Cornish EJ et al. (2009), A microarray screen for direct targets of Zic1 ...

XB-ART-39606

Dev Dyn 2009 May 01;2385:1179-94. doi: 10.1002/dvdy.21953.

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A microarray screen for direct targets of Zic1 identifies an aquaporin gene, aqp-3b, expressed in the neural folds.

Cornish EJ , Hassan SM , Martin JD , Li S , Merzdorf CS .

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The Zic1 transcription factor plays multiple roles during early development, for example, in patterning the early neural plate and formation of the neural crest, somites, and cerebellum. To identify direct downstream target genes of Zic1, a microarray screen was conducted in Xenopus laevis that identified 85 genes upregulated twofold or more. These include transcription factors, receptors, enzymes, proteins involved in retinoic acid signaling, and an aquaglyceroporin (aqp-3b), but surprisingly no genes known to be involved in cell proliferation. We show that both aqp-3 and aqp-3b were expressed in adult tissues, while during early embryonic development, only aqp-3b was transcribed. During neurula stages, aqp-3b was expressed specifically in the neural folds. This pattern of aqp-3b expression closely resembled that of NF-protocadherin (NFPC), which is involved in cell adhesion and neural tube closure. Aqp-3b may also be involved in neural tube closure, since mammalian Aqp-3 promotes cell migration and proliferation.

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Species referenced: Xenopus laevis
Genes referenced: ankrd11 aplnr aqp3 aspm auts2 celf3 cfb cldn12 cldn6.1 cpeb2 crabp2 dgat2 dnmt1 efnb1 eomes evx1 fat1 fgfr2 fibcd1 foxf1 foxo3 gja7 glipr2 gprc5b hes5.5 hesx1 hs3st3a1 itga6 KIAA1671 lgals4.2 marchf8 mrps18b mtus1 myod1 ncoa3 pcdh7 pkdcc.2 pou2f1 prickle1 psmb8 ptch2 ptgds rab40b reep3 rgcc rnf24 sall1 smc5 snai1 son sp7 tbx2 tead1 tek thop1 tiparp top1.2 traf4 vegt wnt11b wnt8a zbtb33 zic1

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	Figure 1. Zic1 regulates the expression of direct target genes in whole embryos. The expression patterns by in situ hybridization are shown. A-C: aqp-3b. D-F: cpl-1. G-I: CRABP-II. J, K: tnrc4. L, M: Xl.25952. N, O: Xl.8933. P, Q: myoD. A, D, G, J, L, N, and P are uninjected embryos illustrating the expression of aqp-3b, cpl-1, CRABP-2, tnrc4, Xl.25952, Xl.8933, and myoD, respectively. B, E, H, K, M, O, and Q show reductions in gene expression produced by injection of RNA for a dominant interfering construct of zic1 (zic1 Delta N, previously called dnzic1; Li et al.,[2006]; Merzdorf and Sive,[2006]). C, F, and I show increases in gene expression resulting from injection of full-length zic1 RNA. One cell of 2-cell embryos was injected with either 100 pg zic1 RNA or 100 pg zic1 Delta N RNA together with 25 pg lacZ RNA. Stage 15-18 neurula stage embryos are shown. Arrowheads mark the injected sides.
	Figure 3. Characterization of aqp-3b expression by whole mount in situ hybridization. aqp-3b is expressed at the edges of the neural plate in early neurula stage 13 embryos (A). As the neural folds begin to rise in stage 14+ embryos, aqp-3b expression extends more posteriorly (B). As the neural folds continue to rise and narrow the gap between them, this is reflected in aqp-3b expression in mid-neurula stage 16 and 17 embryos (D, E). At stage 18, aqp-3b expression almost meets at the midline as the neural folds have nearly come together (G). In late neurula stage 19 embryos, aqp-3b is expressed along the dorsal midline (H). The expression patterns of aqp-3b (B, E, H) are very similar to the neural fold expression of NFPC (C, F, I), while aqp-3b does not share the epidermal ectoderm expression domain of NFPC. During neurulation, aqp-3b (A, E) expression overlaps extensively with zic1 expression (J, K). Transverse sections through stained stage 18 embryos confirmed that aqp-3b is expressed at the tips of the neural folds (L). np, neural plate; nc, notochord; s, somites.
	Figure 4. The aqp-3b and zic1 expression domains overlap. Sibling stage 15 neurula embryos were processed by in situ hybridization for aqp-3b or zic1 expression. A, B: Cryosections of equivalent anterior regions of the embryos showing expression of aqp-3b (A) and zic1 (B). C, D: Cryosections of equivalent more posterior regions of the embryos showing aqp-3b (C) and zic1 (D) expression. Arrowheads point to the neural folds; np, neural plate; nc, notochord.
	Fig. 2. Expression of aqp-3b and aqp-3 in embryos and adult tissues. A: Developmental profile of aqp-3b and aqp-3 expression, which was generated by RT-PCR using RNA from staged Xenopus embryos. aqp-3b was maternally and zygotically expressed and showed increased expression during neurula stages 14�18, when the neural folds rise and the neural tube closes. aqp-3 was not expressed in the embryonic stages tested. B: Expression of aqp-3b and aqp-3 in adult tissues was determined by RT-PCR. aqp-3b was primarily expressed in colon, while aqp-3 was primarily expressed in lung. EF-1 served as loading control and -RT samples indicate the absence of residual genomic DNA.
	dgat2 (diacylglycerol O-acyltransferase 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18 dorsal view, anterior up.
	ptgds (prostaglandin D2 synthase 21kDa (brain)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16 dorsal view, anterior up.
	sp7 (Sp7 transcription factor ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16 dorsal view, anterior up.
	aqp3 (aquaporin 3 (Gill blood group)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16 dorsal view, anterior up.