From timed larvae, whole mount ISH for the midbrain marker, otx2, demonstrated unremarkable expression level and distribution at 24 hours post-fertilization (hpf) in mutants (Figure 3C). However, by 48 hpf mutants displayed weak, spatially restricted expression of otx2, and by 72 hpf, otx2 was not evident in the mutant zebrafish. Since initial otx2 expression was initially unremarkable in mutant animals, our data suggests the progressive loss of expression results from cell loss as opposed to defective patterning. To further differentiate between these possibilities, we performed terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), to detect DNA fragmentation (Chen et al., 2010). We noted a dramatic increase in staining specific to the forebrain and hindbrain (Figure 3D). Therefore, clp1 mutant zebrafish showed evidence of hindbrain neurodegeneration, similar to tsen54 zebrafish morphant (Kasher et al., 2011). The similarity in brain phenotype between the human p.R140H and the zebrafish mutations suggests loss-of-function as the disease mechanism in humans.
