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Figure 1. Characterization of mych and comparison of vertebrates Myc proteins.A. Radiation hybrid mapping determined the location of mych on chromosome 6. B. Nuclear localization of Flag-Mych protein (green) in NIH3T3 cells; nuclei were stained with DAPI (blue). C, D. Evolutionary comparisons. H, human; M, mouse; X, Xenopus; Z, zebrafish. C. Phylogenic tree of Myc family proteins. D. Amino acid sequence alignments of the C-terminal regions containing highly conserved basic helix-loop-helix domains. Amino acid identities are shown in color, with the basic, helix-loop-helix, and leucine-zipper domains marked in green, red, and blue, respectively. The GenBank Accession Number for mych is EU232118.
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Figure 2. Detection of mych transcripts by in situ hybridization.A. Dorsal view of 8-cell stage embryo. B. Double staining of mych (blue) and gsc (red) at the shield stage; arrowhead points to the shield. C-C'. Lateral (C) and anterior dorsal (C') views of 80% epiboly stage. Red asterisk points to the anterior-dorsal area shown in C'. D–I. 3-somite stage embryos. D. Dynamic expression of mych in the anterior brain region. E-E'. Double staining with mab21l2 (red) as a marker for eye and midbrain. F-F'. Double staining with dmbx1a (red) as marker for eye field and midbrain; red arrowhead indicates eye field. G,G'. Co-expression with ctsl1b (red), marking the prechordal plate. H-H'. Dorsal view of HuC-positive neuronal cells (red) and mych staining (blue) in wild type (H) and the mibta52b mutant (H'). I. Rhombomeres 3 and 5 are marked by egr2b (red), while mych stains the anterior part of rhombomere 4. J–K. Dorsal views of 10-somite stage embryo showing mych expression in the eye and midbrain (J) and in trunk somites (K). L. Lateral view of mych expression in the brain and pharyngeal system at 24hpf; mandibular (m), hyoid (h), and branchial (b) arches are indicated. M. Expression of mych in the eye at the 36hpf. Arrowhead points to presumptive photoreceptor cell layer. N. mych expression in the heart and intestine (int; magnified in O) at 72hpf. P–Q. Co-expression with foxd3 (red) at the 4-somite stage. The yellow open square area in P is shown as a section in Q. R–S. Pharyngeal arch marker dlx2a (red in S) is co-expressed with mych at 32hpf. b, branchial arch; e, eye; h, hyoid arch; ht, heart; int, intestine; le, lens; m, mandibular arch; mi, midbrain; ov, otic vesicle; pp, prechordal plate; r, rhombomere; s, somite.
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Figure 3. Mych morphant phenotypes.A–B. Group image of 3-somite stages. Mych MO-injected embryos (B) have reduced anterior and caudal regions compared to control MO-injected embryos (A). C–F. Lateral view of 24hpf MO-injected embryos; (C,D) Control, (E) mych UTR MO, (F) mych SP MO. G–H, J–K. Lateral (G–H) and ventral (J–K) views of control (G,J) and mych UTR MO injected embryos (H,K) at 72hpf. Arrowhead in G points to the eye. Methyl green stained sections of the eye are shown as insets (J',K').Yellow arrowhead in J' indicates ganglion cell layer, and red arrow indicates photoreceptor cell layer of control embryos; mych UTR MO-injected embryos show no retinal layering. F,I. Lateral view of 24hpf mych SP MO injected embryo (F), and embryo rescued by coinjection of mych mRNA (I). L,O. Embryo injected with mych UTR MO (2ng) plus mych SP MO (2.5ng) at 24hpf (L), and rescued embryo after coinjection of mych mRNA (O). M–N. The phenotype of mych UTR MO-injected embryos (M) was rescued by mych mRNA (N), as seen at 24hpf. h, heart.
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Figure 4. Mych MO affects multiple regions at the 3-somite stage.A–P. Lateral views (A–H) and dorsal views (I–P) of control MO-injected (A,C,E,G,I,K,M) and mych MO-injected embryos (B,D,F,H,J,L,N). Mab21l2 (A–B') as eye anlage and midbrain marker; dmbx1a (C–D') as eye territory and tectum marker; barhl2 (E,F) as diencephalon marker; rx3 as eye field marker. (I–J) double label with pax2a (red) and egr2b (blue). (K–P). Embryos were examined using four probes, ctsl1b (red), dlx3b (blue), ntl (blue), and pcdh8 (red). (O–P) mych MO-induced defects were rescued by co-injection with mych mRNA. lnp, lateral neural plate; mi, midbrain; n, notochord; pp, prechordal plate; r, rhombomere; psm, pre somitic mesoderm.
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Figure 5. Reduced expression of early anterior brain markers after mych MO injection.A–J. Dorsal views (A–J) and lateral views (K–L) of control MO-injected (A,C,E,G,I,K) and mych UTR MO-injected embryos (B,D,F,H,J,L) at the 80% epiboly stage. The probes are indicated.
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Figure 6. mych MO impairs early neural crest induction.A–F. Dorsal view of foxd3 expression at 4-somite stage embryos. Embryos injected with 4ng of mych UTR MO (B), 5ng of mych SP MO (C), or 2ng of mych UTR MO (B) plus 2.5ng of mych SP MO (C) showed reduced foxd3 expression. This phenotype was rescued by mych mRNA coinjection (D,F). G–L. Dorsal view of control MO-injected (G,I,K) and mych UTR MO injected embryos (H,J,L) hybridized to snail1a (G,H), sox9b (I,J), and sox10 (K,L). M–N. Sections of the foxd3 expression region of cont MO (M,M') and mych UTR MO (N,N') injected embryos. Red asterisks in A and B indicate the location of transverse sections, and asterisks in M and N indicate the regions magnified in M', N'. h; hindbrain neural crest, m; midbrain neural crest, tnc; trunk neural crest.
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Figure 7. Multiple defects in pharyngeal arch development caused by mych MO.Control MO (A,C,E,G,I,K) and mych UTR MO-injected embryos (B,D,F,H,J,L). A–B. Lateral view of dlx2a expression in mych UTR MO-injected embryos at 26hpf (B). C–D. Ventral view of expression of hand2 at 36hpf. E–F. Ventral view of tbx1 expression at 45hpf. G–H. Lateral view of fli1-eGFP transgenic line at 40hpf. I–L. Lateral (I,J) and ventral (K,L) views of Alcian blue stained day 5 control (I,K) and mych UTR MO-injected embryos (J,L). ch, ceratohyal, b, branchial arch; e, ethmoid plate; g, gill arches; h, hyoid arch; hs, hyosymplectic; ht, heart; m, mandibular arch; mc, Meckel's cartilage; me, mesoderm; pec, pectoral fin; pq, palatoquadrate.
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Figure 8. Mych depletion results in apoptosis in the early neural plate.A–F'. Detection of cell death by TUNEL assay. Lateral views (D,E,F) and dorsal views (A,B,C,D',E',F') of control MO (A,D,D'), mych UTR MO (B,E,E'), and rescued embryos that received mych UTR MO and mRNA (C,F,F') at the bud (A–C) and 3-somite stage (D–F'). Arrows in B point out TUNEL positive cells at the lateral edge of the neural plate. An arrowhead in B indicates TUNEL positive cells in the neural plate. Bracket in D and D' indicates the anterior neural plate region in which TUNEL-positive cells were counted. The results are shown in G. Average numbers of positive cells per embryo were obtained by counting 20 embryos in each group. For both comparisons p<0.01: Cont MO vs. mych MO: p = 5.27e-14; mych MO vs. rescue: p = 4.87e-17.
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