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Depletion of retinoic acid receptors initiates a novel positive feedback mechanism that promotes teratogenic increases in retinoic acid.

D'Aniello E, Rydeen AB, Anderson JL, Mandal A, Waxman JS - PLoS Genet. (2013)

Bottom Line: Here, we report that zebrafish embryos deficient for RA receptor αb1 (RARαb1), a conserved RAR splice variant, have enlarged hearts with increased cardiomyocyte (CM) specification, which are surprisingly the consequence of increased RA signaling.Importantly, depletion of RARαb2 or concurrent depletion of RARαb1 and RARαb2 also results in increased RA signaling, suggesting this effect is a broader consequence of RAR depletion.Concurrent depletion of RARαb1 and Cyp26a1, an enzyme that facilitates degradation of RA, and employment of a novel transgenic RA sensor line support the hypothesis that the increases in RA signaling in RAR deficient embryos are the result of increased embryonic RA coupled with compensatory RAR expression.

View Article: PubMed Central - PubMed

Affiliation: The Heart Institute, Molecular Cardiovascular Biology and Developmental Biology Divisions, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.

ABSTRACT
Normal embryonic development and tissue homeostasis require precise levels of retinoic acid (RA) signaling. Despite the importance of appropriate embryonic RA signaling levels, the mechanisms underlying congenital defects due to perturbations of RA signaling are not completely understood. Here, we report that zebrafish embryos deficient for RA receptor αb1 (RARαb1), a conserved RAR splice variant, have enlarged hearts with increased cardiomyocyte (CM) specification, which are surprisingly the consequence of increased RA signaling. Importantly, depletion of RARαb2 or concurrent depletion of RARαb1 and RARαb2 also results in increased RA signaling, suggesting this effect is a broader consequence of RAR depletion. Concurrent depletion of RARαb1 and Cyp26a1, an enzyme that facilitates degradation of RA, and employment of a novel transgenic RA sensor line support the hypothesis that the increases in RA signaling in RAR deficient embryos are the result of increased embryonic RA coupled with compensatory RAR expression. Our results support an intriguing novel mechanism by which depletion of RARs elicits a previously unrecognized positive feedback loop that can result in developmental defects due to teratogenic increases in embryonic RA.

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Reduction of RA in RARαb1 deficient embryos can rescue developmental defects.(A–H) Control sibling, RARαb1+Cyp26a1 deficient, control sibling treated with DEAB, and RARαb1+Cyp26a1 treated with DEAB embryos. In B and F, arrows indicates loss of the MHB and eng2a expression. Images are lateral views with dorsal right and anterior up. (I) Percentage of control sibling (n = 16), RARαb1+Cyp26a1 deficient embryos (n = 16), control sibling embryos treated with DEAB (n = 13), and RARαb1+Cyp26a1 deficient embryos treated with DEAB (n = 14) that had a MHB based on morphology. (J) Percentage of control sibling (n = 17), RARαb1+Cyp26a1 deficient embryos (n = 14), control sibling embryos treated with DEAB (n = 15), and RARαb1+Cyp26a1 deficient embryos treated with DEAB (n = 12) that had eng2a expression at the MHB. (K–N) Hearts from Tg(-5.1myl7:DsRed-NLS)f2 control sibling, RARαb1 deficient, DEAB treated, and DEAB+RARαb1deficient embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (O) Mean CM number at 48 hpf. (P,Q) Hearts from Tg(-5.1myl7:DsRed-NLS)f2 control sibling embryos and Tg(-5.1myl7:DsRed-NLS)f2 embryos treated with a low concentration of RA. Images are frontal views. Red indicates ventricle. Green indicates atrium. (R) Mean CM number at 48 hpf.
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pgen.1003689-g006: Reduction of RA in RARαb1 deficient embryos can rescue developmental defects.(A–H) Control sibling, RARαb1+Cyp26a1 deficient, control sibling treated with DEAB, and RARαb1+Cyp26a1 treated with DEAB embryos. In B and F, arrows indicates loss of the MHB and eng2a expression. Images are lateral views with dorsal right and anterior up. (I) Percentage of control sibling (n = 16), RARαb1+Cyp26a1 deficient embryos (n = 16), control sibling embryos treated with DEAB (n = 13), and RARαb1+Cyp26a1 deficient embryos treated with DEAB (n = 14) that had a MHB based on morphology. (J) Percentage of control sibling (n = 17), RARαb1+Cyp26a1 deficient embryos (n = 14), control sibling embryos treated with DEAB (n = 15), and RARαb1+Cyp26a1 deficient embryos treated with DEAB (n = 12) that had eng2a expression at the MHB. (K–N) Hearts from Tg(-5.1myl7:DsRed-NLS)f2 control sibling, RARαb1 deficient, DEAB treated, and DEAB+RARαb1deficient embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (O) Mean CM number at 48 hpf. (P,Q) Hearts from Tg(-5.1myl7:DsRed-NLS)f2 control sibling embryos and Tg(-5.1myl7:DsRed-NLS)f2 embryos treated with a low concentration of RA. Images are frontal views. Red indicates ventricle. Green indicates atrium. (R) Mean CM number at 48 hpf.

