Zebrafish assessment
Live embryo phenotypes were analyzed using a Leica MZ12.5 at 24 and
48hpf. The pattern of rescue was determined by the somite shape and eye
color in the rescue experiment. We determined the phenotypic difference
in rescue experiments based on specific abnormal features of smoK/O model: U -type tail and less than half area of retina
pigmentation in the eye region. Embryo phenotypes with partial rescue of
eye shown over half area of retina pigmentation. Embryo phenotypes with
partial rescue of somite shown not curled U -type tail but rather
an unstraight tail. Live embryo images of lateral or dorso-ventral
views, images of gene expression after in situ hybridization and
images for eye area or somite analysis were acquired on a ZEISS AxioCam
HRC Camera and AxioVision SE64 software after embedded embryos in 3%
Methylcellulose (Sigma): Live embryo and eye area or somite analysis; in
Glycerol (Invitrogen): in situ hybridization. Somite angle
measurements were taken at the midpoint between the proximal hindgut and
the anus at 24hpf embryos using ImageJ software. For eye area
measurements, lateral images at 24hpf embryos were quantified using
ImageJ software. For eye and somite analysis, total 45 embryos/group: 15
embryos/group in each triplicate injection, were analyzed.