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Figure 1 c-myc and n-myc are expressed sequentially during early development and in the CMZ. A: c-myc expression from stage 11/12-19. E: n-myc expression from Stage 139. I,J: c-myc expression in the CMZ as visualized on wholemount and tissue sections of stage 41/42 embryos. M, N: n-myc expression in the CMZ on wholemount and tissue sections of Stage 41/42 embryos. K: Double in situ hybridization in stage 32 embryos with c-myc visualized using BM Purple and n-myc visualized using ELF 97. O: Double in situ hybridization in the post-embryonic retina with c-myc visualized using ELF 97 and n-myc visualized using BM Purple. L, P: c-myc and n-myc double in situ hybridization on tissue sections with c-myc visualized using BM Purple and n-myc visualized using Fast Red. Fluorescent images were modified in single channels. Dashed rectangles in the upper panels encompass the region of the eye field. Dashed areas in the lower panels encompass the CMZ regionsn wholemount views (L,K,M,O), this is the annulus created by the two dotted circles. We have not identified the structures outside the eye regions that label with c-myc or n-myc. Bars: A-H 1 mm; I, M 100 lm; J, N 100 lm; K 250 lm; O, L, P 100 lm. See also Figure S1.
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Figure 2 c-myc and n-myc expression as compared with other previously-mapped genes in the CMZ. A: c-myc expression overlaps with that of pax6 at the peripheral CMZ, although some cells located in the RPE express pax6 but not c-myc. B, C: c-myc is expressed more peripherally than Xash1 and Xash3b. Note also c-myc staining in the lens epithelium. E: The second domain of c-myc staining overlaps with Xath5. E: n-myc is expressed more centrally in the CMZ, and some peripheral cells exhibiting neuroepithelial morphology express pax6 but not n-myc. F, G: n-myc expression initiates more peripherally to Xash1 and Xash3b, but also overlaps with them at its more central border. H: n-myc expression terminates before the start of Xath5 expression. I: n-myc expression initiates more peripherally to cyclin D1, cyclin A2, and cdc2. L: n-myc expression terminates before the start of p27Xic1 expression. Bar: A 100 lm.
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Figure 3 Short-term and long-term nucleotide analog labeling show that c-myc+/n-myc␣ cells are slower cycling and long-term label retaining. A: Cells labeled with EdU for 30 min were stained for c-myc and n-myc expression. I: Cells pulsed for three days with EdU (bath-applica- tion) and chased for 14 days without EdU were stained for c-myc and n-myc expression. D,H: DAPI stained nuclei were counted for their EdU signal and rated for their c-myc versus n-myc gene expression. 44.1% 6 4.8% c-myc+/n-myc␣ and 77.5% 6 4.6% n-myc+ cells have incorporated EdU within 30 min. Eleven retinas were counted in total. O: The four cells immediately central to the start of c-myc expression were scored for EdU+ (labeled 1 in I in this example). P: The most peripheral cells were most likely to retain EdU. Fourteen retinas were counted. N: The two cells immediately peripheral to the start of n-myc (1 and 2 in L in this example) and the two cells immediately central to the start of n-myc (3 and 4 in L) expression were scored for EdU retention. Q: Cells peripheral to the start of n-myc expression were much more likely to retain EdU. Eleven retinas were counted in total. Values are given as mean 6 95% confidence intervals. Fluorescent images were modified in single channels. (**) p <0.01. (***)p <0.001. Bars: A 100 lm; I 50 lm. See also Figure S2.
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Figure 4 c-myc is required earlier during eye development compared to n-myc and it is neces- sary for normal n-myc expression. A: c-myc but not n-myc morpholino reduces early pax6 and Xrx1 expression. G: c-myc and n-myc morpholinos both interfere with cyclin D1, cyclin A2, and cdc2 expression. P: c-myc and n-myc morpholinos both interfere with Xash1, Xash3b, and Xath5 expression. A14: n-myc MO does not affect c-myc expression during early retinogenesis. A58: n-myc expression is reduced or abolished in c-myc morpholino injected retinas when examined from Stage 18 onwards. Dotted rectangles indicate the MO containing side of the embryo into which the MO was injected. Bar: A 1 mm. P 1 mm. A18 1 mm. See also Figure S3 and Table S1.
