Shibata M et al. (2001), Systematic screening and expression analysis of...

XB-ART-7830

Dev Biol 2001 Nov 15;2392:241-56. doi: 10.1006/dbio.2001.0428.

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Systematic screening and expression analysis of the head organizer genes in Xenopus embryos.

Shibata M , Itoh M , Ohmori SY , Shinga J , Taira M .

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We describe here a systematic screen of an anterior endomesoderm (AEM) cDNA library to isolate novel genes which are expressed in the head organizer region. After removing clones which hybridized to labeled cDNA probes synthesized with total RNA from a trunk region of tailbud embryos, the 5' ends of 1039 randomly picked cDNA clones were sequenced to make expressed sequence tags (ESTs), which formed 754 tentative unique clusters. Those clusters were compared against public databases and classified according to similarities found to other genes and gene products. Of them, 151 clusters were identified as known Xenopus genes, including eight organizer-specific ones (5.3%). Gene expression pattern screening was performed for 198 unique clones, which were selected because they either have no known function or are predicted to be developmental regulators in other species. The screen revealed nine possible organizer-specific clones (4.5%), four of which appeared to be expressed in the head organizer region. Detailed expression analysis from gastrula to neurula stages showed that these four genes named crescent, P7E4 (homologous to human hypothetical genes), P8F7 (an unclassified gene), and P17F11 (homologous to human and Arabidopsis hypothetical genes) demarcate spatiotemporally distinct subregions of the AEM corresponding to the head organizer region. These results indicate that our screening strategy is effective in isolating novel region-specific genes.

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Species referenced: Xenopus
Genes referenced: btbd6 cer1 chrd dkk1 frzb2 gpr107 hhex mob2 nudt22 ost4 ostc spty2d1 syngr1 tmem52

