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Retinoic acid (RA) signaling is a central pathway regulating anterior-posterior patterning of the embryo through its targets, the Hox genes. RA is produced by two sequential oxidations from vitamin A (retinol) and this biosynthesis has to be regulated temporally, spatially and quantitatively. Mining Xenopus embryonic expression databases identified a novel component of the RA metabolic network, ADHFe1. Using Xenopus laevis embryos as our experimental system we determined the temporal and spatial pattern of AdhFe1 expression. Gain- and loss-of-function of ADHFe1 were induced to study its function and the regulation of the AdhFe1 gene by RA was studied. Expression analysis localized the ADHFe1 protein to the late Spemann's organizer, the trunk organizer. Subsequently, ADHFe1 can be detected in the prechordal mesoderm, the notochord and the lateral plate mesoderm. Manipulation of ADHFe1 levels affects the normal Hox gene expression. The effects of ADHFe1 manipulation can by rescued by increasing the levels of RA or its biosynthesis. Expression of the AdhFe1 gene is regulated by RA itself. ADHFe1 is an enzyme active already during gastrula stages and the protein is still present during neurula stages. ADHFe1 regulates the expression of the Hox genes during the early patterning of the trunk. The effect of ADHFe1 on Hox expression is mediated through regulation of RA levels. ADHFe1 probably reduces retinaldehyde to retinol thereby restricting the availability of retinaldehyde, the substrate needed by retinaldehyde dehydrogenases to produce RA making it a novel regulator of RA concentrations in the embryo and RA homeostasis.
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28621427
???displayArticle.link???Int J Dev Biol ???displayArticle.grants???[+]
Fig. 1. Xenopus laevis ADHFe1 is extensively conserved among vertebrates. The full length open reading frame encoded by the AdhFe1 gene was PCR cloned from embryonic Xenopus laevis RNA samples. (A) Protein sequence alignment of the X. laevis ADHFe1 protein to its orthologs from other vertebrates. Identical protein sequences are in gray boxes. The red line marks the human ADHFe1 sequence used to raise the polyclonal antibody. (B) Phylogenetic tree of the ADHFe1 protein family among representative vertebrates. The percent sequence identity to the X. laevis ADHFe1 sequence are shown.
Fig. 2. ADHFe1 begins expression during mid gastrula in the late Spemann’s organizer. (A) Expression of AdhFe1 was analyzed by qPCR using RNA samples from embryos of different developmental stages. The expression of AdhFe1 was compared to the temporal pattern of Rdh10 and Dhrs3 expression. The spatial distribution of AdhFe1 transcripts was determined by qPCR of RNA samples extracted from embryo fragments isolated from the dorsal, lateral and ventral regions of stage 12 embryos. (B). Marker gene expression was analyzed to determine the accuracy of the dissections. (C) Chordin expression was analyzed as a control for a dorsal-enriched gene. (D) Sizzled was utilized as a ventral-specific marker gene. (E) Expression of MyoD as a marker of the lateral and ventral regions.
Fig. 3. A polyclonal antibody against human ADHFe1 detects the X. laevis ADHFe1 protein. HEK293 cells were transfected with a XADHFe1 expression plasmid (pCS2-XADHFe1). All cells were co-transfected with a GFP expression plasmid to monitor the efficiency of transfection. (A) Protein extracts were prepared from the control (GFP only) and experimental samples and Western blot analysis was performed to detect the Xenopus ADHFe1 protein. (B) RNA samples were prepared from the same transfected cells and analyzed by qPCR to demonstrate the expression of the X. laevis AdhFe1 transcript. (C,D) Two-cell embryos were injected on one-side with the AdhFe1 antisense morpholino oligonucleotide (AdhFe1 MO; D) or with the control MO (C). The samples were co-injected with a lineage tracer. Embryos were processed for immunostaining for the endogenous ADHFe1 protein (purple) and the lineage tracer (turquoise). Dashed lines mark the boundary between the injected sides.
Fig. 4. The ADHFe1 protein localizes to the dorsal midline and the lateral plate mesoderm. The ADHFe1 protein was localized in embryos from various stages utilizing the anti-ADHFe1 antibody. (A) Mid gastrula (stage 11), vegetal view. (B) Late gastrula (stage 12), vegetal view. (C) Late neural fold stage (stage 17), rostral view. (D) Stage 17, dorsal view. (E) Sagital bisected embryo, stage 17. (F) Stage 22, lateral view. (G) Stage 22, dorsal view. (H) Detail of cross section of stage 22 bisected embryo.
Fig. 5. ADHFe1 regulates Hox gene expression through retinoic acid signaling. In order to manipulate the level of ADHFe1 activity, embryos were injected radially with increasing concentrations of AdhFe1 MO (A) or the pCS2-XADHFe1 expression plasmid (B). The changes in Hox gene expression were determined by qPCR. To demonstrate the involvement of RA signaling in the function of ADHFe1 we performed a rescue experiment. Embryos overexpressing ADHFe1 were also treated with the RA precursors, RAL (1mM) or ROL (10mM) and analyzed for changes in HoxA1 (C) and HoxB1 (D) expression by qPCR.
Fig. 6. AdhFe1 expression is negatively regulated by retinoic acid (RA). Embryos were treated with DEAB (100 mM), citral (40 mM) to inhibit the biosynthesis of RA or with ROL (10 mM), RAL (1 mM) and RA (1mM) to increase the levels of RA. All treatments were initiated during late blastula (stage 8) and analyzed by qPCR for changes in expression. Analysis of HoxB1 expression at stage 11 was used as a control to monitor the efficiency of the treatments (A). AdhFe1 expression was determined during mid- (stage 11)(B) and late gastrula (stage 12)(C).
adhfe1 (alcohol dehydrogenase, iron containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, vegetal view, dorsal up.
adhfe1 (alcohol dehydrogenase, iron containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, dorsal view, anteriorleft.
adhfe1 (alcohol dehydrogenase, iron containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anteriorleft, dorsal up.
