Nakata K et al. (1997), Xenopus Zic3, a primary regulator both in neura...

XB-ART-15895

Proc Natl Acad Sci U S A 1997 Oct 28;9422:11980-5. doi: 10.1073/pnas.94.22.11980.

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Xenopus Zic3, a primary regulator both in neural and neural crest development.

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

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Xenopus Zic3 is a Xenopus homologue of mouse Zic and Drosophila pair-rule gene, odd-paired. We show here that Zic3 has significant roles both in neural and neural crest development in Xenopus embryo. Expression of Zic3 is first detected in prospective neural plate region at gastrulation. Onset of the expression was earlier than most proneural genes and followed chordin expression. The expression was induced by blockade of BMP4 signal. Overexpression of Zic3 resulted in hyperplastic neural and neural crest derived tissue. In animal cap explant, the overexpression of Zic3 induced expression of all the proneural genes and neural crest marker genes. These findings suggest that Zic3 can determine the ectodermal cell fate and promote the earliest step of neural and neural crest development.

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Species referenced: Xenopus
Genes referenced: actl6a bmp4 chrd hoxa9 hoxc9 ncam1 neurod1 neurog1 snai2 tbxt twist1 zic3

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	Figure 1 The spatial expression pattern of Zic3 in Xenopus embryos. Series of embryo were hybridized with digoxigenin-labeled antisense Zic3 RNA. (A) Stage 10.25. (B) Stage 10.5. (C) The expression of Zic3 in dorsal lip (arrow) and prospective neural plate (arrowheads) was confirmed in the cross section of the same embryo in B. (D) Stage 12. (E) Stage 14. Asterisk indicates prospective region of mesencephalon and rhombencephalon. (F) Stage 16. White and black arrowheads indicate the lateral edge of neural plate and neural crest, respectively. (G and H) Stage 20. (I) Stage 30. In H and I, large black arrowhead, small black arrowhead, arrow, and white arrowhead indicate the telencephalon, diencephalon, mesencephalon, and rhombencephalon, respectively. Small arrow in I indicates the staining in a part of lateral mesoderm of tail. (J) Transverse section through the head region of same embryo as in I. Staining in the gut is nonspecific one, because it was detected also by the sense strand probe(data not shown). A, B, and I are the lateral views. The upper side of the panel is the animal side in A and B and the dorsal side in I. D are the dorsal views, the anterior side is toward left. H is the anterior view. The upper side is dorsal. b, blastopore; e, eye; nt, neural tube; fg, fore-gut.
	Figure 2. The temporal expression profiles of Zic3 and neural marker genes during development. (A) RNA was extracted from embryos at the indicated stage of development and the contents of Zic3 and neural marker genes (XASH-3, XATH-3, XlPOU 2, NeuroD, NCAM, N-tubulin) mRNA were measured by RT-PCR. (B) To compare the onset of the expression of Zic3 and neural inducer, chordin, during development, RNA was extracted from embryos at the indicated time after artificial fertilization and the expression level was determined by RT-PCR. The ubiquitous marker, Histone H4 served as a control for both A and B. H.A.F.; hours after fertilization.
	Figure 3. Zic3 was induced by blockade of BMP4 signaling. (A) Expression of Zic3 and other marker genes in the dispersed animal cap explants were measured by RT-PCR. The dispersed caps (Dispersed) differentiated into neural cells expressing NCAM whereas not dissociated caps (Intact) differentiated into epidermal cells expressing epidermal keratin (E. keratin). Zic3 expression was detected only in the dispersed animal caps. Sibling control embryos served as a positive control (Embryo) and PCR on the same RNA without reverse transcription was done to check the absence of genomic DNA (RT−). (B) Zic3 was induced by the injection of dnBMPR mRNA. Zic3 expression was detected by whole-mount in situ hybridization of early gastrula (stage 10.25) embryos injected with control LacZ mRNA (Upper) or dnBMPR mRNA (Lower) into the ventromarginal region at two-cell stage. The embryo injected with dnBMPR mRNA showed an ectopic Zic3 expression in the ventromarginal zone (arrows in Lower) in addition to the proper site (arrowheads in Lower), which was also found in the control embryo (arrowheads in Upper)
