Takahashi N et al. (2005), Systematic screening for genes specifically exp...

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Int J Dev Biol 2005 Jan 01;498:939-51. doi: 10.1387/ijdb.052083nt.

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Systematic screening for genes specifically expressed in the anterior neuroectoderm during early Xenopus development.

Takahashi N , Tochimoto N , Ohmori SY , Mamada H , Itoh M , Inamori M , Shinga J , Osada S , Taira M .

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A cDNA library derived from the anterior neuroectoderm (ANE) of Xenopus late-gastrula embryos was systematically screened to isolate novel developmental regulatory genes involved in early brain development. We isolated 1,706 5 expressed sequence tags (ESTs), which were subdivided into 1,383 clusters and categorized into 19 classes based on predicted functions according to their similarities to other known genes. Of these, 757 clusters that were considered possible novel regulatory genes or unknown genes were subjected to expression pattern analysis using whole-mount in situ hybridization. Genes from 69 clusters (9%) were expressed in the ANE region. Based on their expression patterns and predicted amino acid sequences, 25 genes were selected for further analysis as novel Xenopus genes expressed broadly or region-specifically in the ANE. Eighteen genes were expressed in postulated patterning centers in the neuroectoderm, including the anterior (four genes) and lateral (nine genes) neural ridges, the midbrain-hindbrain boundary region (one gene) and the midline region of the neural plate (two genes), whereas 13 genes were expressed in the eye anlagen. Therefore, early regionalization of the neuroectoderm appears to occur mainly in those neural patterning centers and the eye anlagen. We determined the entire coding regions of p54nrb, Semaphorin 6D and a novel gene designated scribble-related protein 1 (SCRP1). Interestingly, Semaphorin 6D is expressed in the mesoderm with a dorsoventral gradient, as well as in the ectoderm at the gastrula stage, implying a new role for this protein in development other than in axon guidance.

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Species referenced: Xenopus laevis
Genes referenced: cdkn2aip cirbp cripto.3 crx eif4a3.1 epha2 etv1 fgfr4 foxa4 fzd7 gmnn hes4 hesx1 lemd3 lhx5 lrrc1 nono nppa nrp1 otx1 otx2 pax2 pdcd10 prickle1 psen1 ran rax rbpj rhoa rpl32 rrm1 rrm2.2 sema6d sfrp2 six3 sox11 sox2 sox3 tbx2 tcf3 utp15 XB972976 zic1 zic3

