Abnormal phalloidin staining in skeletal muscle from <i>caf</i> mutant embryos.

<p>Wild type (WT) and <i>caf</i> mutants were staining with phalloidin to illuminate filamentous actin and then visualized whole mount by confocal microscopy. (A) WTs show the expected pattern of staining at 24 hpf (n = 5). (B) In muscle from <i>caf</i> mutants, there is an accumulation of intense staining in the region of the myotendinous junction (arrow) (n = 5). Scale bar = 10 um.</p

Coiling deficiency is not observed in the zebrafish model of Duchenne muscular dystrophy.

<p><i>Sapje</i> mutants, the zebrafish model of Duchenne muscular dystrophy, do not present with a coiling phenotype at 23 hpf (WT sibs: 6.823 ± 0.4642, n = 62; <i>cafs</i>: 5.000 ± 0.9244, n = 11; p = 0.1088) or 24 hpf (WT sibs: 7.347 ± 0.4950, n = 72; <i>cafs</i>: 8.063 ± 1.066, n = 16; p = 0.9997). Bars represent mean ± SEM.</p

Muscle fiber detachment of <i>caf</i> mutants can be observed with birefringence.

<p>Under plane polarized light, muscle from WT siblings (A) appears uniformly bright and white, consistent with normal muscle organization. In contrast, <i>Caf</i> mutants (B-F_ can be identified as having stochastic patterns of muscle degeneration and detachment with birefringence as early as 2 dpf. This is seen in the muscle compartment as dim white areas (thinned or atrophied fibers) and black spots (presumed areas of muscle fiber detachment). Of note, genotype for all depicted animals was confirmed by Sanger sequencing. Bars represent mean ± SEM.</p

Heterozygous <i>lama2</i>^<i>+/-</i> embryos have no coiling deficiency.

<p>Heterozygous (<i>lama2</i>^<i>+/-</i><i>)</i> embryos do not display any coiling deficiency compared to their <i>lama2</i>^<i>+/+</i> WT siblings at 24 hpf after manual dechorionation (WT: 13.20 ± 2.037, n = 10; Het: 11.80 ± 1.654, n = 15; p = 0.5984). Bars represent mean ± SEM.</p

Two Dynamin-2 Genes Are Required for Normal Zebrafish Development

<div><p>Dynamin-2 (DNM2) is a large GTPase involved in clathrin-mediated endocytosis and related trafficking pathways. Mutations in human <em>DNM2</em> cause two distinct neuromuscular disorders: centronuclear myopathy and Charcot-Marie-Tooth disease. Zebrafish have been shown to be an excellent animal model for many neurologic disorders, and this system has the potential to inform our understanding of DNM2-related disease. Currently, little is known about the endogenous zebrafish orthologs to human DNM2. In this study, we characterize two zebrafish dynamin-2 genes, <em>dnm2</em> and <em>dnm2-like</em>. Both orthologs are structurally similar to human DNM2 at the gene and protein levels. They are expressed throughout early development and in all adult tissues examined. Knockdown of <em>dnm2</em> and <em>dnm2-like</em> gene products resulted in extensive morphological abnormalities during development, and expression of human <em>DNM2</em> RNA rescued these phenotypes. Our findings suggest that <em>dnm2</em> and <em>dnm2-like</em> are orthologs to human DNM2, and that they are required for normal zebrafish development.</p> </div

Structure and expression of <i>dnm2</i> and <i>dnm2-like.</i>

<p>(A) Molecular intron-exon organization of human <i>DNM2,</i> zebrafish <i>dnm2</i> and zebrafish <i>dnm2-like.</i> (B) Protein structure of zebrafish Dnm2 and Dnm2-like compared to human DNM2. Percent identity between zebrafish and human protein domains was calculated using BLASTP. PH, pleckstrin homology domain; GED, GTPase effector domain; PRD, proline-rich domain. (C) RT-PCR was used to assay spatial expression levels of <i>dnm2</i> and <i>dnm2-like</i> in tissues isolated from adult zebrafish. Primers for <i>ef1α</i> were used as an internal control. (D) RT-PCR was used to assay temporal expression levels of <i>dnm2</i> and <i>dnm2-like</i> between 0 hpf and 24 hpf. All classical dynamins appear to be deposited as maternal mRNAs and expressed throughout early development.</p

Morpholino-mediated knockdown of <i>dnm2</i> and <i>dnm2-like</i> expression results in morphological changes.