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	FIG. 3. Isolated cDNA clones expressed in the AEM region and notochord. (A) Clone names and similar genes or proteins in the public databases are indicated on the left side of each panel. Results of whole-mount in situ hybridization for early neurula (stage 13) are shown in dorsal (left) and lateral (right) view. (B) Northern blot analysis. Clone names and their closest genes or proteins in the public databases are indicated on the left side of each panel and the deduced mRNA size (kb) of each clone on the right (see Table 2 for P11F3). The developmental stages are indicated on the top of each column by numerical figures according to Nieuwkoop and Faber (1967). 18S rRNA stained with ethidium bromide is for loading control. Ten micrograms of total RNA from stage-9, -10, -12.5, and -15 embryos were electrophoresed in each lane, blotted, and hybridized with probes derived from each clone except for crescent. Crescent probe was hybridized to the same blot as previously used (Hikasa and Taira, 2001) with a wider range of stages as indicated.
	FIG. 5. Detailed expression pattern analysis of P7E4, P8F7, and P17F11 by whole-mount in situ hybridization. Whole or hemisectioned embryos were hybridized with P7E4 (A), P8F7 (G), P17F11 (L), or crescent (Q, R) probe. Other halves of hemisectioned embryos were used for dkk-1, cerberus (cer), chordin (chd), or XHex probe as indicated. Stained embryos were treated with BB/BA to become transparent for observation (B, F, K, P, right panel). Sections were made from stained whole embryos (C, H), and are shown in bright field and with DAPI staining. (A) P7E4 (homologous to human hypothetical proteins, BDPL and KIAA0952). (A) Sagittal hemisections of early gastrulae (stage 10.5) stained for P7E4 (upper panel) and dkk-1 (lower panel). P7E4 expression was detected around the Brachet cleft (arrowhead) and weakly in the mesoderm region (arrow). Dorsal to the right. (B) Dorsal view of early neurula. Anterior to the left. Cross sections were made at the indicated positions (C) which correspond to the following panels (C). (C) Sections of the same embryo as shown in (B). P7E4 was expressed in the archenteron roof as well as the bilateral domains including the neuroectoderm and mesoderm region (arrowheads) (C). The bilateral expression domains were notable in the mesodermal region at position D. In the posterior region, expression was seen in the notochord (E). (F) Dorsal view of sagittally hemisectioned neurula embryos stained for P7E4 and chordin. Anterior is up. (G) P8F7 (unknown). (G) Sagittal hemisections of early gastrula stained for P8F7 (top) and dkk-1 (middle), and vegetal view of the hemisectioned embryo (bottom). Right panels, higher magnification of the left panels. Dorsal is to the right (top, middle) or up (bottom). Arrow, the surface layer of the dorsal blastopore lip, expressing P8F7 but not dkk-1; arrowhead, punctuated expression of P8F7 in the epithelial layer of the dorsal ectoderm. (H) Vegetal view of early gastrula (upper panel, dorsal is up) and a sagittal section (lower panels, dorsal to the right). Arrowhead, dorsal blastopore lip; arrow, anterior archenteron. (I) Sagittal hemisections of midgastrula stained for P8F7 and chordin. Dorsal to the right. Arrowhead, expression of P8F7 in the anterior archenteron roof. (J) Sagittal hemisections of early neurula stained for P8F7 and XHex. Anterior to the left. Arrow, expression in the anterior endoderm; arrowhead, expression in the anterior archenteron (P8F7) or the prechordal plate (XHex). (K) Dorsal view of sagittally hemisectioned neurula stained for P8F7 and chordin. Anterior is up. (L) P17F11 (related to human MGC13045 and Arabidopsis CAB90947). (L) Sagittal hemisections of late blastula stained for P17F11 and chordin. (M) Sagittal hemisections of early gastrula stained for P17F11 (top) and dkk-1 (middle), and vegetal view of the hemisectioned embryo (bottom). Dorsal is to the right (top, middle), or up (bottom). Arrowhead, the surface layer of dorsal blastopore lip, expressing P17F11 but not dkk-1. (N, O) Sagittal hemisections of late gastrulae stained for P17F11, chordin, or XHex. Dorsoanterior to the right. (P) Sagittal hemisections of early neurula stained for P17F11 (upper) and chordin (lower), and dorsal view of the hemisectioned embryo (right). Partial breakage of the archenteron roof shows interior staining in the anterior mesoderm as indicated by af. Anterior is to the left (left panels), or up (right panel). (Q, R) crescent. (Q) Sagittal hemisections of early gastrula stained for crescent (upper) or dkk-1 (lower), and vegetal view of the hemisectioned embryo (right). Dorsal is to the right (left panels), or up (right panel). (R) Sagittal hemisections of early gastrula stained for crescent and cerberus. Dorsal is to the right. Arrowheads, the surface layer of dorsal blastopore lip, expressing crescent but not dkk-1 or cerberus. Numerical figures in the panels indicate the developmental stages according to Nieuwkoop and Faber (1967). Pictures of hemisections stained for dkk-1, cerberus, chordin, and XHex were flipped for comparison. af, artifact; en, endoderm; nc, notochord; ne, neuroectoderm; sm, somitic mesoderm.
	btbd6 (BTB (POZ) domain containing 6) gene expression in Xenopus laevis embryos, NF stage 10.5, as assayed by in situ hybridization. Bisected gastrulae: Dorsal to the right.
	nudt22 (nudix hydrolase 22) gene expression in Xenopus laevis embryos, NF stage 10.5, as assayed by in situ hybridization. Bisected gastrulae: Dorsal to the right.
	gpr107 (G protein-coupled receptor 107) gene expression in Xenopus laevis embryos, NF stage 13, as assayed by in situ hybridization. Left image: dorsal view, anterior left. Right image: lateral view of bisected embryo: anterior left, dorsal up.
	mob2.1 (MOB kinase activator 2) gene expression in Xenopus laevis embryos, NF stage 13, as assayed by in situ hybridization. Left image: dorsal view, anterior left. Right image: lateral view of bisected embryo: anterior left, dorsal up.
	nudt22 (nudix hydrolase 22) gene expression in Xenopus laevis embryos, NF stage 13, as assayed by in situ hybridization. Left image: dorsal view, anterior left. Right image: lateral view of bisected embryo: anterior left, dorsal up.
	spty2d1 (SPT2 chromatin protein domain containing 1) gene expression in Xenopus laevis embryos, NF stage 13, as assayed by in situ hybridization. Left image: dorsal view, anterior left. Right image: lateral view of bisected embryo: anterior left, dorsal up.
	syngr1 (synaptogyrin 1) gene expression in Xenopus laevis embryos, NF stage 13, as assayed by in situ hybridization. Left image: dorsal view, anterior left. Right image: lateral view of bisected embryo: anterior left, dorsal up.