 mRNA in embryos. One cell embryos obtained from a cross between two heterozygote patchytail fish were injected with wild-type zebrafish dag1 mRNA. In non-injected clutches, 21.5 ± 2.8% of the embryos showed the mutant phenotype and 78.5 ± 2.8% showed normal phenotype at 4 dpf as seen by birefringence assay. Embryos injected with dag1 mRNA showed a reduction in the mutant phenotype to 10 ± 2.65% of embryos with 90 ± 2.65% of embryos exhibiting normal phenotype (Fig. F). Rescue of the mutant phenotype on overexpression of dystroglycan mRNA strongly indicates that the dystrophic phenotype in patchytail fish is a result of dag1 mutation.
Dystroglycan deficiency leads to destabilization of the DGC complex
Members of the DGC complex are required not only for maintaining stability of the complex but also for the protein stability of their interacting partners. Therefore, the effect of the lack of dystroglycan on stability of other members of DGC complex was evaluated by studying their expression in dag1-mutated fish by immunofluorescence at 7 dpf. In zebrafish embryos, like human embryos, members of the DGC complex are expressed initially at the peripheral ends of myofibers known as myotendenous junctions or myosepta. Immunofluorescence using α- and β-dystroglycan showed that in wild-type embryos these proteins are mainly localized at myosepta in skeletal muscles during embryonic development (Fig. A and C). In dag1-mutated embryos, however, a complete absence of α-dystroglycan and β-dystroglycan proteins was observed (Fig. B and D). Expression of laminin α2 was observed at the ECM at myosepta between adjacent somites in wild-type embryos. In dystroglycan-deficient embryos reduced expression of laminin α2 was observed compared with wild-type. Moreover, the area of laminin α2 expression at myosepta appeared widened in comparison with wild-type myosepta (Fig. E and F, arrow). Dystrophin expression was also detected at the myosepta in wild-type embryos. The expression of dystrophin in dystroglycan-deficient fish was seen to be highly reduced (Fig. G and H). These studies demonstrate that dystroglycan deficiency results in destabilization of their interacting partners in both extracellular as well as the intra-cellular side of the myoseptum.
Figure 4. Dystroglycan is required for stability of its interacting partners in myoseptum. Immunofluorescence analysis of dystroglycan and interacting proteins (green) and nuclei [stained blue with 4′,6-diamidino-2-phenylindole (DAPI)] in wild-type and (more ...)
Dystroglycan deficiency leads to ECM defects in skeletal muscles
To evaluate the loss of dag1 on structure of affected skeletal muscles, toluidine blue-stained longitudinal sections of wild-type and dystroglycan-deficient fish were examined at 7 dpf (Fig. A–D). Myofibers in both wild-type and mutant fish looked well organized. However, the myosepta in mutant fish appeared to be thickened in width compared with wild-type siblings. Higher magnification views revealed gaps at myosepta between adjacent somites (Fig. D, arrow). Dystroglycan-deficient fish also showed gaps between adjacent myofibers, suggesting fiber detachment at ECM (Fig. D, arrowhead).
Figure 5. Dystroglycan deficiency affects the structure of myosepta. (A–D) Histology of skeletal muscles stained with toluidine blue in wild-type and dystroglycan-deficient fish at 7 dpf. In comparison with wild-type fish (A and C), dystroglycan-deficient (more ...)
Chimeric mice deficient for dystroglycan exhibit a neuromuscular junction defect with fewer and fragmented nerve synapses in the affected muscles (39
). Therefore, the effect of dystroglycan deficiency on zebrafish neuromuscular junctions was also evaluated. Whole-mount labeling for postsynaptic acetylcholine receptors using fluorescent-conjugated α-bungarotoxin was performed for dystroglycan-deficient as well as wild-type embryos (Fig. E and F). Acetylcholine receptors were found to be localized to myofiber ends adjacent to myoseptum as well as with in the somites in wild-type embryos (Fig. E). Similarly, dystroglycan-deficient embryos showed well-developed postsynaptic receptor clusters. There were no discernable differences between wild-type and dystroglycan-deficient fish in the number and size of clusters, which suggests that dystroglycan may not be required for neuromuscular junction formation in zebrafish muscles.