Mentions: Although one interpretation of the functional interaction of RARαb1 and Cyp26a1 depletion is that there is increased embryonic RA levels in these embryos, we wanted to further test this hypothesis using additional assays. First, we sought to use a distinct readout of embryonic RA, so we made a novel stable transgenic RA sensor line which incorporated the RARαb ligand binding domain (RLBD) fused to the Gal4 DNA binding domain (GDBD) expressed under the β-actin promoter (Figure S15A–S15G) 31. Previous studies have found that similar GDBD fusions with nuclear hormone receptor LBDs create an effective reporter of nuclear hormone activity 6,32,33. We observed a dramatic increase in reporter expression when RARαb1 and Cyp26a1 were depleted together in Tg(β-actin:GDBD-RLBD); Tg(UAS:EGFP) embryos (Figure 5M–5P, 5R) 34. Second, our hypothesis predicted that reducing embryonic RA levels should be able to rescue teratogenic phenotypes found in RARαb1+Cyp26a1 and RARαb1 deficient embryos. Consistent with this hypothesis, DEAB treatment of RARαb1+Cyp26a1 deficient embryos was able to rescue the loss of MHB (Figure 6A–6J). Additionally, treatment of RARαb1 deficient embryos with DEAB partially rescue the enlarged heart phenotype and restored atrial CM number (Figure 6K–6O). Lastly, our hypothesis predicts that exogenous treatment with a concentration of RA that causes a minor increase in RA signaling should result in aberrant heart phenotypes that are similar to RARαb1 deficient embryos. Indeed, embryos treated with low concentrations of exogenous RA (lower than we had reported using previously 27) had enlarged hearts with an increase in both atrial and ventricular CM number at 48 hpf (Figure 6P–6R). Altogether, our results suggest that increases in embryonic RA, coupled with compensatory RAR expression, contribute to the developmental defects found in RARαb1 deficient embryos.


Depletion of retinoic acid receptors initiates a novel positive feedback mechanism that promotes teratogenic increases in retinoic acid.

D'Aniello E, Rydeen AB, Anderson JL, Mandal A, Waxman JS - PLoS Genet. (2013)

Reduction of RA in RARαb1 deficient embryos can rescue developmental defects.(A–H) Control sibling, RARαb1+Cyp26a1 deficient, control sibling treated with DEAB, and RARαb1+Cyp26a1 treated with DEAB embryos. In B and F, arrows indicates loss of the MHB and eng2a expression. Images are lateral views with dorsal right and anterior up. (I) Percentage of control sibling (n = 16), RARαb1+Cyp26a1 deficient embryos (n = 16), control sibling embryos treated with DEAB (n = 13), and RARαb1+Cyp26a1 deficient embryos treated with DEAB (n = 14) that had a MHB based on morphology. (J) Percentage of control sibling (n = 17), RARαb1+Cyp26a1 deficient embryos (n = 14), control sibling embryos treated with DEAB (n = 15), and RARαb1+Cyp26a1 deficient embryos treated with DEAB (n = 12) that had eng2a expression at the MHB. (K–N) Hearts from Tg(-5.1myl7:DsRed-NLS)f2 control sibling, RARαb1 deficient, DEAB treated, and DEAB+RARαb1deficient embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (O) Mean CM number at 48 hpf. (P,Q) Hearts from Tg(-5.1myl7:DsRed-NLS)f2 control sibling embryos and Tg(-5.1myl7:DsRed-NLS)f2 embryos treated with a low concentration of RA. Images are frontal views. Red indicates ventricle. Green indicates atrium. (R) Mean CM number at 48 hpf.
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Related In: Results  -  Collection

