Exon 5-targeted <i>bbs9</i> splice morpholino affects eye development independent of p53 pathway.

<p>(<b>A</b>) At 24 hpf, the <i>p53</i>-atgMO (1.5 ng) alone injection did not elicit a phenotype. The <i>bbs9</i>-spMO (1 ng) injection alone caused developmental defects in the eye, brain and tail of morphants. However, co-injection of <i>p53</i>-atgMO reduced the defects seen by the <i>bbs9</i>-spMO injection alone, though mild eye defect remained the tail becomes normal (bottom panel). (<b>B</b>) Higher magnification of morphants' head region. Top, middle and bottom rows are 24-, 48- and 72-hpf, respectively. Left and right column of panels are <i>p53</i>-atgMO without and with <i>bbs9</i>-spMO, respectively. At 48 hpf the effect of <i>bbs9</i>-spMO injection on eye size visible (compare the arrows). The <i>bbs9</i>-spMO injection also resulted in hydrocephalous (compare the arrow heads). The defects seen at 48 hpf are weaker at 72 hpf. (<b>C</b>) The gel photograph of RT-PCR showing exon-skipping by <i>bbs9</i>-spMO. mRNA isolated from individual embryos was used for RT-PCR. U, C (4 and 6 ng) and B (1, 4, 6 ng) represent un-injected, control, and <i>bbs9</i>-spMO, respectively. Splice blocking gave an additional smaller (marked e5skip) band along with the original WT band. The bottom panel shows β-actin control for respective samples. (<b>D</b>) Quantification of the effect of morpholino(s) injection on eye size. X-axis shows the morpholinos used and time (hpf) of scoring. Y-axis shows eye size in pixels (mean ± SEM).</p

Human mRNA rescues zebrafish <i>bbs9</i>-spMO phenotype.

<p>h<i>W</i> and h<i>M</i> represent wild type and mutant human mRNA, respectively. The arrows indicate eye phenotype. (<b>A</b>) The uninjected control (top) and <i>bbs9</i>-spMO alone injected (bottom) zebrafish at 72 hpf. (<b>B</b>) Rescue of <i>bbs9</i>-spMO eye phenotype by hW 100 pg (top), but not by lower dose of 50 pg (bottom). (<b>C</b>) The <i>bbs9</i>-spMO phenotype is not rescued by h<i>M</i> as the eye defect remains in the morphants. (<b>D</b>) The quantification of embryos' eye size at 72 hpf in rescue experiment using human mRNAs co-injected with <i>bbs9</i>-spMO. X-axis shows category of embryos scored. Y-axis shows the eye size in pixels. Data are presented as mean ± SEM. Statistically significant and non-significant observations are indicated with p value and n.s., respectively.</p

Zebrafish <i>bbs9</i> gene: structural comparison and expression pattern.

<p>(<b>A</b>) A comparison of <i>BBS9</i> exon:intron structure between human (<i>H. sapiens</i>, top blue), mouse (<i>M. musculus</i>, middle black) and zebrafish (<i>D. rerio</i>, bottom gray/black). The filled and open boxes indicate coding exons and UTRs, respectively. The blue and black boxes represent validated exons. The gray boxes represent exons present in provisional sequence XM_002664792.1. Exons 2 to 8 are highly conserved across species (boxed area within hatched square). The yellow arrow points to yellow mark on exon 5, which represents the missense mutation G→A (p.G141R) in human BBS9 protein. Under the zebrafish <i>bbs9</i> transcript, the red line represents <i>bbs9</i>-spMO targeting site at intron4:exon5 boundary. (<b>B</b>) The protein sequence alignment (clustalW) between human (NP_940820.1), mouse (NP_848502.1) and predicted zebrafish BBS9 (904 amino acids). Exon 5 is highlighted (yellow), and the position of missense mutation (p.G141R) in human is highlighted by a black rectangle. The bar coding on top of the sequences represents degree of conservation (red and blue represent maximum and minimum conservation, respectively). (<b>C, D</b>) <i>In situ</i> hybridization analysis at 11 hpf and 15 hpf. Left and right panels represent the sense and anti-sense probes generated from <i>bbs9</i> cDNA. (<b>E</b>) <i>In situ</i> hybridization analysis at 48 hpf. Expression of <i>bbs9</i> in the eye, brain and somites gives a strong signal with the anti-sense probe compared to the background signal from the sense probe. Compare the strong signal in the head regions (arrows). Left and right panels represent lateral and dorsal views, respectively.</p