	Figure 4. Zic3 overexpression induces neural tissue hyperplasia and ectopic pigment cell expression. A total of 100 pg of Zic3 mRNA (A) was injected into one blastomere of two-cell stage embryos. (A) Uninjected control side (stage 27). (B) Zic3 mRNA injected side (stage 27). Clusters of pigment cells appeared in the anterior region (arrow). (C) Dorsal view of anterior region of the injected embryo (stage 36). (D) Transverse sections of the embryo shown in C. The levels of sections are indicated in C. The injected side was characterized by the hyperplastic neural walls, distorted eye, and unidentified tissue, probably derived from the neural crest (asterisk in D, E, and F, see Results). A total of 100 pg of Zic3 mRNA or control LacZ mRNA was injected into dorsoanimal (G) or ventroanimal (H) two blastomeres of eight-cell stage embryo, and cultured to stage 36 (G), stage 25 (H and I), or stage 20 (J). Lateral view of control LacZ mRNA injected embryos (G and H,Upper) and Zic3 mRNA injected embryos (G and H,Lower). The eye abnormalities were observed in the embryos injected with Zic3 mRNA into dorsal blastomeres (G). Remarkable clusters of ectopic pigment cells appeared in the embryos injected with Zic3 mRNA into ventral blastomeres (H). (I) Higher magnification of the pigment cell clusters in (H). (J) Xtwi expression in the embryos (ventral view) injected with LacZ mRNA (Upper) or Zic3 mRNA (Lower) into ventroanimal two blastomeres at eight-cell stage. The Xtwi expression was observed in the head neural crest of the control embryo (Upper, arrowhead). However, the Zic3 mRNA injection induced the ectopic Xtwi expression (Lower, arrow) near the ectopic clusters of pigment cells (white arrowheads; the brown staining indicates the remnants of the pigments) in the ventral side of embryo. The expansion of the Xtwi expressing cephalic neural crest (Lower, black arrowheads) was also observed in this embryo.
	Figure 5 Zic3 induced NCAM, Xtwi, and Xslu expression but reduced epidermis in early stage embryo. A total of 100 pg of Zic3 mRNA was injected into one blastomere of two-cell stage embryos (A). In situ hybridization was performed with NCAM (A), Xtwi (B), and Xslu (C) probe, and immunohistochemistry was performed with EpA mAb (D). (A) Dorsal view of a stage 14 embryo. NCAM, Xtwi, and Xslu expressing regions expand laterally in the injected side (A). EpA staining in the epidermis is reduced in the injected side (D).
	Figure 6 Zic3 induced neural and neural crest marker genes without mesoderm induction in animal cap explant. Embryos were injected with 100 pg of Zic3 or control LacZ mRNA at the two-cell stage. Animal caps were explanted at stage 9 and cultured. (A) Zic3 induced all the neural and neural crest marker genes tested. When the sibling embryos reached stage 20, the expression of neural marker (NCAM, Neurogenin, NeuroD, XASH-3, XATH-3, XlPOU 2) and neural crest marker (Xtwi, Xslu) were examined by RT-PCR. Although uninjected (Uninj.) or LacZ injected (LacZ) caps do not express any of these markers, animal caps injected with Zic3 mRNA (Zic3) express all of the neural markers and neural crest markers we tested. (B) Zic3 did not induce mesodermal marker. An early mesodermal marker, Xbra, and a dorsal mesodermal marker, M.actin, expression were examined by RT-PCR when the sibling embryos became stage 10.5 and stage 20, respectively. (C) Zic3 induced anterior neural character. Expression of the neural markers, which is expressed differentially along the anteroposterior axis, was examined by RT-PCR when the sibling embryos became stage 27. Anterior neural marker, En-2, but not the posterior neural marker, XlHbox6, was expressed in the Zic3 overexpressed animal cap. In each experiment, sibling control embryos served as a positive control (Embryo) and PCR on the same RNA without reverse transcription was done to check the absence of genomic DNA (RT−).

References [+] :

Aruga, A novel zinc finger protein, zic, is involved in neurogenesis, especially in the cell lineage of cerebellar granule cells. 1994, Pubmed