Merzdorf CS and Sive HL (2006), The zic1 gene is an activator of Wnt signaling.

XB-ART-71

Int J Dev Biol 2006 Jan 01;507:611-7.

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The zic1 gene is an activator of Wnt signaling.

Merzdorf CS , Sive HL .

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The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, we asked whether Xenopus Zic1 acted through the Wnt pathway. Using Wnt signaling inhibitors, we demonstrate that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated here. Together, our data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates.

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Species referenced: Xenopus laevis
Genes referenced: actl6a bmp4 chrd en2 fgf4 fgf8 gsk3b nog otx2 wnt1 wnt11 wnt3a wnt4 wnt5a wnt7b wnt8a wnt8b zic1

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	Fig. 1. The zic1 expression domain overlaps with those of both en- 2 and wnt8b. In situ hybridization of stage 17 neurula embryos. (A) zic1 expression domain. (B) Double in situ hybridization with zic1 and en-2 probes shows overlap between the two expression domains. The bracket indicates en-2 expression. (C) wnt8b expression domain in the midbrain. (D) Double in situ hybridization with zic1 and wnt8b probes indicates that their expression domains overlap. The bracket indicates wnt8b expression.
	Fig. 2 (Left). Zic1 acts through the Wnt pathway to activate en-2. Both cells of 2-cell stage embryos were injected with the in vitro synthesized RNAs listed along the top. A C-terminal truncation of the zic1 coding sequence was used (zic1δC). Animal caps were isolated at stage 9 and cultured until sibling embryos reached stage 22. Total RNA was isolated and subjected to RT-PCR analysis with the primers shown on the left. (Lanes 1,2), β-globin or zic1δC injected animal caps did not show expression of neural markers. (Lane 3) Noggin mRNA induced the anterior neural marker otx2 and the general neural marker N-CAM. (Lane 4) Co-injected zic1δC plus noggin induced expression of the midbrain/hindbrain boundary marker en- 2. Induction of en-2 expression is inhibited by dnWnt (lane 5) or GSK3 (lane 6), which are inhibitors of the Wnt pathway. This demonstrates that Zic1 requires an active Wnt pathway to induce en-2 expression. N-CAM and otx2 were expressed in all samples that received noggin (lanes 3-6). (Lane 7) Whole embryos at stage 22 served as positive control. Muscle actin controlled for mesodermal contamination, EF-1α served as loading control and -RT samples controlled for DNA contamination. After culture to the equivalent of stage 22, animal caps expressing β-globin and zic1 constructs were always completely round. Noggin-expressing animal caps were occasionally elongated, but the amount of noggin used was low enough that most explants were round. Co-injection of dnWnt or GSK3 RNA did not influence the shape of the animal caps beyond the effects of noggin. Injections were as follows: 200 pg β-globin, 200 pg zic1δC, 5 pg noggin, 150 pg dnWnt8, 80 pg GSK3.
	Fig. 3 (Right). Zic1 induces wnt expression. Embryos were injected into both blastomeres at the 2-cell stage with the indicated RNAs. zic1δC was tested in addition to full length zic1. Animal caps were isolated at stage 9 and cultured until sibling embryos reached stage 22. Total RNA was isolated and subjected to RT-PCR analysis with the primers shown on the left. (Lane 1) β-globin injected animal caps. (Lane 2) Noggin-injected. (Lane 3) zic1δCinjected. (Lane 4) zic1δC plus noggin-injected. (Lane 5) Full length zic1-injected. (Lane 6) Full length zic1 plus noggin-injected. (A) wnt1, wnt4 and wnt8b expression was induced by zic1δC plus noggin (lane 4). zic1δC alone induced wnt1 expression (lane 3), while full length zic1 plus noggin did not induce wnt4 expression (lane 6). (B) zic1δC induced wnt8 expression (lane 3). (C) wnt3a and wnt7b were expressed in β-globin, zic1δC and zic1- injected animal caps (lanes 1, 3 and 5), but not in samples that had been co-injected with noggin (lanes 2, 4 and 6). (D) Muscle actin controlled for mesodermal contamination, EF-1α served as loading control and -RT samples controlled for DNA contamination. Injections were as follows: 200 pg β-globin, 200 pg zic1δC, 200 pg zic1, 5 pg noggin.
	Fig. 4. Zic1 induces ectopic wnt8b expression. One cell of 2-cell albino embryos was injected with (A) 100 pg zic1δC RNA or with (B) 100 pg full length zic1 RNA together with 25 pg lacZ RNA as tracer. In situ hybridization with wnt8b probe at neurula stage 18 showed that zic1δC and full length zic1 upregulate wnt8b expression on the injected (arrowheads) side.
	Fig. 5. Wnt1, Wnt3a and Wnt8 induce en-2 expression. Embryos were injected at the 2-cell stage into both cells with the sense RNAs listed along the top. Animal caps were isolated at stage 9 and cultured to the equivalent of stage 22. RT-PCR analysis was performed with the primers shown on the left. (Lanes 1,2) β-globin or noggin injected animal caps did not express en-2. (Lanes 3-5) 25 pg wnt1, 25 pg wnt3a and 25 pg wnt8 RNA induced en-2 expression. (Lane 6) 50 pg wnt8b failed to induce en- 2 expression. All wnt RNAs were co-injected with 3 pg noggin RNA. The shape of the animal caps was round in all cases since very low levels of noggin and wnt RNAs were used. Muscle actin controlled for mesodermal contamination, EF-1α served as loading control and -RT samples control for DNA contamination.
	Fig. 6. Zic1 is required for en-2 induction. (A) One cell of 2-cell albino embryos was injected with 100 pg zic1δC RNA, resulting in an increase of en- 2 expression levels within the en-2 expression domain. Embryos injected with 100 pg of a dominant interfering zic1 construct (dnzic1) showed very significant decrease (B) in en-2 expression and (C) in wnt1 expression. All embryos were co-injected with 25 pg lacZ RNA as tracer and the injected sides are shown on the right.
	Fig. 7. Model of en-2 induction by Zic1. zic1 expression is activated after inhibition of BMP signaling by Noggin or Chordin. Zic1 and possibly other Zic proteins synergize with other factors to activate expression of wnt1 and other wnt genes. Wnt proteins activate expression of en-2. Wnt3a may act via a Zic1-independent pathway. An alternate pathway, by which en-2 expression is activated, may involve Fgf8 signaling. The activation events indicated by arrows need not necessarily be direct.