Arima K et al. (2005), Global analysis of RAR-responsive genes in the ...

XB-ART-2547

Dev Dyn 2005 Feb 01;2322:414-31. doi: 10.1002/dvdy.20231.

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Global analysis of RAR-responsive genes in the Xenopus neurula using cDNA microarrays.

Arima K , Shiotsugu J , Niu R , Khandpur R , Martinez M , Shin Y , Koide T , Cho KW , Kitayama A , Ueno N , Chandraratna RA , Blumberg B .

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Retinoid signaling is important for patterning the vertebrate hindbrain and midaxial regions. We recently showed that signaling through retinoic acid receptors (RARs) is essential for anteroposterior patterning along the entire body axis. To further investigate the mechanisms through which RARs act, we used microarray analysis to investigate the effects of modulating RAR activity on target gene expression. We identified 334 up-regulated genes (92% of which were validated), including known RA-responsive genes, known genes that have never been proposed as RA targets and many hypothetical and unidentified genes (n = 166). Sixty-seven validated down-regulated genes were identified, including known RA-responsive genes and anterior marker genes. The expression patterns of selected up-regulated genes (n = 45) were examined at neurula stages using whole-mount in situ hybridization. We found that most of these genes were expressed in the neural tube and many were expressed in anterior tissues such as neural crest, brain, eye anlagen, and cement gland. Some were expressed in tissues such as notochord, somites, pronephros, and blood islands, where retinoic acid (RA) plays established roles in organogenesis. Members of this set of newly identified RAR target genes are likely to play important roles in neural patterning and organogenesis under the control of RAR signaling pathways, and their further characterization will expand our understanding of RA signaling during development.

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Species referenced: Xenopus
Genes referenced: 1a11 acsl1 agr2 aldh1a2 cdx4 cldn1 cyp11a1 cyp26a1 dhrs3 dusp6 epc2 etv1 fetub fgf2 fli1 foxa1 frzb gata2 gata4 gata5 gbx2.2 gdf3 gsc gse1 hnf1b hoxa1 hoxd4 hrg lmo2 lmo4.1 mdk meis1 meis3 mxi1 ncoa3 ndrg1 nkain4 nop58 nptn nr5a2 nr6a1 otx2 pitx1 pkdcc.1 pkdcc.2 prph rab40b rara sall1 sema3fl shd slc12a1 slc27a2 slc38a8 sox3 tcaf1 tcaf2 tfcp2l1 tll2 tmem150c tmem38a tubb4a ubp1 xa-1 xepsin

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	Figure 4. Expression patterns of selected retinoic acid receptor (RAR) target genes. Expression pattern of selected upregulated genes at stage 18 and stage 22 were determined by whole-mount in situ hybridization. Embryos were hybridized with probes made from indicated expressed sequence tag (EST) clones. The staining patterns in whole and sectioned embryos were summarized and used to categorize the genes broadly into four groups (group 1, 2, 3, and 4) by expression patterns. The signature staining of group 1 is present in the neural tube, neural crest, and optic anlagen; the staining patterns of group 2 are similar to those of group 1 but absent from the neural crest. Group 3 genes show staining in the neural tube but not in the optic anlagen. Group 4 genes have staining in the lateral plate mesoderm where RALDH2 is predominantly expressed. A, B, and C are in group 1; D, E, and F are in group 2; G, H, and I are in group 3; and J, K, and L are in group 4. A1-L1, A3-L3, M1-R1, and M2-R2: dorsal view; A2-I2 and A4-I4: anterior view; C6 and J2-L2: lateral view; C7: ventral view; C5: J4-L4 and M3-R3: sagittal section of stage 22 embryos. VLCS, very-long-chain acyl-CoA synthetase; Hypothetical, similar to ESTs from other organisms; Unidentified, no significant similarity to database sequences from any organism; Sm, somites; rp, roof plate of the neural tube; no, notochord; nt, the neural tube.
	Fig. 2. Northern blot analysis. Equal amounts of total RNA isolated from controls or embryos treated with TTNPB or AGN 193109 were electrophoresed, blotted to nylon membranes, and hybridized with probes derived from each clone. The name of each clone used for probe-templates and its closest match found in public databases are given on the left of each panel. Fold and (PCR) indicate change in microarray analysis and a validation result of quantitative real-time reverse transcriptase-polymerase chain reaction (QRT-PCR). C, T, and 109 indicate control, TTNPB, and AGN193109 treatment, respectively. The asterisks indicate known RA target and/or posterior marker genes, and ISH indicates clones used as probes for whole-mount in situ hybridization. MK, Midkine; VLCS, very-long-chain acyl-CoA synthetase; SDR1, short-chain dehydrogenase/reductase; LCS, long-chain acyl-CoA synthetase.
	Fig. 5. The effects of modulated retinoid signaling on expression pattern of retinoic acid receptor (RAR) target genes. The effects of AGN 193109, TTNPB, or vehicle control on expression pattern of three expressed sequence tags (ESTs), XL016p20 (S1�S6), XL049b08 (T1�T6), and XL043c08 (U1�U6), were determined in stage 18 embryos by whole-mount in situ hybridization. AGN193109 treatment reduced posterior neural expression of all three genes compared with controls (S1 vs. S2, T1 vs. T2, and U1 vs. U2) but increased medial staining of XL016p20 (S1 vs. S2), XL049b08 staining in the eye anlagen (T4 vs. T5), and XL043C08 expression in the forebrain (U1 vs. U2). In contrast, TTNPB treatment increased expression of XL016P20 throughout the embryo (S3, S6 vs. S2, S5) and reduced Xl043C08 staining in the forebrain and presumptive trigeminal ganglion (U3, U6 vs. U2, U5). S1�S3, T1�T3, and U1�U3: dorsal view; S4�S6, T4�T6, and U4�U6: anterior view; TTNPB, TTNPB-treated embryos; Control, vehicle control embryo; AGN193109, AGN193109-treated embryos.
	cyp11a1 (cytochrome P450, family 11, subfamily A, polypeptide 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior left.
	nkain4 (Na+/K+ transporting ATPase interacting 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, anterior view, dorsal up.
	pkdcc.2 (protein kinase domain containing, cytoplasmic) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, dorsal up, anterior left.
	shd (Src homology 2 domain containing transforming protein D) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior left.
	slc27a2 (solute carrier family 27 (fatty acid transporter), member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
	tfcp2l1 (transcription factor CP2-like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior left.
	tmem38a (transmembrane protein 38A) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior left.
	tmem150c (transmembrane protein 150C) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior left.
	1a11 (71 kDa protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, dorsal up (J1 left), and lateral view (J2, right) anterior left.
	1a11 (71 kDa protein) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, dorsal up (J3 left), and lateral view (J4, right) anterior left.