<p>(A) Splice targeting morpholinos were designed against intron-exon boundaries within the <i>dnm2</i> and <i>dnm2-like</i> genes. (B) Knockdown in morpholino injected embryos was verified using RT-PCR. Embryos were injected with a scrambled control morpholino (Ctl MO; 0.3 mM), <i>dnm2</i> MO (0.3 mM), or <i>dnm2-like</i> MO (0.1 mM). Arrows indicate the alternative splice product induced by <i>dnm2</i> MO and <i>dnm2-like</i> MO injection. <i>dnm2</i> MO injection also resulted in an additional higher weight band due to activation of a cryptic splice site (*). (C) At 2 dpf, <i>dnm2</i> MO-injected embryos exhibit shortened body length, upward curled tails, pericardial and yolk edema, and reduced head size when compared to control morpholino injected embryos. By contrast, embryos injected with <i>dnm2-like</i> MO have small muscle compartments, pigmentation defects, and mild tail curvature. (D) Percent of affected embryos at 2 dpf (ctl MO vs. <i>dnm2</i> MO p<0.0001, ctl MO vs. <i>dnm2-like</i> MO p<0.0001; Fisher’s exact test). The total number of embryos is noted above each bar.</p

Human DNM2 RNA rescues <i>dnm2</i> and <i>dnm2-like</i> morphant phenotypes.

<p>Rescue of <i>dnm2</i> and <i>dnm2-like</i> morphants at 2 dpf. (A) Co-injection of human DNM2 RNA can rescue morphological abnormalities in both morphants. (B) RT-PCR of human DNM2 expression in <i>dnm2</i> or <i>dnm2-like</i> morphants at 3 dpf. (C) The percentage of normal appearing larvae is significantly increased in both <i>dnm2</i> and <i>dnm2-like</i> rescue conditions, but not in control larvae (<i>dnm2</i> p<0.0001, <i>dnm2-like</i> p<0.0001, ctl p = 0.30; Fisher’s exact test). The total number of embryos is noted above each bar.</p

Western blot.

<p>Lysates of knock-out homozygous <i>aldh7a1</i> zebrafish embryos (at 8 dpf) western blot analysis showing no aldh7a1 protein compared to wild-type.</p

Knockdown of <i>dnm2</i> results in motor deficits and abnormal muscle histology.

<p>(A) Quantitation of spontaneous embryo coiling at 1 dpf. On average, control morphants coiled 35.7 times in 60 seconds, while <i>dnm2</i> morphants coiled only 9.5 times. (B-C) Touch-evoked swimming was measured in 3 dpf morphants. Most control and <i>dnm2-like</i> morphants responded to tail taps with a rapid escape response, while <i>dnm2</i> morphants exhibited impaired escape responses. (D) Toluidine blue stained semi-thin sections from 3 dpf morphants. Somites from <i>dnm2</i> morphants are small with highly disorganized myofibers. Scale bar is equal to 50 µm. (E) Quantification of myofiber length in 3 dpf embryos. Average myofiber size in control embryos equaled 87.8 µm, while <i>dnm2-like</i> morphants equaled 76.8 µm and <i>dnm2</i> morphants equaled 66.0 µm (*p<0.05 ctl to <i>dnm2-like</i>, *p<0.01 ctl to <i>dnm2,</i> p = 0.056 <i>dnm2</i> to <i>dnm2-like</i> morphants; ANOVA ). (F) Representative electron micrographs from larval <i>dnm2</i> morphant muscle. Irregular membrane structures were found throughout the muscle (black arrow). Scale bar is equal to 1 µm.</p