Koide T , Downes M , Chandraratna RA , Blumberg B , Umesono K .

GM-60572 NIGMS NIH HHS , R01 GM060572 NIGMS NIH HHS

	Figure 1. Developmental expression of corepressors and xRARαs. (A) RT–PCR analysis during development (stages indicated above the lanes) shows maternal and zygotic transcription of xSMRT, xN-CoR, xRARα1, and xRARα2 compared with histone H4 control. (B) Whole-mount in situ hybridization analysis of xRARαs, xSMRT, and xN-CoR transcripts. (St. 10+) Dorsal view of early gastrula embryos. xRARαs and both corepressors are expressed in the ectoderm and dorsal blastopore lip. (St. 10.5, St. 12.5) Embryos were cut sagittally and oriented with the animal side up and dorsal lip to the right. xRARαs, xSMRT, and xN-CoR are expressed in the ectoderm, dorsal and ventral mesoderm. (St. 14) Dorsal side is up, anterior side is left. xSMRT and xN-CoR are expressed in the anterior neuroectoderm, but xRARαs are not expressed in this region.
	Figure 2. Transcriptional repression by RARα inXenopus embryos. Gal4UAS reporter plasmid was injected either alone (left), together with mRNA encoding Gal4–xRARα (200 pg) (middle, GAL–RAR), or together with Gal4–xRARα (200 pg) and c-SMRT (4 ng) mRNAs (right, GAL–RAR+C-SMRT) into both dorsal blastomeres at the 8-cell stage. Embryos were collected at stage 16 and luciferase assays performed. Similar results were obtained from three independent experiments.
	Figure 3. Effects of c-SMRT mRNA and morpholino oligonucleotide microinjection on Xenopus development. (a) Treatment with 10−6 RA causes anterior truncations; (b) c-SMRT overexpression causes anterior truncation similar to those elicited by RA treatment; (c) embryos microinjected with 10 ng of a control morpholino antisense oligonucleotide; (d) embryos microinjected with 10 ng of a morpholino antisense oligonucleotide directed against xRARα1; (e) embryos microinjected with 10 ng of a morpholino antisense oligonucleotide directed against xRARα2; (f) embryo microinjected with 10 ng of a morpholino antisense oligonucleotide directed against both xRARα1 and xRARα2; (g) 10 ng of the xRARα2 morpholino was rescued by coinjection with 500 pg of xRARα2 mRNA; (h) 10 ng of the xRARα2 morpholino was rescued by coinjection with 500 pg of dominant-negative xRARα mRNA; (i) 10 ng of the xRARα2 morpholino was not rescued by coinjection with 500 pg of constitutively active VP16–xRARα mRNA.
	Figure 4. Changes in anterior neural marker expression elicited by removal of RAR-mediated repression. Whole-mount in situ hybridization analysis of Otx2 (a,c,i,k) or BF1/Xen2 (b,d,j,l). (a,b,e,f) Control embryos injected with GFP mRNA. (c,d,g,h) Embryos injected with the control morpholino oligonucleotide. (i,j) Embryos injected with 4 ng of C-SMRT and 200 pg of GFP mRNAs. (k,l) Embryos injected with 10 ng of morpholino antisense oligonucleotide directed against xRARα2 and 200 pg of GFP mRNA. (e–h,m–p) Fluorescence view of the embryos shown in a–d and i–l, revealing the location of the GFP lineage tracer. Embryos were fixed at stage 16–18 and are shown with dorsal upward.
	Figure 5. Effect of inverse agonist (AGN193109) on Xenopus development and expression of anterior markers. (a) The effects of AGN193109 were tested on 10−6 M RA-treated embryos. RA treated embryos (top), 10−6M RA + 10−7 M AGN193109 (second), 10−6 M RA + 10−6 M AGN193109 (third), 10−6 M RA + 10−5 M AGN193109 (fourth). (b) The effects of AGN193109 on normal Xenopus development. AGN193109 causes enlargement of head and short trunk (top three) compared with control embryo (bottom). (c) Dorsal view of embryo treated with AGN193109 (top) and control embryo (bottom). (d) Effects of RA and AGN193109 on anterior neural markers. Embryos were treated with RA (lane 1, RA), with AGN193109 (lane 3, 109), solvent control (lane2, sol) and all were analyzed by RNase protection assay at the neural late stage (stage 20) for the expression of Xotx2, XBF1, and XANF1. Whereas RA suppressed the expression of all three markers (lane1, RA), AGN193109 enhanced expression of the same markers (lane 3, 109).
	Figure 6. Modulating RAR signaling affects head induction bycerberus. (a) An ectopic head is induced in an embryo injected with 100 pg of cerberus mRNA in the D4 blastomere at 32-cell stage. (b) Ectopic head formation by cerberusis enhanced when embryos are treated with AGN193109 or (d) 1 ng of DN–RAR mRNA is coinjected with 100 pg of cerberus mRNA into the D4 blastomere. (c) The formation of ectopic heads bycerberus is inhibited when 1 ng of c-SMRT mRNA is coinjected with 100 pg of cerberus mRNA into the D4 blastomere. (e) Control embryo.

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