Fig. 7

Fig. 7 (A) Live imaging of 48 hpf Mnpc2^+/m and MZnpc2^m/m embryos injected with either vehicle (Mnpc2^+/m n=53, MZnpc2^m/m n=76) or 100 pmol of 2HPβCD (Mnpc2^+/m n=81, MZnpc2m/m n=80) at 2.5 hpf (n=3 experiments). Many 2HPβCD-injected MZnpc2^m/m embryos showed milder defects, including more normal head/brain appearance, restored red blood cell circulation, and less-curved body axis than observed in vehicle-injected MZnpc2^m/m embryos. Two representative images of 2HPβCD-injected MZnpc2^m/m embryos are shown with their corresponding corrected phenotype. Phenotype composite scores of vehicle and 2HPβCD-treated MZnpc2^m/m embryos are shown (Score 3: improved head/brain appearance, restored red blood cell circulation, and less-curved body axis; Score 1: less-curved body axis). Individuals exhibiting all four defects (the lack of otoliths, abnormal head/brain development, absent circulating red blood cells and a curved/twisted body axis) received a score of 0, and individuals with none of the four defects received a score 4. Experiments were repeated three times. All Mnpc2^+/m embryos injected with either vehicle or 2HPβCD displayed normal phenotype, and all MZnpc2^m/m injected with vehicle displayed abnormal phenotypes including all defects described above. Scale bars: 500 μm. (B) Magnifications of the images in A showed that although 2HPβCD was able to partially rescue some defects in MZnpc2^m/m embryos, otoliths were still not seen in 2HPβCD-treated MZnpc2^m/m embryos despite some of embryos displaying more organized otic vesicles (black arrowheads). Some 2HPβCD-treated MZnpc2^m/m embryos displayed improved head/brain appearance (red arrowheads) as they did not exhibit the enlarged hollow compartment (white arrowheads) found in vehicle-treated MZnpc2^m/m embryos. Red blood cells were found in the pericardial region of some 2HPβCD-injected MZnpc2^m/m embryos (white asterisks) but not in vehicle-injected MZnpc2^m/m embryos and some more severe 2HPβCD-injected MZnpc2^m/m embryos (black asterisks), consistent with restored circulation. Scale bars: 200 μm. (C) 2HPβCD-injected 48 hpf MZnpc2^m/m embryos displayed improved phenotype in head/brain structure, body axis and circulation but the absent otolith phenotype was still largely unchanged. n=76 (vehicle-injected MZnpc2^m/m embryos) and n=80 (2HPβCD-injected MZnpc2^m/m embryos) (n=3 experiments. ****P<0.0001 (χ2, Fisher's exact test). ns, not significant. (D) Distribution of phenotype composite scores of vehicle and 2HPβCD-treated MZnpc2^m/m embryos. Most 2HPβCD-treated MZnpc2^m/m embryos showed improvement in one to three individual findings.