Fujimi TJ et al. (2006), Xenopus Zic4: conservation and diversification ...

XB-ART-95

Dev Dyn 2006 Dec 01;23512:3379-86.

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Xenopus Zic4: conservation and diversification of expression profiles and protein function among the Xenopus Zic family.

Fujimi TJ , Mikoshiba K , Aruga J .

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We compared the expression and function of Xenopus Zic4 with those of the other four Xenopus laevis Zic family members (Zic1, Zic2, Zic3, and Zic5). Zic4 expression was detected mainly in the neural plate border, dorsal neural tube, and somites, and was similar to that of Zic1, which is adjacent to Zic4 on the same chromosome. Injection of wild-type or mutant Zic4 RNA caused the induction of neural crest marker gene expression, hyperplastic neural tissue, and ectopic pigment cell formation, indicating that Zic4 can induce neural and neural crest tissue, as can other Xenopus Zic genes. Deletion analysis showed that the zinc-finger domain is critical for many Zic4 functions, but the C-terminal region is differently involved in induction of two neural crest markers, Slug and Sox10. The protein function as determined by the animal cap explant assay was similar to that of Zic5, but different from those of Zic1, Zic2, and Zic3, suggesting that Xenopus Zic genes can be divided into two groups based on function. These results indicate that the five Xenopus Zic genes cooperatively regulate both neural and neural crest development, despite significantly diverged expression profiles and functions.

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Species referenced: Xenopus laevis
Genes referenced: dct myc snai2 sox10 zic1 zic2 zic3 zic4 zic5

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	Figure 2. Expression profiles of Xenopus laevis Zic genes during embryonic development. Whole-mount in situ hybridization (WMISH) analyses with digoxigenin-labeled probes were performed to determine the spatial expression patterns of each Zic gene during development. Uppercase labels represent embryos stained with a Zic probe (A, Zic1; B, Zic2; C, Zic3; D, Zic4; E, Zic5). Lowercase labels indicate embryo stages (a, stage 11.5; b, 15; c, 19; d and e, 23; f, 26; g-i, 33). In b, arrows indicate the prospective region of hyoid crest and branchial crest, which have high Zic1 and Zic4 expression, and arrowheads show the anterior neural plate border region, with high levels of Zic5. e is the frontal view of the embryo shown in d. Arrowheads in d and e indicate the expression in eye (red), somite (green), lateral mesoderm (blue), and olfactory placode (white). h is a higher-magnification view of the embryo shown in g. i shows the transverse section of the WMISH sample of stage 33. The position of the section in i is indicated by a thin line in g. nt, neural tube; nc, notochord; ey, eye.

	zic1 (Zic family member 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 15, dorsal view, anterior up
	zic1 (zic family member 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 19, dorsal view, anterior up.
	zic1 (Zic family member 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.

	zic2 (Zic family member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior up.
	zic2 (Zic family member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
	zic2 (Zic family member 2) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
	zic4 (Zic family member 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior up
	zic4 (Zic family member 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.

	zic5 (Zic family member 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, anterio-dorsal view, dorsal up.
	zic5 (Zic family member 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up. I

	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.