Taverner NV et al. (2005), Microarray-based identification of VegT targets...

XB-ART-2180

Mech Dev 2005 Mar 01;1223:333-54. doi: 10.1016/j.mod.2004.10.010.

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Microarray-based identification of VegT targets in Xenopus.

Taverner NV , Kofron M , Shin Y , Kabitschke C , Gilchrist MJ , Wylie C , Cho KW , Heasman J , Smith JC .

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The Xenopus T box family member VegT is expressed maternally in the vegetal hemisphere of the embryo. Mis-expression of VegT in prospective ectodermal tissue causes ectopic activation of mesodermal and endodermal markers, and ablation of VegT transcripts prevents proper formation of the mesendoderm, with the entire embryo developing as epidermis. These observations define VegT as a key initiator of mesendodermal development in the Xenopus embryo, and in an effort to understand how it exerts its effects we have used microarray analysis to compare gene expression in control animal caps with that in ectodermal tissue expressing an activated form of VegT. This procedure allowed the identification of 99 potential VegT targets, and we went on to study the expression patterns of these genes and then to ask, for those that are expressed in mesoderm or endoderm, which are direct targets of VegT. The putative regulatory regions of the resulting 14 genes were examined for T domain binding sites, and we also asked whether their expression is down-regulated in embryos in which VegT RNA is ablated. Finally, the functions of these genes were assayed by both over-expression and by use of antisense morpholino oligonucleotides. Our results provide new insights into the function of VegT during early Xenopus development.

