Yoon J , Kim JH , Kim SC , Park JB , Lee JY , Kim J .

	Fig. 1. PV.1 suppresses neural gene expression. (A) PV.1 RNAs of PV.1 was injected as indicated at the one-cell stage. Phenotypical changes were observed at stage 33. DAI means the dorso- anterior index. (B) PV.1 (500 pg) was either injected alone or co-injected with DNBR (1 ng) at the one-cell stage. Animal cap explants were dissected at stage 8, incubated until stage 10 and then RT-PCR was performed for the analysis of relative gene expression. ODC, loading control; noRT, control reaction without reverse transcriptase; WE, Whole embryo as a positive control; chordin, Dorsal mesodermal marker; Xbra, mesodermal marker; FoxD5b and Zic3, early neural marker. (C) The temporal expression pattern of FoxD5b and PV.1 were analyzed using RT-PCR at various developmental stages as indicated. (D) PV.1 RNAs were injected into one side of each of the two cell embryos. These embryos were processed for whole mount in situ hybridization with anti-sense probe of FoxD5b at stage 10. The injected side of the embryos is indicated by Beta-gal staining. The blue dotted line indicates the dorsal lip.
	Fig. 2. PV.1 reduces FoxD5b and Zic3 expression. (A, B) Embryos were injected with RNAs (PV.1, 500 pg and DNBR, 1 ng) at the one-cell stage as indicated. Animal explants were dissected at stage 8 and incubated in the animal cap media containing DMSO or cyclohexamide (CHX, 5 ng/ml) until stage 10. RT-PCR was performed to analyze the relative FoxD5a and Zic3 expressions. Data are shown as the means ± the S.D. of the values from at least 3 independent experiments. Differences were considered significant at P < 0.05.
	Fig. 3. PV.1 reduces the promoter activity of FoxD5b. (A) Embryos were co-injected with the -1336 construct (20 pg) and PV.1 (500 pg) at the one-cell stage and incubated until stage 10. Luciferase activity was measured as described in the “Materials and Methods”. (B) The graph shows regions of high similarity based upon the NCBI-BLAST results. (C) Schematic representation of the serially truncated FoxD5b promoter constructs. (D) The embryos were injected with FoxD5b promoter alone or with PV.1 as indicated. Luciferase activities were measured at stage 10. The data are shown as the means ± the S.D. of the values from at least three independent experiments. Differences were considered significant at P < 0.05.
	Fig. 4. PV.1 regulates FoxD5b expression indirectly via the putative Hox binding site. (A) Embryos were co-injected -1336 construct with RNAs (PV.1, 500 pg and DNBR, 1 ng) at the one-cell stage as indicated. Embryos were harvested at stage 10 for analyses of the relative luciferase gene expressions using RT-PCR. (B) Schematic representation of -301_(m)Hox construct (C) The -301 or -301_(m) Hox constructs were either injected alone or co-injected with PV.1 at the one-cell stage. Luciferase activity was measured at stage 10. (D) RNAs of PV.1 (500 pg) were injected at the one-cell stage. Animal caps were dissected at stage 8 and incubated until stage 10. RT-PCR was performed for the analysis of the expression of the indicated genes. The data are shown as the means ± the S.D.s of the values of at least three independent experiments. Differences were considered significant at P < 0.05.

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