To visualize the ultrastructure of skeletal muscles, transmission electron microscopy was performed. Dystroglycan-deficient fish display a mild dystrophic phenotype during early development (3 dpf) followed by severe impairment of locomotive behavior by 7 dpf. Therefore, to understand the pathological progression of muscle degeneration, ultrastructure of dag1-mutated fish was examined at these two time points during zebrafish development. Early in development, myospeta, in most of the somites of the dystroglycan-deficient fish, were organized similar to wild-type siblings (Fig. A and B). In some somites of the mutant fish, initiation of tearing, identified by the lack of electron dense areas in a myoseptum, was observed at this stage (Fig. B, arrow). Despite this minor tearing, myofibers were largely intact in the mutant fish (Fig. B, arrowhead). By 7 dpf, ultrastructure of dystroglycan-deficient fish showed highly distorted and irregular-shaped myospetal boundaries in most of the somites (Fig. C and D). The myosepta of mutant embryos appeared to be fragmented with regions of separated ECM (Fig. D, arrowhead).
Figure 6. Transmission electron micrograph of skeletal muscles in wild-type and dag1-mutated embryos at 3 and 7 dpf. Myosepta in wild-type embryos (A) are compact and have electron dense structures, whereas myosepta of dystroglycan-deficient fish (B) show mild (more ...)
Around 7 dpf during normal development, expression of DGC components becomes prominent in the sarcolemma of myofibers (40
). To investigate whether the loss of dystroglycan affects sarcolemmal integrity, ultrastructure of sarcolemma of wild-type and dag1
-mutated embryos were evaluated at 7 dpf. The cross-sectional view of wild-type zebrafish muscle fibers showed well-organized sarcolemmal boundaries between adjacent myofibers (Fig. E). However, the sarcolemmal membranes in dag1
-mutated embryos appeared to be grossly disorganized when compared with wild-type muscles (Fig. F). High magnification examination revealed widening of the area between myofibers of two adjacent fibers on the ECM side (Fig. H, arrows). However, no such gaps were observed between wild-type myofibers (Fig. G, arrows). The sarcolemmal integrity was evaluated in vivo
using Evans blue dye that labels cells with damaged plasma membranes. No uptake of dye was observed in either wild-type or dystroglycan-deficient fish, suggesting that the sarcolemma remains largely intact in dag1
-mutant fish. These data demonstrate that severe dystrophy observed in dystroglycan-deficient fish is due to massive detachment in extracellular junctions at myoseptum as well as disruption of extracellular integrity at the sarcolemmal membranes.
Myofibers in dystroglycan-deficient fish have abnormal t-tubule structures
Further analysis of myofiber ultrastructure in dystroglycan-deficient fish showed normal organization of the contractile apparatus with no necrotic fibers or apoptotic nuclei through 7 dpf (Fig. A–D). While the contractile apparatus was structurally normal appearing in mutant fish, higher magnification views revealed that t-tubules in dag1-deficient fish were smaller and disorganized in comparison with wild-type controls even during earlier stages of development (3 dpf) (Fig. B, arrow). Disorganized t-tubules were very rare in wild-type controls, but were observed even in somites of patchytail fish where the myoseptum was seen to be intact, suggesting that these effects are independent of each other. No significant changes in sarcoplasmic reticulum were observed at 3 dpf. These observations suggest that t-tubule disorganization is an early event in the pathogenesis of the disease. During the larval stages at 7 dpf, most of the myofibers continued to have highly disorganized t-tubules (Fig. D, arrow). Additionally, terminal cisternae of sarcoplasmic reticulum, which are localized adjacent to t-tubules, also appeared to be collapsed in most of the myofibers in comparison with control fish (Fig. D, arrowhead). No significant abnormality was detected in the longitudinal vesicles of the sarcoplasmic reticulum.
Figure 7. Ultrastructure of t-tubules in wild-type and dag1-mutated embryos at 3 and 7 dpf. (A and B) t-Tubules are well formed in wild-type muscles but not in dystroglycan-deficient muscles at 3 dpf (B, arrow). (C and D) Wild-type muscles have well-developed t-tubules (more ...)
