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Figure 3. Effects of Zic4 truncation. A: Schema of the structures of Zic4 (MT-Z4) and C- or N-terminal truncated mutants (MT-Z4δC and MT-Z4δN). All constructs were designed with 6 Myc tags at the N-terminus. Gray boxes illustrate ZF. B: Top panels are WMISH analyses of MT-Z4 or truncated-mutant-injected embryos (St. 17) with antisense digoxigenin-labeled Slug probe. RNA (200 pg) was injected in the right side of the embryo (inje.) with EGFP RNA (400 pg). Injected constructs are shown on the top of each panel. EGFP indicates that the control embryos were injected with EGFP only. The left side of the embryo is the uninjected control. Bottom panels are sections of the head region of each injected embryo (St. 30 to 32). Injected constructs and their amounts are the same as in the top panels. The typical sections were stained by hematoxylin and eosin. Arrowheads indicate the abnormality of the injected embryo (white, hyperplasia of neural tube; black, eye formation disturbance; red, neural tube closure defect). nt, neural tube; nc, notochord; ey, eye. C: Top (injected side) and middle (control side) panels are embryos (St. 40 to 42) in which MT-Z4 or truncated mutants were injected. We used less RNA (MT-Z4, 150 pg; MT-Z4δC, 150 pg; MT-Z4δN, 100 pg) than in B to increase the survival rate. The eye abnormalities were less frequently observed (MT-Z4, 5/13; MT-Z4δN, 5/9) at this dose, and normal ones are presented. Yellow arrowheads indicate ectopic melanocytes induced by MT-Z4 RNA injection. We also noticed an increase in small brown pigment cells (Fig. 4C, red arrowheads) in almost all cases (MT-Z4, 10/13; MT-Z4δC, 5/9; and MT-Z4δN, 9/9). The small cells could be seen at stages before melanocytes were formed, but the cells were not stained by whole-mount in situ hybridization using Dct (DOPA chrome tautomerase, an enzyme related to the eumelanin synthesis) probe (data not shown). Bottom panels are WMISH analyses of MT-Z4 or truncated-mutant-injected embryos (St. 17) with antisense digoxigen-labeled Sox10 probe. The embryos shown in the bottom panels are siblings of embryos shown in the top panels, used in the same experiments. RNA (MT-Z4 and MT-Z4δC, 150 pg; MT-Z4δN, 100 pg) was injected in the right side of embryos with EGFP RNA (400 pg). Injected constructs are shown at the top of each panel. EGFP means that the control embryos were injected only with EGFP. The left side of the embryo is the uninjected control. |