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Species referenced: Xenopus
Genes referenced: admp2 alpl aplnr atp1a1 best2 bix1.3 cdk5r1l cdx4 chrd ckm copa cxcr4 cyp26a1 dhcr7 dhrs3 epha4 esr-5 exosc5 flrt3 foxb1 foxc1 fzd7 gdf3 ggt1 has2 hes5.7 hes7.2 hey1 kif26a krt18.1 krt8.1 lin28a mdn1 mid1ip1 msx1 myc myod1 nradd nrf1 pdlim5 pnhd post prickle1 pygm pzp rap2b rdh10 rhov rnf19a rpl24 rsl24d1 set slc12a3l slc16a6 slc2a1 slc6a14.2 snai1 sp5l spry1 stx6 tmod4 tp63 txnl4a utp4 vcan vegt ventx1 ventx1.2 wnt11b wnt5b XB5748694 zic2 zswim4
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	Fig. 1. A hormone-inducible version of VegT (VegT-GR) causes animal caps to undergo convergent extension (inset) and to activate expression of Bix4. No effect is observed in the absence of dexamethasone. Animal caps were dissected from embryos injected with 200 pg RNA encoding VegTGR. They were cultured to mid-blastula stage 8.5 and animal pole regions were dissected and cultured in the presence or absence of 2 mM dexamethasone (Dex) until the equivalent of early gastrula stage when they were frozen and assayed by real-time RT-PCR for expression of the VegT target Bix4. Two animal caps were maintained in culture to confirm, by the onset of convergent extension movements, that induction had occurred (inset).
	Fig. 2. Microarray analysis of VegT targets. (A) Scatter plots of Cy3 fluorescence (control animal caps; Y axis) against Cy5 fluorescence (dexamethasonetreated caps; X axis). Pink spots were removed from further analysis because signal intensities were too low. Red spots represent genes that are differentially expressed in response to VegT. (B) Classification by function of genes that are up-regulated by VegT.
	Fig. 3. In situ hybridisation analysis of genes that are up-regulated by VegT. Embryos were fixed at early gastrula stage 10, bisected, and subjected to in situ hybridisation. The first image shows the expression pattern of VegT/Antipodean and the second a schematic representation of the three germ layers, colourcoded as in Table 3, with ectoderm shaded red, mesoderm yellow, and endoderm blue. The images are labelled according to the clone identification number of the gene in question.
	Fig. 4. Classification of VegT targets as ‘Direct’, ‘Indirect’, or ‘Induced by Cycloheximide’. Bix4 is classified as a direct target because it is activated by VegTGR and dexamethasone in the presence of cycloheximide (CHI). Mixer is classified as indirect because it is not activated by VegT-GR and dexamethasone in the presence of cycloheximide. Dkk1 is activated by cycloheximide alone, and no conclusions can be drawn as to its response to VegT. Animal caps were dissected from embryos injected with 200 pg RNA encoding VegT-GR as described in the legend to Fig. 1 and treated with cycloheximide (CHI: 10 mM) or dexamethasone (Dex: 2 mM) or both, as indicated, and cultured to the equivalent of early gastrula stage 10–10.25, when they were assayed by real-time RTPCR for expression of Bix4, Mixer and Dkk1.
	Fig. 5. The promoter regions of Bix4 (Casey et al., 1999) and XtBix, illustrating the similar arrangements of T box binding sites.
	Fig. 6. Analysis of the expression of direct VegT targets in embryos lacking VegT. RNA was extracted at the equivalent of early gastrula stage 10.5 from control embryos or from embryos depleted in maternal VegT mRNA. Expression of the indicated genes was analysed by real-time RT-PCR, with Xbra, Xsox17a, Bix4 and Xlim-5 acting as positive controls; expression of the first three is known to be down-regulated in embryos lacking VegT, while expression of Xlim-5 is enhanced.
	Fig. 7. Phenotypes of Xenopus tropicalis embryos over-expressing direct VegT targets. Xenopus tropicalis embryos were injected with 0.5 ng of the indicated RNAs at the one-cell stage, and the embryos were allowed to develop to tadpole stages. The identities of the injected RNAs are indicated. The results illustrated in (A–H) were obtained in one experiment, and those in (I–P) in another. The frequencies with which the illustrated phenotypes were obtained are as follows. (A) Uninjected (normal development): 30/30; (B) b-galactosidase (normal development): 28/30; (C) XtBix (positive control): 29/29; (D) XtVent-1: 23/27; (E) XtEsr5 (development disrupted): 14/28; (F) XtEsr5 (normal development): 13/28; (G) XtHesr1: 28/28; (H) XtSnail: 19/29; (I) b-galactosidase (normal development): 29/30; (J) XtHesr1: 19/19; (K) XtSnail: 17/26; (L) XtEsr4: 26/27; (M) XtPinhead: 28/29; (N) XtPrickle: 29/29; (O) XtSDR: 22/26; (P) XtAngio1: 27/30.
	Fig. 8. Inhibition of proper splicing of XtSnail by an antisense morpholino oligonucleotide. (A) The structure of Xenopus tropicalis Snail. The red region indicates the SNAG domain that is characteristic of the Snail family, while the blue regions represent the zinc fingers. The antisense morpholino oligonucleotide is designed to bind to the splice donor site of intron 2, which is positioned immediately after the region encoding zinc finger 3. (B) RT-PCR analysis indicates that normal splicing is inhibited by the antisense morpholino oligonucleotide. RNA was extracted from control or injected embryos at stage 10 and stage 13, as indicated, and analysed by RT-PCR. Injection of the specific oligonucleotide prevented proper splicing, represented by the lower arrow, and instead caused the production of several PCR products, the largest of which (upper arrow) results from retention of intron 2.
	Fig. 9. Phenotypes of Xenopus tropicalis embryos expressing antisense morpholino oligonucleotides directed against previously unstudied direct targets of VegT. Antisense morpholino oligonucleotides (15 ng) were injected into Xenopus tropicalis embryos at the one-cell stage, and embryos were allowed to develop to tadpole stages, when they were fixed and photographed. The results illustrated in (A–F) were obtained in one experiment, and those in (G–L) in another. The frequencies with which the illustrated phenotypes were obtained are as follows. (A) Uninjected (normal development): 11/11; (B) control oligonucleotide (normal development): 7/8; (C) XtAngio1: 10/10; (D) XtEsr4: 11/14; (E) XtEsr5: 17/18; (F) XtSDR: 8/8; (G) uninjected (normal development): 11/14; (H) control oligonucleotide (normal development): 12/13; (I) XtEdd: 11/11; (J) XtEsr4CXtEsr5: 11/11; (K) XtHesr: 9/11; (L) XtSnail: 9/11.
	copa (coatomer protein complex subunit alpha) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, animal up, dorsal right.
	exosc5 (exosome component 5) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, animal up, dorsal right.
	kif26a (kinesin family member 26A) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, animal up, dorsal right..
	pygm (phosphorylase, glycogen, muscle) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, animal up, dorsal right.
	rnf19a (ring finger protein 19A, RBR E3 ubiquitin protein ligase) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, animal up, dorsal right.
	rpl24 (ribosomal protein L24) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, animal up, dorsal right.
	txnl4a (thioredoxin like 4A) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, animal up, dorsal right.
	best2 (bestrophin 2) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, animal up, dorsal right.
	utp4 (UTP4 small subunit processome component ) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, dorsal right.
	stx6 (syntaxin 6) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, dorsal right.
	mdn1 (midasin AAA ATPase 1) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, dorsal right.
	ggt1 (gamma-glutamyltransferase 1) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, horizontal view, dorsal right.