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Fig. S1. Additional defects in the kof and rgd mutant embryos in the developing central nervous system. (A,B) Ventral views of the head region of 72-hpf wild-type (A) and kof (B) embryos. Anterior, top. The retinal axons were stained with the anti-acetylated α-tubulin antibody. In the wild-type embryos, the optic chiasm was formed in the ventral midline (A, arrowhead). In the kof embryos, the position of the optic chiasm was randomly shifted toward the left or right near the midline (B, arrowhead). (C,D) Lateral views of the trunk region of 72-hpf wild-type (C) and the rgd (D) embryos. Dorsal, top; anterior, left. The trunk muscles were visualised with rhodamine-phalloidin. In the wild-type embryos, Isl1-GFP-positive spinal motor axons extended to the dorsal part of the trunk muscles (C, arrowheads). In the rgd embryos, Isl1-GFP-positive spinal motor axons grew ventrally and changed their growth pathway posteriorly after reaching the level of the myoseptum (D, arrowheads). (E,F) Transverse views of the hindbrain at r6 of the 72-hpf wild-type (E) and rgd (F) embryos. Dorsal, top. In the wild-type embryos, there were no Isl1-GFP-positive cells in the lateral part of the hindbrain (E). In the rgd embryos, Isl1-GFP-positive cells were ectopically observed in the lateral part of the hindbrain (F, arrowhead).
Fig. S2. Abnormal axonal pathfinding of putative motoneurons in the spinal cord of vmc Isl1-GFP embryos at 36 hpf. (A,B) Dorsal, top; anterior, left. The axons of these neurons were visualised by stochastic expression of Kaede using the HuC-Kaede construct with direct fluorescent microscopy. Their axons grew anteriorly within the spinal cord, then extended out of the spinal cord through abnormal exit points (arrows).
Fig. S3. Sequential time-lapse 3D imaging of the multiple mutant embryos with a Tanaka-Okamoto (TO) chamber. (A) Embryo embedding procedure. (1) The TO chamber was made from a plexiglass plate (30×20×5 mm) with a central hole (15 mm in diameter), and had two indentations at opposite ends of the hole to prevent rotation of the agarose. The TO chamber was attached to the glass slide using silicon grease. (2) The hole of the TO chamber was filled with 0.6% low melting point agarose (Agarose-LM Sieve; Nakalai) in the 10 mM HEPES-buffered embryo medium (Westerfield, 2000) and the surface of the molten agarose was then covered with a cover slip. A small plastic cube (2×2×1 mm) was attached at the centre of the cover slip to make a cavity for the embryo in the agarose. (3) When the agarose had set, the cover slip was removed. The agarose in the perimeter region of the hole was scraped out of the TO chamber using a surgical knife, forming a moat. Then, the space was filled with 10 mM HEPES-buffered embryo medium containing 0.01% antibiotic (cat. no. 15240, Gibco) and anaesthetic (0.01% tricaine, Nakalai). (4) The anesthetised embryo was placed in the cavity and embedded in the same 0.6% low melting point agarose. (5) The TO chamber was covered with the cover slip and sealed with silicon grease. (B) The 6-well TO chamber (65×45×5 mm) for sequential time-lapse recording. (C) The laser-scanning microscope (LSM510; Zeiss) equipped with an electric motor-driven stage (MCU28; Zeiss). The laser-scanning microscope was used in a darkroom with the ambient temperature regulated at 28°C during time-lapse recording.
Movie 1. Time-lapse images (lateral views) of 36-48 hpf wild-type embryos. Composite stacks of serial optical images were made every 15 minutes at 36-48 hpf; dorsal is top, anterior is left.
Movie 2. Time-lapse images (lateral views) of 36-48 hpf vmc embryos. Composite stacks of serial optical images were made every 15 minutes at 36-48 hpf; dorsal is top, anterior is left.
Movie 3. Time-lapse images (lateral views) of 60-72 hpf wild-type embryos. Composite stacks of serial optical images were made every 15 minutes at 60-72 hpf; dorsal is top, anterior is left.
Movie 4. Time-lapse images (lateral views) of 60-72 hpf vmc embryos. Composite stacks of serial optical images were made every 15 minutes at 60-72 hpf; dorsal is top, anterior is left.
Movie 5. Time-lapse images (ventral views) of 52-72 hpf wild-type embryos. Composite stacks of serial optical images were made every 15 minutes at 52-72 hpf; anterior is top.
Movie 6. Time-lapse images (ventral views) of 52-72 hpf vmc embryos. Composite stacks of serial optical images were made every 15 minutes at 52-72 hpf; anterior is top.
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