Figure 4.

Knockdown of slc25A46 in zebrafish causes brain maldevelopment, loss of spinal motor neurons, and abnormal increase in mitochondrial length in neurites. (A) Zebrafish embryos injected with slc25A46-specific antisense morpholinos AUG (targeted against the start codon) or SPL (targeted against the splice-donor site for exon 3) compared to those injected with non-specific control demonstrate abnormal indentation at the midbrain-hindbrain junction on lateral view (red arrowhead) as well as an abnormal gap between the optic tectum on dorsal view (black arrowhead). (B) Zebrafish embryos from the ET2 line (in which GFP is specifically expressed in the caudal primary motor neurons) injected with AUG morpholino demonstrate under epifluorescent microscopy decreased abundance of spinal motor neurons (arrows) on lateral view compared to embryos injected with control morpholinos. (C) Visualization of dsRed-labelled mitochondria in GFP-labelled neurons isolated from morpholino-injected embryos. Cells were dissociated from the brain dissected from 1–2 days post-fertilization zebrafish embryos injected with different morpholinos and maintained in culture for 1–2 days. (D) Distribution of the length of mitochondria in neurites. AUG morpholino injection led to an increase in length with a rightward shift in distribution compared to the results from control morpholino injection. Co-injection of the wild-type but not the mutant slc25a46 mRNA reverses mitochondrial length. (E) Summary of mitochondrial length measurements in cultured neurons isolated from zebrafish embryos injected with different constructs. Over 100 neurons were used in each condition, with 400–800 mitochondria being measured. Both AUG and SPL morpholino injection led to an increase in the length of mitochondria, which was reversed by the coinjection of the wild-type but not the mutant slc25a46 mRNA. Box = 25–75th percentile; whisker = 5–95th percentile; line = median; circle = mean; ^*P < 0.0001, two-tailed Mann-Whitney U-test. MO = morpholino.