Nakata K et al. (1998), Xenopus Zic family and its role in neural and n...

XB-ART-14263

Mech Dev 1998 Jul 01;751-2:43-51.

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Xenopus Zic family and its role in neural and neural crest development.

Nakata K , Nagai T , Aruga J , Mikoshiba K .

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We characterized two new members of the Zic family, Xenopus Zic1 and Zic2. They are very similar to mouse Zic1 and Zic2 in the protein coding region including the zinc finger domain. In early gastrula, Zic1 expression was restricted to the prospective neural plate region whereas Zic2 was expressed widely in the ectoderm. We observed enhanced neural and neural crest-derived tissue formation in the Zic1 or Zic2 overexpressed embryos and neural and neural crest marker induction in the Zic1 or Zic2 overexpressed animal cap explants. Our findings suggest that Zic1 and Zic2 have essentially the same properties as Zic3 and that the Xenopus Zic family may act cooperatively in the initial phase of neural and neural crest development.

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Species referenced: Xenopus laevis
Genes referenced: en2 h4c4 hoxb9 krt12.4 myc ncam1 snai2 zic1 zic2 zic3
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	Fig. 2. Temporal expression profiles of Xenopus Zic1, Zic2 and Zic3 during Xenopus development. RNA was extracted from embryos at the indicated stage of development and Zic1, Zic2 and Zic3 mRNA expression levels were measured by RT-PCR. The ubiquitous marker Histone H4 served as a control.
	Fig. 3. Spatial expression patterns of Zic1 and Zic2 in Xenopus embryos. Series of embryos were hybridized with digoxigenin-labeled antisense Zic1 (A–L) and Zic2 (M–X) RNA. (A,M) blastula stage (stage 9). In the blastula stage, Zic1 and Zic2 were expressed throughout the ectoderm (A, animal side; V, vegetal side). (B,C,N,O) mid-gastrula stage (stage 10.5). (C) The expression of Zic1 in the prospective neural plate was confirmed in a cross-section of the same embryo as in B (arrowhead). However, Zic2 was expressed in a broader region of the ectoderm (N,O). (D,P) Stage 11–12. (E,Q) Stage 12.5–13.5. (F,R) Stage 14–15. White and black arrowheads indicate the lateral edge of the neural plate and the neural crest, respectively. Arrows indicate the neural plate border region of the prospective rhombencephalon. (G,H,S,T) Stage 20. The black arrowhead, white arrowhead and arrow indicate the telencephalon, diencephalon and mesencephalon, respectively. (I–L,U–X) Stage 30. Transverse section through the head (J,V) and trunk region (L,X) of the same embryo as in (I,U). (K,W) magnified views in the head region of I and U. (A,M,I,U) are lateral views. The upper side of the panel is the animal side in (A,M). (B,N) Dorsal-vegetal views. (D–G,P–S) Dorsal views. (D–F,P–R) anterior side is upper side. (G,S) The anterior side is toward the left. (H,T) Anterior views of (G,S). The upper side is dorsal. dl, dorsal lip; bp, blastopore; s, somite; nt, neural tube; ov, optic vesicle; tel, telencephalon; di/mi, diencephalon and midbrain boundary; rh, rhombencephalon; e, eye; ev, ear vesicle; cg, cement gland.
	Fig. 4. Zic1 or Zic2 overexpression induces ectopic pigment cell expression in embryo, as does overexpression of Zic3. A total of 100 or 500 pg of MT-Zic3 (B), MT-Zic1 (C, D), or MT-Zic2 (E, F) mRNA was injected into two blastomeres of 2-cell stage embryos, which were then cultured to stage 28–29. Clusters of pigment cells appeared in MT-Zic3, MT-Zic1 or MT-Zic2 mRNA-injected embryos (B,D,E,F, arrowheads). Transverse sections of MT-Zic1 (G) or MT-Zic2 (H) mRNA-injected embryos. A total of 250 pg of MT-Zic1 (G) or 125 pg of MT-Zic2 (H) mRNA was injected into one blastomere of 2-cell stage embryos and embryos were cultured to stage 35–36. Ectopic expression of presumptive mesenchymal tissues was observed in the MT-Zic1 or MT-Zic2 mRNA-injected side (G,H, arrowheads). (I) Western blot analysis of each Zic protein level in MT-Zic3, MT-Zic1 or MT-Zic2 mRNA-injected embryos. A total of 100 pg of MT-Zic3, MT-Zic1 or MT-Zic2 mRNA was injected into two blastomeres of 2-cell stage embryos and cultured to stage 10.5. A total of 10 μg of protein prepared from whole embryo was analyzed by Western blot analysis using an anti-myc antibody.
	Fig. 5. The pigment cells expressed by Zic1 or Zic2 overexpression in animal cap explant. (A) Uninjected animal cap explant. A total of 250 pg of MT-Zic1 (B) or a total of 125 pg MT-Zic2 (C) mRNA was injected into two animal blastomeres of 2-cell stage embryos obtained by the mating between albino female and wild type males. Animal caps were explanted at stage 9 and cultured. (D) A magnified view of the abdominal region of the control embryo at the same time as amimal cap explant (stage 45). The pigment cells appeared in the MT-Zic1 or MT-Zic2 overexpressed animal cap explants are similar to the melanocytes which are derived from neural crest.
	Fig. 6. Zic1 and Zic2 induced NCAM and Xslu expression but reduced epidermis in early stage embryos. A total of 250 pg of MT-Zic1 mRNA (A–C) or 125 pg of MT-Zic2 (D-F) was injected into one blastomere of 2-cell stage embryos. In situ hybridization was performed with a pan-neural marker gene (NCAM; A,D), a neural crest marker (Xslu; B,E) probe, and immunohistochemistry was performed with an epidermal marker (EpA) monoclonal antibody (C,F). Dorsal view of a stage 13–14 embryo. NCAM and Xslu expressing regions show lateral expansion of the injected side (A,B,D,E). EpA staining in the epidermis is reduced on the injected side (C,F).
	Fig. 7. Zic1 and Zic2 induced anterior neural marker genes and a neural crest marker gene without mesoderm induction in animal cap explants, as seen with Zic3 Embryos were injected with MT-Zic3 or MT-Zic1 mRNA at the two-cell stage. Animal caps were explanted at stage 9 and cultured. When sibling embryos reached stage 25, the expression of anterior-posterior marker genes (En2 and HoxB9 (=Xlhbox6)), a pan-neural marker gene (NCAM), a neuronal differentiation marker (N-tubulin), a neural crest marker (Xtwi), and a dorsal mesodermal marker (M. actin; muscle actin) were examined by RT–PCR. Although uninjected (Uninj.) caps expressed none of these markers, animal caps injected with Zic1, Zic2, or Zic3 mRNA expressed the anterior marker (En2) and the neural crest marker (Xtwi) while expressing neither the posterior marker (HoxB9) nor the dorsal mesodermal marker (M. actin). In each experiment, sibling control embryos served as a positive control (Embryo) and PCR on the same RNA without reverse transcription was done to verify the absence of genomic DNA (RT-PCR).