Complementary regulation of rbp7a and aldh1a2 by Nodal signaling.

[A-F] Lateral views of whole mount in situ stains of rbp7a in wild type embryos at 4hpf [A, C] 6hpf [B,D], 9hpf [E] and 10hpf [F]; [C-D] vibratome sections of 4hpf and 6hpf embryos. rbp7a signals are found in marginal cell [A-D], YSL, and in forerunner cells (arrowhead in [D-F]). [G-J] Marginal expression of aldh1a2 is strongly reduced in 6hpf MZsur [H] and MZoep [I] embryos; [J] RT-qPCR verified reduced expression levels of aldh1a2 in MZsur, MZoep as compared to wild type embryos. [K-P] Whole mount in situ hybridization for rbp7a (arrow heads) and goosecoid (gsc) (white asterisks) performed at 6 and 8 hpf in Msur^+/- as control [K, N], MZsur [L, O] and MZoep [M, P] embryos. Note that expression levels of rbp7a around the YSL were similar among the gsc positive embryos and the gsc negative MZsur [B, E] and MZoep [C, F] mutants. [Q-S] rbp7a expression at 24hpf. Arrows mark rbp7a signals in the posterior midbrain that were present in MZsur [R] and MZoep embryos [S] but not in control embryo [Q]. [T] RT-qPCR results for rbp7a in of 6hpf and 24hpf embryos. Graphed is the mean and SEM from triplicate experiments. Error bars indicated the SEM. Unpaired T-test was used to test the significance (*P<0.05).

rbp7a is a direct target of FoxH1.

[A] Schematic drawing of the rbp7a gene highlighting 4 potential FoxH1 binding sites (triangles: S1, S2, S3, and S4). Exons are indicated by boxes, the numbers correspond to the distance form the transcriptional start site. [B] Sequence comparison of the mouse FoxH1 consensus log [37] with the four potential sites in rbp7a. [C] In vitro EMSA studies with translated FoxH1 protein with oligonucleotides containing the four potential FoxH1 binding sites (sequences and positions were shown in [A, B]). Competition experiments with unspecific (unsp.comp) and specific (self.comp) unlabeled oligonucleotides were added to verify the binding specificity. [D] In vivo chromatin immunoprecipitation (ChIP-qPCR) experiments performed with 6hpf eGFP-foxH1 mRNA injected MZsur embryos. Bars show the enrichments of DNA fragments in the regions of FoxH1 binding sites in relation to a negative control region (rhodopsin promoter region) that was lacking FoxH1 binding sites (*P<0.05; error bars indicated the SEM). [E] Induced and depleted rbp7a expression in 5hpf wild type embryos after injection of fkh-vp16 and fkh-en mRNA, respectively.

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Subcellular localization of Nmnat1, Rbp7a and Nmnat1-Rbp7a proteins.

[A] PSORT based predictions for sub-cellular location of Nmnat1, Rbp7a and Nmnat1-Rbp7a fusion. [B] Confocal image scans of HEK cells transfected with indicated fusion proteins. Note the strictly cytoplasmic and nuclear GFP-signals for Rbp7a-GFP and Nmnat1-Rbp7a-GFP, respectively. Nmnat1-Rbp7a-GFP transfected cells show weak GFP signals throughout the cytoplasm and stronger focal signals in association with the nuclei and cellular protrusions. [C] Confocal image scans of 6hpf embryo injected with mRNA encoding indicated GFP-tagged proteins. Co-injected mRNA encoding H2B-RFP (nuclear RFP, middle column) was used as loading control and to outline nuclei. Control injections of H2B-GFP and memGFP (membrane GFP) were used to document GFP/RFP co-expression. Rbp7a-GFP and Nmnat1-Rbp7a-GFP both showed nuclear and cytoplasmic localizations; but Nmnat1-GFP was only found in the nucleus.