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First published online 5 May 2004
doi: 10.1242/dev.01129

Development 131, 2653-2667 (2004)
Published by The Company of Biologists 2004
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Multiple points of interaction between retinoic acid and FGF signaling during embryonic axis formation

Jason Shiotsugu1,*, Yu Katsuyama1,*,{dagger}, Kayo Arima1, Allison Baxter1, Tetsuya Koide1, Jihwan Song2, Roshantha A. S. Chandraratna3 and Bruce Blumberg1,{ddagger}

1 Department of Developmental and Cell Biology, University of California, Irvine, CA 92697-2300, USA
2 Laboratory of Stem Cell Biology, Cell & Gene Therapy Research Institute, Pochon CHA University College of Medicine, Seoul 135-081, Korea
3 Retinoid Research, Departments of Chemistry and Biology, Allergan, Irvine, CA 92623, USA



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  		Fig. 1. Developmental expression of XRAR{alpha}. Whole-mount in situ hybridization was performed on embryos from stage 9 to stage 25 using a probe that recognizes all isoforms of XRAR{alpha}. (A) Dorsal (left) and ventral (right) view of a stage 9 embryo. (B,D,F,H,J) Frontal views. (C) A dorsal view of the stage 10 embryo. (E) A vegetal view of the stage 10 embryo. Note the sharp anterior border of strong staining.

 


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  		Fig. 2. XRAR{alpha}2.2 loss-of-function leads to axial truncations and reduction of HOXB9 expression. (A-D) Microinjection of RAR-MO causes anterior and posterior truncations at highest frequency when expressed in the head region (A) or dorsally (B). (C) Phenotypes are mild to undetectable when the lineage tracer is distributed laterally or ventrally. (D) Phenotypes are rescued by co-injection of XRAR{alpha}2 mRNA, irrespective of where the lineage tracer is located. (E,F) Neither XBRA (E) nor XWNT8 (F) expression is affected by RAR-MO injection. (G-K) Effects of RAR-MO on the expression of HOXB9. (H-J) The types of phenotypes obtained. (K) HOXB9 expression was restored by co-injecting XRAR mRNA and RAR-MO.

 


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  		Fig. 3. Modulating retinoid signaling affects the expression of XCAD3 but not XCAD1 or XCAD2. (A-C,F-H,K-M) Embryos were treated with the indicated compound or ethanol solvent controls from the early blastula stage (stage 7) until harvesting when control embryos reached stage 18. Embryos were fixed and processed for whole-mount in situ hybridization with the indicated probes. (A,F,K) 10–6 M AGN193109 (RAR-selective antagonist), (B,G,L) ethanol solvent control, (C,H,M) 10–6 M TTNPB (RAR-selective agonist). RAR-MO was injected unilaterally at the two-cell stage with ß-galactosidase lineage tracer alone (D,I,N) or together with 1 ng XRAR{alpha}2 mRNA (E,J,O). Embryos were fixed when controls reached stage 18, stained for ß-galactosidase activity and processed for whole-mount in situ hybridization with the indicated probes. Some embryos were used for RNA extraction and QRT-PCR analysis as described in the text.

 


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  		Fig. 4. FGF8 cannot rescue the effects of XRAR{alpha}2.2 loss-of-function on posterior marker genes. Embryos were microinjected at the two-cell stage with the indicated reagents, allowed to develop until controls reached stage 18 and processed for whole-mount in situ hybridization with either HOXB9 (A-E) or XCAD3 (F-J) probes.

 


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  		Fig. 5. A constitutively active RAR, but not RA, can rescue the effects of FGF gene loss-of-function on posterior markers. Embryos were microinjected unilaterally at the two cell stage with ß-galactosidase mRNA as lineage tracer and (A,D) 1 ng of XFD mRNA, (B,E) 1 ng of XFD mRNA then treated with 10–6 M atRA, or (C,F) 1 ng of XFD and 1 ng VP16-XRAR{alpha}2 mRNA. When control embryos reached stage 18, the embryos were fixed, stained for ß-galactosidase activity and processed for whole-mount in situ hybridization with either HOXB9 (A-C) or XCAD3 (D-F) probes.

 


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  		Fig. 6. FGF gene loss of function alters the expression of RAR signaling pathway components in microinjected embryos. Embryos were microinjected unilaterally into one blastomere at the two- or four-cell stage with 1 ng of XFD mRNA and ß-galactosidase mRNA as lineage tracer. Embryos were allowed to develop until controls reached stage 11 then fixed and processed for whole-mount in situ hybridization with the probes (A,B) XCAD3, (C,D) XRAR{alpha}, (E,F) RALDH2 or (G,H) CYP26.

 


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  		Fig. 7. FGF8 and RA induce members of the other signaling pathways in animal caps. Embryos were microinjected with mRNA encoding FGF8 mRNA. Caps were cut from FGF8 or control embryos at or before stage 9 and allowed to develop until untreated sibling embryos reached stage 20 in the presence or absence of 10–6 M all-trans RA. RNA was prepared from the caps and control embryos and QRT-PCR analysis performed with the indicated primer sets. Experiments were performed in triplicate and reproduced in independent experiments (Student's paired t-test). P<0.03 for all data sets.

 


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  		Fig. 8. XRAR{alpha}2.2 loss-of-function alters the expression of FGF8, FGFR4 and FGFR1. Embryos were microinjected unilaterally with ß-galactosidase mRNA plus 10 ng RAR-MO or 10 ng RAR-MO plus 1 ng XRAR{alpha}2 mRNA. Embryos were fixed when control uninjected embryos reached stage 18, stained for ß-galactosidase activity and then processed for in situ hybridization with FGF8 (A-F), FGFR4 (G-L) or FGR1 (M-R) probes.

 


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  		Fig. 9. XRAR{alpha}2.2 is required for XCAD3-mediated upregulation of HOXB9. Embryos were microinjected with 10 ng RAR-MO (B), 1 ng XCAD3 mRNA (C) or 10 ng RAR-MO plus 1 ng XCAD3 mRNA (D-F), then allowed to develop until untreated embryos reached stages 18 (A-D,F) or 23 (E), fixed and processed for whole mount in situ hybridization with HOXB9.

 


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  		Fig. 10. XCAD3 expression requires RAR at early but not late stages. Embryos were treated with either TTNPB (E-H) or AGN193109 (I-L) at the blastula stage and cultured until controls (A-D) reached the indicated stages, fixed and processed for in situ hybridization with XCAD3. (A,C,E,G,I,K) Lateral views; (B,D,F,H,J,L) dorsal views.

 


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  		Fig. 11. Schematic model of the interactions among FGF, RAR and XCAD3 signaling pathways.

 

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© The Company of Biologists Ltd 2004