|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
| ||||||||||||||||||||
Files in this Data Supplement:
Fig. S1. Tissue patterning and proliferation are normal in mutant retinas. (A) The proliferation rate of retinal progenitors (RPCs; a,b), as well as the specification of the lens (c,d) are normal in opo mutant retinas, as assessed by anti phospho-Histone3 staining and α-A-crystallin expression, respectively. (B) The ventral retina is correctly specified in opo mutants, as assessed by the expression of known ventral markers, such as vax2 (a,b) and pax2 (c,d), detected both in wild-type and mutants by in situ hybridization (ISH).
Fig. S2. Morphogenetic defects observed in opo mutant tissues. (A) Neural crest cells are correctly specified in mutants as assessed by snail1b expression, but they fail to migrate/delaminate and remain in the dorsal neural tube, as indicated by the red arrow (a,e). Tunnel staining reveals that neural crest precursors die by apoptosis in opo mutants (b,f). Subsequently, most neural crest derivatives are greatly reduced in opo mutants. These include both the melanocytes (c,g), here labelled by the transgenic line Tyr:GFP (3 kb of the medaka tyrosinase promoter fused to GFP), and craniofacial skeletal elements (d,h) detected by Alcian Blue staining. (B) Brain sutures do not close in the mutant (arrow in b) despite the correct specification of the brain territories, here revealed by the expression of the marker pax6 (a,b). (C) tbx5 expression at stage 26 reveals that the fin buds are correctly specified in opo mutants (a,b). However, later pectoral fins do not progress during development and appear greatly reduced at later stages (c-f).
Fig. S3. opo locus in vertebrates. (A) Vista alignment shows opo locus conservation in several vertebrate genomes. Medaka exons are indicated as a reference in purple. Non-coding conserved regions are indicated in pink. Note that the Danio rerio sequence is incomplete in this locus. (B) Schematic shows the membrane topology of the protein, as predicted by Phobious and Sousi algorithms. The positions of the conserved exons in the protein are indicated in red. Red dotted lines indicate the portion of the protein absent from the short isoform.
Fig. S4. Ocular phenotype in zebrafish opo morphants. (A) The retinal phenotype (at 46 hpf) of zebrafish embryos injected with morpholinos (300 µM) against the exon 3 splice donor of the opo ortholog resembles the medaka mutant phenotype. (B) RT-PCR shows the amplification of zebrafish exons 3 and 4 (control, 120-bp band, blue arrow) and the band shift induced by the injection of the morpholino Mo-3d (Mo-3d, 440-bp band, red arrow). The morpholino-induced bands correspond to the inclusion of the 320-bp intron 3 into the mature transcript, as confirmed by sequencing. See quantitative data in Table S4 in the supplementary material.
Fig. S5. opo expression pattern and protein localization. (A-D) opo expression in the retina of stage 26 (A,B) and stage 23 (C) medaka embryos, and stage HH13 chick embryos (D) by ISH. Chicken transcripts are also localized (arrows) to basal side of the retina (nr). (E-G). opo was detected by ISH in all tissues affected by the mutation, both in wild-type (E,G) and mutant (F) embryos. Expression was detected in migrating neural crest cells (nc; arrows, E,F) and the apical ectodermal ridge (aer, arrow in G). (H-K) Imunostaining reveals the basal localization of the Opo protein in wild-type (H,I), but not in opo mutant (J,K), retinae. Scale bars: 50 µm in E-G; 10 µm in H-K.
Fig. S6. Basal feet mophology and cellular organization in opo mutant retinae. (A-B′) The morphology of neuroblast feet is revealed by membrane rx2::mYfp and DAPI nuclear staining in stage 25 sections from wild-type (A,A′) and mutant (B,B′) retinae. The abnormal morphology of the basal feet in opo mutants is highlighted in red (A′,B′). (C) Feet widths were measured and quantitative data are provided. Note: 10 pixels equal approximately 3 µm. (D,E) Mosaic retinae generated by homotypic transplantation of rx2::eGFP cells into unlabelled hosts show that both wild-type (D) and opo mutant (E) neuroblasts extend across the entire neuroepithelium. zne, zone of nuclear exclusion. Scale bar: 20 µm.
Fig. S7. Basal lamina integrity and vesicle accumulation in opo mutant neuroblasts. (A) Laminin A staining of stage 23 wild-type (a) and opo mutant (b) embryos shows that basal lamina (bl) deposition is normal in the mutants. Scale bar: 20 µm. (B) EM analysis of stage 23 wild-type (a) and opo mutant (b) retinas. Arrows indicate the basal lamina. Scale bar: 500 nm.
Fig. S8. Time-lapse analysis of neuroblast basal surface dynamics. (A-O) Time series taken from Movies 3 and 4 showing wild-type (0 to 8 minutes, A-E; 20 to 28 minutes, F-J) and mutant (0 to 8 minutes K-O) retinas labeled with rx2::mYfp. Extension of short filopodia (red arrows) and apical and basal surfaces (yellow and white dotted lines, respectively) are depicted. (P,Q) Superposition of apical and basal (color-coded) outlines from a wild-type time series (P corresponds to the projection in A-E, and Q to the projection in F-J) shows basal surface dynamics. Vertical displacements of the basal surface are indicted by red numbers in P,Q. (R) Superposition of apical and basal (color-coded) outlines from an opo mutant time series shows no apparent displacement of the surface (corresponding to the projection in K-O). Scale bar: 15 µm. See also Movies 3 and 4 in the supplementary material.
Movies 1, 2. SPIM analysis of optic cup folding in wild-type and opo mutant embryos. Medaka rx2::eGFP wild-type (Movie 1) and mutant (Movie 2) retinas were recorded laterally through 40 and 33 hours, respectively (stages 22 to 27), using SPIM microscopy. At each time point (time resolution 10 minutes), several central planes, spanning approximately 9 µm, were integrated by maximum projection. See also Fig. 1G,H.
Movies 3, 4. Analysis of wild-type and opo cell shape dynamics during optic cup folding. Medaka rx2::mYFP wild-type (Movie 3) and mutant (Movie 4) retinas were recorded laterally through 200 minutes during optic cup folding (stage 23) by confocal time-lapse microscopy. At each time point (time resolution 2 minutes), three optical sections spanning approximately 3 µm were captured and integrated by maximum projection. See also Fig. S8 in the supplementary material.
| ||||||||||||||||||||
