Tanibe M et al. (2008), Retinoic acid metabolizing factor xCyp26c is sp...

XB-ART-38796

Int J Dev Biol 2008 Jan 01;527:893-901. doi: 10.1387/ijdb.082683mt.

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Retinoic acid metabolizing factor xCyp26c is specifically expressed in neuroectoderm and regulates anterior neural patterning in Xenopus laevis.

Tanibe M , Michiue T , Yukita A , Danno H , Ikuzawa M , Ishiura S , Asashima M .

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Anterior-posterior neural patterning is determined during gastrulation when head structure is induced. Induction of anterior neural structures requires inhibition of Wnt signaling by several Wnt antagonists. We performed microarray analysis to isolate genes regulated by canonical Wnt signaling and abundantly expressed in the anterior neuroectoderm at the early neurula stage. We identified xCyp26c, a Cyp26 (RA-metabolizing protein)-family gene. In situ hybridization showed xCyp26c expression restricted to the anterior region of neurula, while xCyp26a was expressed in both anterior and posterior regions. At the tadpole stage, xCyp26c was also expressed in restricted sets of cranial nerves. Microarray, RT-PCR and in situ hybridization analyses revealed decreased xCyp26c expression with overexpression of beta-catenin, suggesting regulation by Wnt/beta-catenin signaling. We also assessed the effects of retinoic acid (RA) on xCyp26c expression. Embryos treated with 10(-7) M RA showed an anterior shift in the spatial expression of xCyp26c, reflecting a posteriorization effect. Conversely, expression patterns in embryos treated with more than 10(-6) M RA were less affected and remained restricted to the most anterior region. Moreover, injection of xCyp26c mRNA into animal poles caused head defects, and exogenous expression of xCyp26c rescued the posteriorizing effect of RA treatment. Taken together, these results implicated a role for xCyp26c in anterior patterning via RA signaling.

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Species referenced: Xenopus laevis
Genes referenced: cyp26a1 cyp26c1 egr2 en2 gal.2 otx2 rax