Thus far, expression of dystroglycan has been reported at the sarcolemma in skeletal muscles cells. To understand whether the t-tubule defects are a direct result of loss of function of dystroglycan, indirect immunofluorescence was performed to evaluate the subcellular localization of dystroglycan in developing zebrafish muscles. In wild-type fish, α- and β-dystroglycan are expressed both at the sarcolemma and within myofibers in a striated pattern at 7 dpf (Fig. E and F). Co-immunostaining for dystroglycan and the t-tubule marker, Dhpr, showed that α- and β-dystroglycan co-localize with Dhpr in wild-type myofibers (Fig. G and I). As expected, no expression of α- and β-dystroglycan was observed in myofibers from patchytail fish. Interestingly, Dhpr levels appeared to be reduced significantly in the dag1-mutant fish (Fig. H and J). Co-immunostaining for ryanodine receptors, Ryr1, and Dhpr showed that, as expected, they co-localized at t-tubules and sarcoplasmic reticulum junction (i.e. the triads) in wild-type fish. While moderate levels of Ryr1 expression were detected in dystroglycan-deficient fish, expression of Dhpr was again highly diminished (Fig. K and L). These data suggest that, in addition to expression at the sarcolemma, a significant fraction of dystroglycan is also localized at the t-tubules in wild-type fish where it may play a role in organizing or stabilizing Dhpr calcium channels.
Lack of dystroglycan affects eye structure in zebrafish
Dystroglycan is widely expressed in different organs in addition to skeletal muscles in mammals. To study the expression of dystroglycan in different organs in zebrafish, whole-mount immunofluorescence was performed. Immunofluorescence studies showed that besides expression at the myosepta in skeletal muscles, dystroglycan is also expressed in the eyes, CNS and heart in wild-type fish (Fig. A). In dag1-mutated fish, a complete absence of dystroglycan protein was observed in all organs (Fig. B). Owing to strong expression in the eyes and CNS and the involvement of these organs in a wide range of dystroglycanopathies, the effect of dystroglycan deficiency on these organs was evaluated in patchytail fish.
Figure 8. Dystroglycan-deficient fish show ocular defects. (A and B) Whole-mount immunofluorescence of wild-type and dystroglycan-deficient fish (5 dpf) with β-dystroglycan antibody shows expression of dystroglycan in the brain, eye and heart (5 dpf). ( (more ...)
Upon gross examination, the eyes of 5 dpf patchytail embryos appeared to be normal (Fig. C and D); however, histological examination revealed striking abnormalities (Fig. E and F). At this stage, wild-type eyes are well organized into photoreceptor, inner and outer plexiform layers, ganglion cell layer, lens, and well-developed cornea. In dystroglycan-deficient fish, differentiated cell layers were also present in the eyes. However, in the posterior chamber, cells within the ganglion layers were loosely packed in comparison with wild-type fish and there were gaps present between cells in the ganglion layer in dystroglycan-deficient fish (Fig. F, arrow).
The anterior chamber of the mutant eyes showed the most drastic changes relative to wild-type fish. By 5 dpf, wild-type fish lens fibers have differentiated, lost their nuclei and become crystalline. The lens in dag1-mutated fish, however, was cellular with several inclusion bodies present (Fig. F, asterisk). The cornea of the eye was absent in dag1-mutated fish suggesting either it was not formed or degenerated due to dystroglycan deficiency.
Loss of dystroglycan results in brain abnormalities in zebrafish
The effect of dystroglycan loss on CNS development was also studied in patchytail. Histology of control brains showed that by 5 dpf, the CNS is well organized into different chambers; forebrain (telencephalon), midbrain (tectum) and hind brain (cerebellum and myelencephalon) (Fig. A). When compared with wild-type brain, all four chambers were seen in dystroglycan-deficient fish as well (Fig. B). However, tectal cells in midbrain and granular cell layer in cerebellum appeared to be less organized in dystroglycan-deficient fish when compared with wild-type (Fig. C–F, arrows). The mutant brain did not show any hydrocephalus or neuronal heterotopia at this stage of development as reported in dystroglycan-deficient mice.
Figure 9. Histology of wild-type and patchytail zebrafish brains stained with toluidine blue. (A and B) Sagittal section of wild-type and patchytail brains at 5 dpf. Tectum and cerebellum showed cell disorganization in dystroglycan-deficient fish when compared (more ...)