Opa1 Is Required for Proper Mitochondrial Metabolism in Early Development

<div><p>Opa1 catalyzes fusion of inner mitochondrial membranes and formation of the cristae. <i>OPA1</i> mutations in humans lead to autosomal dominant optic atrophy. OPA1 knockout mice lose viability around embryonic day 9 from unknown reasons, indicating that OPA1 is essential for embryonic development. Zebrafish are an attractive model for studying vertebrate development and have been used for many years to describe developmental events that are difficult or impractical to view in mammalian models. In this study, Opa1 was successfully depleted in zebrafish embryos using antisense morpholinos, which resulted in disrupted mitochondrial morphology. Phenotypically, these embryos exhibited abnormal blood circulation and heart defects, as well as small eyes and small pectoral fin buds. Additionally, startle response was reduced and locomotor activity was impaired. Furthermore, Opa1 depletion caused bioenergetic defects, without impairing mitochondrial efficiency. In response to mitochondrial dysfunction, a transient upregulation of the master regulator of mitochondrial biogenesis, <i>pgc1a</i>, was observed. These results not only reveal a new Opa1-associated phenotype in a vertebrate model system, but also further elucidates the absolute requirement of Opa1 for successful vertebrate development.</p> </div

Phenotypic analyses of MMC (A, C, E, G, I, K, M, O) and TB (B, D, F, H, J, L, N, P) morphant embryos and larvae.

<p>Opa1 morphants at 24 hpf (<b>B</b>) have increased density or ‘graininess’ in the brain region (arrow) and smaller eyes. At 48 hpf, Opa1 morphants have hindbrain ventricle enlargements (arrow) and smaller eyes (<b>D</b>). Opa1 morphants at 48 hpf also have impaired circulation compared with MMC morphants and often has blood accumulation below the heart (arrow) (<b>F</b>). At 72 hpf, Opa1 morphants have larger yolk cells, smaller eyes, smaller hearts, small pectoral fin buds (<b>H</b>) and pericardial edema (<b>J</b>). Many Opa1 morphants had unlooped hearts (<b>L</b>). (<b>N</b>) is the same image as (<b>L</b>) with the heart margins outlined (solid line) and the midline indicated by a dashed line. By 96 hpf, the edema is still present and can involve the eyes (<b>P</b>).</p

Antisense morpholino sequences.

<p>Antisense morpholino sequences.</p

Western blot analysis.

<p><b>A.</b> Representative Western blot for Opa1 yolk-less protein in MMC and Opa1 morphants. Opa1 protein is reduced at 24, 48, and 72 hpf. Differences were isoform specific. Samples from 24 hpf were imaged from a separate blot. Contrast was adjusted to improve visualization. *indicates isoforms selected for densitometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059218#pone-0059218-g001" target="_blank">Figure 1B</a>). Western blot results have been replicated with at least four independent injections <b>B.</b> Quantification of most intense Opa1 isoforms identified by Western blot. All values were first normalized to β-actin protein levels.</p

Primers for RT-PCR.

<p>Primers for RT-PCR.</p

Primers for XL-PCR for mtDNA deletion assay.

<p>Primers for XL-PCR for mtDNA deletion assay.</p

Gene expression changes in MMC morphants (black) and Opa1 morphants (grey) normalized to MMC morphant levels.

<p>Significant increases in gene expression <i>pgc1a</i> (a, p = 0.02) and <i>peo1</i> (b, p = 0.002) were observed in <i>opa1</i> morphants compared to MMC morphants. Error bars are shown +/- SEM, n = 5. P-values obtained by ANOVA.</p

Eye area and heart rate analyses for MMC (black) and Opa1 morphants (grey).

<p><b>A.</b> Eye area was measured by tracing the circumference of individual eyes using AxioVision software. N = 9–12. *p-value <0.05, **p-value <0.01 by Student's 2-tailed t-test. <b>B.</b> Heart rates were measured by counting beats per min for individuals injected with MMC or TB morpholino. N = 34 (48 hpf), n = 44 (72 hpf). *p-value <0.01 by Student's 2-tailed t-test.</p

Gene expression changes of mitochondrial morphology genes in MMC morphants (black) and Opa1 morphants (grey) normalized to MMC morphant levels.

<p>Significant increases in gene expression of mitochondrial fusion proteins (<b>A</b>) <i>opa1</i> (a, p = 0.003), (<b>B</b>) <i>mfn1</i> (b, p = 0.001) and (<b>C</b>) <i>mfn2</i> (c, p = 0.01) were observed in Opa1 morphants compared to MMC morphants. No differences were observed for (<b>D</b>) <i>drp1</i>, a mitochondrial fission protein. Error bars are shown +/− SEM, n = 5. P-values obtained by ANOVA.</p

Pharmacological inhibitors used for deconvolution of total respiration, the mechanism of action, and the final concentrations used.

<p>*1.875 µM FCCP for 3–12 hpf, 2.5 µM FCCP for 24–48 hpf.</p>#<p>1.25 mM sodium azide for 48 hpf, 6.25 mM sodium azide for 3–30 hpf.</p