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	Fig. 1. Strategy for systematic screening of genes specifically expressed in the Xenopus ANE. Screening for eliminating non-specific clones in the ANE region from an ANE library was performed along this scheme. Dissection of late-gastrula (stage 12-12.5) embryos was performed for isolation of ANE and AEM regions. Dissected pieces were treated with collagenase and separated into ANE and underlying AEM. A region-specific cDNA library was constructed from the ANE RNA. Further detailed processes are described in the text. AEM, anterior endomesoderm; ANE, anterior neuroectoderm.
	Fig. 2. Clustering of ESTs and classification of their sequences and expression patterns. (A) Result of clustering. One thousand seven hundred six clones from the ANE cDNA library formed 1,383 clusters (ANE clusters). Of them, 1,264 (91%) are ANEâspecific clusters, which do not mingle with previously reported AEM clones (Shibata et al., 2001). When ESTs from the ANE and AEM libraries were combined, total 2,963 ESTs formed 2,179 clusters. The number of clones in each single cluster was counted from total 2,179 clusters or 1,264 ANE-specific clusters. Ordinate, the number of cluster; abscissa, the number of clones in a single cluster; green bar, the number of clusters based on total clusters; red bar, the number of clusters based on ANE-specific clusters. (B) Classification of predicted proteins and expression patterns of ANE clusters. (a) ANE clusters (1,383) were classified according to sequence similarity to known genes or proteins of Xenopus or other species in public databases. Clusters were divided into three classes as indicated. (b) ANE clusters (1,383) were also classified according to functions of genes (see Table 1). (c) Out of 968 clusters which are functionally unclassified or possible developmental regulators (Classes B, C and D), 757 clones from 729 clusters were analyzed for expression patterns by WISH and classified into three categories as indicated. Values besides each column are given as the number of clusters and percentages in parentheses.
	Fig. 3. Expression patterns of possible novel genes. The left column shows cluster (gene) names and the right panels, show expression patterns visualized by WISH. Developmental stages according to Nieuwkoop and Faber (1967) (Nieuwkoop and Faber, 1967) and orientations of embryos are indicated on the bottom of each panel: D, dorsal; L, lateral; A, anterior. (A) Genes which were expressed in the anterior neural plate or anterior neural ridge, or both. (B) Genes which were expressed in eye anlagen together with or without the neural ridge. (C) Genes which were widely expressed in the neural plate or neural ridge, or both. (D) Genes which were expressed in the neural tissue as well as other tissues.
	Fig. 4. Alignments of the predicted amino acid sequences of isolated clones and their orthologous proteins in other species. (A) SCRP1 (N3A9) and human LRRC1 (AAH03193). Green line, LRR region; red line, PDZ domain binding sequence. (B) Xenopus p54nrb (N3B10) and the human ortholog. Green underline, DBHS domain; red overline, RRM1 and RRM2. (C) Xenopus Sema6D4 (N30H6) and the human ortholog. Green line, signal sequence; red line, sema domain; blue line, transmembrane domain.
	Fig. 5. Temporal expression profiles of Xenopus SCRP1 and XSema6D4 by Northern blot analysis. (A) SCRP1. The upper panel shows 3.8- and 2.7-kb transcripts of SCRP1 (arrow heads). (B) XSema6D4. The upper panel shows about 6.2 and 5.8-kb transcripts of XSema6D4 (arrow heads). Numbers, Nieuwkoop and Faber developmental stages; 18S rRNA stained with ethidium bromide, loading control.
	Fig. 6. WISH analysis of XSema6D4 (N30H6) during early development. (A) Late blastula stage (lateral view; animal pole to the top). (B) Late blastula stage (transversal section; animal pole to the top). (C) Mid gastrula stage (dorsal view; anterior to the top). (D) Sagittal section of mid gastrula. Arrows indicate the organizer (C,D). (E) Mid neurula stage (dorsal view; anterior to the top). Arrowheads indicate the blastopore (D,E). (F) Sagittal hemisection of mid neurula (dorsal to the top; anterior to the left). (G) Late neurula stage (anterior view; dorsal to the top). An arrow indicates the presumptive otic vesicle. (H) Tailbud stage (dorsal view; anterior to the left). (I) Late tailbud stage (lateral view; anterior to the left). Embryos were treated with BB/BA to become transparent (A,D,E,G~I). Developmental stages and orientations are indicated on the bottom of each panel: d, dorsal; l, lateral; a, anterior; t, transversal; s, sagittal. Abbreviations: anp, anterior neural plate; ba, branchial arches; bc, blastocoel; br, brain; n, notochord; ov, otic vesicle; p, pronephros; sc, spinal cord; sl sensorial layer; sm somites; tg, trigeminal ganglion; yp, yolk plug.
	lrrc1 (leucine rich repeat containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage20, lateral view, anterior left, dorsal up.
	pdcd10 ( programmed cell death 10 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, dorsal view, anterior left.
	pdcd10 ( programmed cell death 10 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, lateral view, anterior left, dorsal up.
	nono (non-POU domain containing, octamer-binding) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17 dorsal view, anterior left.
	nono (non-POU domain containing, octamer-binding) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, lateral view, anterior left, dorsal up.
	nono (non-POU domain containing, octamer-binding) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12.5 dorsal view, anterior left.
	cdkn2aip (CDKN2A interacting protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, anterior view, dorsal up.
	cdkn2aip (CDKN2A interacting protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, lateral view, anterior left, dorsal up.
	rpl 32 (ribosomal protein L32) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
	rpl 32 (ribosomal protein L32) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, anterior view, dorsal up.
	utp15 (UTP15, U3 small nucleolar ribonucleoprotein, homolog) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
	utp15 (UTP15, U3 small nucleolar ribonucleoprotein, homolog) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, anterior view, dorsal up.