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	Fig. 2. Temporal and spatial pattern of xCyp26c expression. (A) Temporal pattern of xCyp26c expression was examined by RT-PCR analysis with cDNAs prepared from embryos at various stages. XCyp26c expression increased from the mid-late gastrula stage. This pattern is different from xCyp26a, which is ubiqui- tously expressed. (B-K) The spatial pattern of xCyp26c (B,C,F,G,J) and xCyp26a (D,E,H,I,K) expression was analyzed by whole-mount in situ hybridization. Differ- ent from xCyp26a, xCyp26c was expressed only in the presumptive anterior neural region at the early neurula stage (B,C, compared with D,E). At the mid- neurula stage, the midline of xCyp26c expression decreased (F,G). By the late neurula stage, xCyp26c expression was restricted to a set of rhombencepharic cells (J), whereas xCyp26a expression was greatly reduced (K). (L-Q) Double in situ hybridization. Brown staining indicates xCyp26c expression, while the light blue staining indicates xCyp26a (L,M), xOtx2 (N,O), and xkrox20 (P,Q) staining, respectively. Anterior view (L,N,O,P). Dorsal view (M). (Q) Magnified image of (M). r2-5 show rhombomeres 2 to 5, respectively. These data indicated that xCyp26c- positive cells were mainly identical with r2, r3, and r4.
	Fig. 3. Spatial expression of xCyp26c after tailbud stage. (A-B, D-E, G-H) XCyp26c expression. (C,F,I) XCyp26a expresion. Stage 24 (A-C), stage 28 (D-F), and stage 36 (G- I). XCyp26c was expressed in restricted sets of cranial nerves (GC(VII) and GC(IX)). XCyp26c was also expressed in the pharyngeal pouch (PP) and rhombomeric cells (r2/3 and r6). These patterns were obviously different from those of xCyp26a (C,F,I).
	Fig. 4. Regulation of xCyp26c expres- sion by canonical Wnt signaling. (A) RT-PCR analysis with cDNAs synthesized using β-catenin-injected embryos. Injec- tion of β-catenin mRNA into the animal pole region of embryos decreased xCyp26c expression in a dose-dependent manner. XCyp26a expression was not clearly decreased by β-catenin injection. (B-E) Expression of xCyp26c in mid-neu- rula embryos injected into the animal pole with nothing (B,C) or β-catenin (D,E). β-catenin clearly inhibited xCyp26c ex- pression (D,E).
	Fig. 5. Effect of retinoic acid (RA) on xCyp26c expression. (A-X) Embryos were treated with various doses of RA at stage 8, cultured until stage 13, and then prepared for WISH analysis. No treatment (A-C). RA treatment (D-X). Concentration of RA (M) is shown on left side. Expres- sion pattern of xCyp26c (A-E), xCyp26a (I-P) and xOtx2 and xkrox20 (Q- X). Moderate doses of RA increased xCyp26c expression, but narrowed the area of expression (A-M). Over 10-6 M of RA, xCyp26c expression became restricted in the anterior neuroectoderm and underwent no further change (F-H). XCyp26a was expressed throughout the embryo (O,P), whereas xOtx2 expression completely disappeared (V-X). (Y) Real-time PCR analysis with whole embryos treated with RA. These graphs are expressed as ratio of expression level of RA-treated embryo (lane 2-7) to that of no-RA treated embryo (lane 1).
	Fig. 6. Overexpression of xCyp26c could attenuate retinoic acid (RA)-induced posteriorization. (A-F) Phenotype of tad- pole injected with xCyp26c mRNA. (A-C) Normal tadpoles. (D- F) Tadpoles injected with 1 ng of xCyp26c mRNA into the animal pole region. (B,C,E,F) Histological section of xCyp26c-injected tadpoles. Sections were stained by hematoxylin-eosin. (B,E) Sagittal sections, whereas (C,F) show transverse sections of (A,D), respectively. (G-I) Head defect by RA treatment was rescued by xCyp26c injection. Embryos not injected (G) or xCyp26c mRNA-injected embryos (H) were treated with 10-6 M of RA. In xCyp26c-injected embryos, loss of eye structure was rescued (arrow). These results are summarized in graph (I). White box, dotted box, and filled box show normal eye, small eye, and no eye structure, respectively. Point out that the size of eye vesicle reflects the severity of posteriorizaion by RA treatment. (J) RT-PCR analysis with cDNAs synthesized from whole embryo injected with β-gal (1 ng; lanes 1-3) or xCyp26c (1 ng; lanes 4-6). Embryos were treated with 10-7 M (lane 2, 5) or 10-6 M (lane 3, 6) of RA, or untreated (lane 1, 4). Overexpres- sion of xCyp26c attenuated the inhibition of xBF-1 (telencepha- lic marker; column 1) and xRX1 (diencephalic marker; column 2) expression by RA treatment (lane 6). (K-N) Change of expres- sion patterns of En2 and xkrox20 by xCyp26c injection. (L,N) are magnified views of (K,M), respectively. Injected side (inj. In (K) and (M)) was indicated by lineage tracer (Red-gal). (K,L) show no RA-treated embryos and (M,N) show 10-7 M RA-treated embryos. Normal embryos showed a slight posterior shift of xkrox20 expression in the xCyp26c-injected area (white arrow- head, compared with noninjected region (black arrowhead)). RA-treated embryos showed an obvious difference in xkrox20 expression patterns between injected and noninjected regions.
	cyp26a1 (cytochrome P450, family 26, subfamily A, polypeptide 1 ) gene expression in Xenopus laevis embryos, NF stage 13, as assayed by in situ hybridization, dorsal view, anterior up.
	cyp26a1 ( cytochrome P450, family 26, subfamily A, polypeptide 1) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	cyp26a1 (cytochrome P450, family 26, subfamily A, polypeptide 1) gene expression in Xenopus laevis embryo, NF stage 36, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	cyp26c1 (cytochrome P450, family 26, subfamily C, polypeptide 1) gene expression in Xenopus laevis embryos, NF stage 14, as assayed by in situ hybridization, anterior view, dorsal up.
	cyp26c1 (cytochrome P450, family 26, subfamily C, polypeptide 1) gene expression in Xenopus laevis embryos, NF stage 18, assayed by in situ hybridization, anterior view, dorsal up.
	cyp26c1 ( cytochrome P450, family 26, subfamily C, polypeptide 1 ) gene expression in Xenopus laevis embryo, NF stage 24, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	cyp26c1 (cytochrome P450, family 26, subfamily C, polypeptide 1) gene expression in Xenopus laevis embryos, NF stage 34, assayed by in situ hybridization, lateral view, anterior left, dorsal up.