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Figure 5 c-myc expression is downregulated more quickly by growth factor inhibitors compared with n-myc. A, B: Application of SU5402 (FGFR1 inhibitor), AG1478 (EGFR inhibitor), and picro- podophyllin (IGF-1R inhibitor) for 6 h abolishes the first domain of c-myc staining, while leaving the second domain intact. E, F: Application of the three inhibitors for 6 h does not obviously affect n-myc expression. C, D: After 24 h, the CMZ is clearly reduced in size, but the second domain of c-myc expression is still present (G, H) n-myc expression is also abolished after 24 h. I: EdU was bath applied from 6 to 18 h after initiation of growth factor inhibitor treatment. After 18 h, n-myc expression is only retained in the ventral retina, but cells in the CMZ have taken up EdU during this period. Bar: 100 lm. See also Figure S4.
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Figure 6 myc family members exhibit sequential expression patterns in the zebrafish retina. AC: myc family members exhibit distinct expression patterns in four-day-old zebrafish CMZ, with myca being the most peripherally expressed, mycn being the next most peripherally expressed, and mycl1a being most centrally expressed. The peripheral tip of the CMZ is indicated by a white asterix in the neighboring pigmented epithelium.
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Figure 7. Representation of c-myc+ and n-myc+ cell proliferation in the lower vertebrate CMZ. In the Xenopus ret- ina, growth-factor-dependent c-myc+ cells residing at the furthest periphery of the CMZ cycle slowly and undergo self-renewal while giving rise to n-myc+ progenitors that cycle more quickly and undergo more limited rounds of divisions to produce differentiated cells of the central retina.
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Supplementary Figure 4. Growth factor signalling is redundant in the CMZ (A-H) Application of individual and combinations of growth factor inhibitors for 24 hours affects c-myc expression to different degrees, with the triple inhibitor treatment generating the most dramatic phenotype. (I-P) n-myc expression is also affected differently by different combinations of inhibitors after 24-hour-treatment. Bar: 100μm.
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Supplementary Figure 3. c-myc is necessary for eye formation whereas n-myc is necessary for neuronal differentiation (A, B, D, E) Compared to control MO injected embryos, c-myc MO injected embryos often exhibit complete failure in eye formation. (C, F) n-myc MO injected embryos often exhibit reduced eye size but never lose their eyes completely (3 separate experiments of approximately 20 embryos each for c-myc and n-myc MOs). (G-L) Ganglion cell differentiation is inhibited in n-myc MO injected cells as shown by the absence of hermes staining, especially when compared to control MO injected cells. Fluorescent images were modified in single channels. Bar: A-C 1mm, D-F 100μm, G-L 100μm.
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Supplementary Figure 2. Some n-myc+ cells are weakly positive for EdU signal after long-term labelling (A-D) Following long-term labelling, some retinal sections contain n-myc+ cells that contain low levels of EdU. However, signals in these cells are often weaker than in their n-myc- neighbours, and the signal appears to decrease further as cells are positioned closer to the central retina. The more peripheral, presumably c-myc+ cells that retain higher levels of EdU are outlined. Bar: A-D 100μm.
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Supplementary Figure 1. c-myc and n-myc are expressed in many of the same tissues during early, mid- and late neurogenesis (A-F) c-myc and n-myc are both expressed in various tissues, especially those of the central nervous system, during development. b: brain, op: optic vesicle, mnc: migrating neural crest, tel: telencephalon, di/mi: diencephalon/midbrain, mhb: midbrain/hindbrain boundary, rh: rhombencephalon, e: eye, pa: pharyngeal arches, ov: otic vesicle, s: somites, fb: forebrain, mi: midbrain. Bars: A-B 1 mm, C-D 1mm, E-F 100μm.
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