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PLoS One
2015 Sep 01;109:e0138588. doi: 10.1371/journal.pone.0138588.
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Rdh10a Provides a Conserved Critical Step in the Synthesis of Retinoic Acid during Zebrafish Embryogenesis.
D'Aniello E
,
Ravisankar P
,
Waxman JS
.
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The first step in the conversion of vitamin A into retinoic acid (RA) in embryos requires retinol dehydrogenases (RDHs). Recent studies have demonstrated that RDH10 is a critical core component of the machinery that produces RA in mouse and Xenopus embryos. If the conservation of Rdh10 function in the production of RA extends to teleost embryos has not been investigated. Here, we report that zebrafish Rdh10a deficient embryos have defects consistent with loss of RA signaling, including anteriorization of the nervous system and enlarged hearts with increased cardiomyocyte number. While knockdown of Rdh10a alone produces relatively mild RA deficient phenotypes, Rdh10a can sensitize embryos to RA deficiency and enhance phenotypes observed when Aldh1a2 function is perturbed. Moreover, excess Rdh10a enhances embryonic sensitivity to retinol, which has relatively mild teratogenic effects compared to retinal and RA treatment. Performing Rdh10a regulatory expression analysis, we also demonstrate that a conserved teleost rdh10a enhancer requires Pax2 sites to drive expression in the eyes of transgenic embryos. Altogether, our results demonstrate that Rdh10a has a conserved requirement in the first step of RA production within vertebrate embryos.
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26394147
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Fig 2. Rdh10a deficient embryos have decreased expression of RA signaling responsive genes.(A-F) ISH for cyp26a1, hoxb5b, and dhrs3a expression in control sibling and Rdh10a deficient embryos. In A and B, views are lateral with dorsal rightward. In C-F, views are dorsal. In all images anterior is up. Arrows in B, D and F indicate decreased expression relative to control sibling embryos. (G) RT-qPCR for RA signaling responsive genes.
Fig 3. Rdh10a deficient embryos have increased CM number.(A, B) Hearts from control sibling (n = 10) and Rdh10a deficient (n = 10) Tg(-5.1myl7:DsRed2-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (C) Mean CM number at 48 hpf. (D) RT-qPCR for CM differentiation marker gene expression at 48 hpf.
Fig 4. Rdh10a depletion enhances defects in nls/aldh1a2 mutant embryos.(A, D) WT control and Rdh10a deficient embryos. (B,E) Control and Rdh10a deficient embryos heterozygous for the nls allele. (C,F) Control and Rdh10a deficient embryos homozygous for the nls allele. Nls mutant embryos (C) show anteriorization of nervous system (black arrowhead), increases in the size of the head (black bracket), and pericardial edema. Rdh10a deficient; nls mutant embryos (F) have an accentuated anteriorization of the nervous system (arrowhead), larger and dysmorphic head (red bracket), and enhanced pericardial edema compared to nls embryos. Dorsal is up and anterior is to the right.
Fig 5. Rdh10a overexpression promotes embryonic RA signaling.(A, F) Control DMSO treated, (B,G) rdh10a mRNA (300 pg), (C,H) ROL treated (15 μM), and (D,I) ROL treated, rdh10a mRNA injected embryos. (A-D) At 48 hpf, DMSO and rdh10a mRNA injected embryos were overtly normal. 15 μM ROL produced overtly normal embryos, except some have a modestly shortened, kinked tail (arrow in C). ROL treated, rdh10a mRNA injected embryos were severely truncated. Views are lateral with dorsal up and anteriorright. (E) Percentage of truncated embryos at 48 hpf in A-D. For each condition n = 19 embryos. (F-I) ISH for egfp expression in Tg(12XRARE-ef1a:EGFP)sk72 embryos. In F, pn indicates pronephros and sc indicates spinal cord. In G, arrowhead indicates anterior neural expression. Views are lateral with dorsal right and anterior up.
Fig 6. Differential abilities of ROL, RAL and RA to promote RA signaling.(A-D) EGFP fluorescence in Tg(12XRARE-ef1a:EGFP)sk72 embryos after treatment with 15 μM ROL, 1 μM RAL, and 0.5 μM RA beginning at 24 hpf. (E-H) ISH for egfp expression in Tg(12XRARE-ef1a:EGFP)sk72 embryos after treatment with 15 μM ROL, 1 μM RAL, and 0.5 μM RA beginning at 24 hpf. Arrows indicate the boundaries of expression. Views are lateral with dorsal right and anterior up.
Fig 7. Comparison of RA, RAL, and ROL teratogenicity.(A-J) Embryos at 48 hpf that were treated from sphere stage through 24 hpf with DMSO, ROL, RAL, and RA. (D,G) Low concentrations of RAL (0.1 μM) and ROL (10 μM) did not cause significant overt defects. (B,E,H) Intermediate concentrations of RAL (1 μM) and ROL (20 μM) and the lower concentration of RA (0.025 μM) produced truncated embryos that still had eyes (arrows). (C,F,I) 0.05 μM RA, 2 μM RAL, and 40 μM ROL produced severely truncated embryos without eyes.
Fig 8. ROL, RAL, and RA eliminate CMs.(A-D) Hearts from Tg(-5.1myl7:DsRed-NLS)f2 embryos treated with DMSO, 20 μM ROL, 1 μM of RAL and 0.025 μM RA. Images are frontal views. Red indicates ventricle. Green indicates atrium. (E) Mean CM number at 48 hpf.
Fig 9. Zebrafish Pax2 proteins bind a putative Rdh10a enhancer.(A) Alignment of vertebrate rdh10 genes to the zebrafish rdh10a taken from the UCSC genome browser. (B) Sequence alignment of the E2 enhancer in teleosts. Red nucleotides indicate the predicted Pax2 binding site. Blue indicates the Pea3 binding site (C-E) Representative images from transient transgenic embryos with rdh10a E2-enhancer expression in the notochord, heart, and eye. (F) Mutations that were made in the promoter of the rdh10a E2-enhancer and the oligos used for EMSA. (G,H) EMSA with the WT and mutated Pax2 sites and zebrafish Pax2a and Pax2b.
Fig 1. Rdh10a deficient embryos have decreased RA signaling.(A) Schematic of the metabolic pathway that synthesizes RA during development and negative regulation of Rdh10 and Aldh1a2 expression from mice and Xenopus studies [23, 28, 29]. (B) Control sibling embryo. (C) Embryos injected with rdh10a MOs. Arrow indicates pericardial edema. Arrowhead indicates yolk extension. (D-E) ISH for egfp expression in Tg(12XRARE-ef1a:EGFP)
sk72 control sibling and Rdh10a deficient embryos. Brackets indicate the length of egfp expression in the spinal cord. (F) Measurements of egfp expression length in the spinal cord using arbitrary units (AU) from in situ hybridization (ISH) of Tg(12XRARE-ef1a:EGFP)
sk72 control embryos (n = 16) and Rdh10a deficient embryos (n = 15). (G) RT-qPCR for egfp expression at 24 hpf in control sibling and Rdh10a deficient embryos. In all graphs, asterisks indicate a statistically significant difference (p<0.05) and bars indicate standard deviation.
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