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Files in this Data Supplement:
Movie 1. AC neurites are added progressively to the forming IPL. In vivo time-lapse recording of a line 220 eye, showing progressive addition of GFP+ neurites to the IPL. Each image frame is a maximum projection of the confocal reconstruction of 19 mm sections through the retina. Images were acquired every hour from 50-76 hpf.
Movie 2. Sublamination of the GFP projection to the IPL. In vivo time-lapse recording showing sublamination in the IPL of a line 220 retina. Each image is the maximum projection of optical planes encompassing a total depth of 3 mm of retina. Images were acquired every hour between 61 and 74 hpf.
Movie 3. Formation and sublamination of the IPL imaged in single embryos. Time-lapse movies showing GFP-expressing amacrine cells of line 220 in wild-type (A) and lak mutants (B). Age-matched siblings were followed in parallel from the early stages of IPL innervation through sublamination. These are from a different experiment than the fish depicted in Fig. 3. In each movie, each image represents image planes encompassing 4-5 mm depth of retina, acquired every hour for a total of 39 hours, beginning around 45 hpf. The wild-type IPL (S3A) shows orderly innervation by ACs. Note the vigorous movements of AC somata during the period of sublamination. In lak mutants (S3B), IPL formation is characterized by the dynamic clustering and de-clustering of AC somata and neurites. There is also a failure of sublamination during the recording period.
Movie 4. Defects in the lak mutant IPL at 70 hpf. Digital rotation of an image stack (30 mm total depth) encompassing the disorganized region of the retina depicted in Fig. 4E. Rotation of the image stack shows irregular organization of the GFP+ amacrine cell neurites in this particularly badly perturbed region. B scrolls through this image stack step-wise. (C-E) Rotations of image stacks (total 30 mm, 1 mm steps) of the retina of a wild type (C) and two lak mutants (D,E) imaged in vivo at around 70 hpf. The three animals were siblings and were imaged concurrently. Although the GFP+ processes form a smooth plexus in wild type (C), gaps and neurite clusters are characteristic of the lak mutant IPL at this age (D,E).
Movie 5. AC neurites grow towards each other. Time-lapse movie of GFP+ ACs of line 243 in the lak background showing AC-AC neuritic interaction. Each frame represents image planes encompassing 20 mm depth of retina, acquired every 10 minutes for a total of 3 hours, beginning at 69 hpf. The imaged cells are within a severely disorganized region of lak retina, with one cell positioned at the ILM border. Note that individual GFP+ cells can be resolved in lak mutants because of laminar disorganization. AC neurites did not grow towards landmarks such as the ILM or the normal position of the IPL; rather, the neurites of individual cells were seen to approach each other and form a stable neuritic plexus.
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