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pgen.1003689-g006: Reduction of RA in RARαb1 deficient embryos can rescue developmental defects.(A–H) Control sibling, RARαb1+Cyp26a1 deficient, control sibling treated with DEAB, and RARαb1+Cyp26a1 treated with DEAB embryos. In B and F, arrows indicates loss of the MHB and eng2a expression. Images are lateral views with dorsal right and anterior up. (I) Percentage of control sibling (n = 16), RARαb1+Cyp26a1 deficient embryos (n = 16), control sibling embryos treated with DEAB (n = 13), and RARαb1+Cyp26a1 deficient embryos treated with DEAB (n = 14) that had a MHB based on morphology. (J) Percentage of control sibling (n = 17), RARαb1+Cyp26a1 deficient embryos (n = 14), control sibling embryos treated with DEAB (n = 15), and RARαb1+Cyp26a1 deficient embryos treated with DEAB (n = 12) that had eng2a expression at the MHB. (K–N) Hearts from Tg(-5.1myl7:DsRed-NLS)f2 control sibling, RARαb1 deficient, DEAB treated, and DEAB+RARαb1deficient embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (O) Mean CM number at 48 hpf. (P,Q) Hearts from Tg(-5.1myl7:DsRed-NLS)f2 control sibling embryos and Tg(-5.1myl7:DsRed-NLS)f2 embryos treated with a low concentration of RA. Images are frontal views. Red indicates ventricle. Green indicates atrium. (R) Mean CM number at 48 hpf.
Mentions: Although one interpretation of the functional interaction of RARαb1 and Cyp26a1 depletion is that there is increased embryonic RA levels in these embryos, we wanted to further test this hypothesis using additional assays. First, we sought to use a distinct readout of embryonic RA, so we made a novel stable transgenic RA sensor line which incorporated the RARαb ligand binding domain (RLBD) fused to the Gal4 DNA binding domain (GDBD) expressed under the β-actin promoter (Figure S15A–S15G) 31. Previous studies have found that similar GDBD fusions with nuclear hormone receptor LBDs create an effective reporter of nuclear hormone activity 6,32,33. We observed a dramatic increase in reporter expression when RARαb1 and Cyp26a1 were depleted together in Tg(β-actin:GDBD-RLBD); Tg(UAS:EGFP) embryos (Figure 5M–5P, 5R) 34. Second, our hypothesis predicted that reducing embryonic RA levels should be able to rescue teratogenic phenotypes found in RARαb1+Cyp26a1 and RARαb1 deficient embryos. Consistent with this hypothesis, DEAB treatment of RARαb1+Cyp26a1 deficient embryos was able to rescue the loss of MHB (Figure 6A–6J). Additionally, treatment of RARαb1 deficient embryos with DEAB partially rescue the enlarged heart phenotype and restored atrial CM number (Figure 6K–6O). Lastly, our hypothesis predicts that exogenous treatment with a concentration of RA that causes a minor increase in RA signaling should result in aberrant heart phenotypes that are similar to RARαb1 deficient embryos. Indeed, embryos treated with low concentrations of exogenous RA (lower than we had reported using previously 27) had enlarged hearts with an increase in both atrial and ventricular CM number at 48 hpf (Figure 6P–6R). Altogether, our results suggest that increases in embryonic RA, coupled with compensatory RAR expression, contribute to the developmental defects found in RARαb1 deficient embryos.

Bottom Line: Here, we report that zebrafish embryos deficient for RA receptor αb1 (RARαb1), a conserved RAR splice variant, have enlarged hearts with increased cardiomyocyte (CM) specification, which are surprisingly the consequence of increased RA signaling.Importantly, depletion of RARαb2 or concurrent depletion of RARαb1 and RARαb2 also results in increased RA signaling, suggesting this effect is a broader consequence of RAR depletion.Concurrent depletion of RARαb1 and Cyp26a1, an enzyme that facilitates degradation of RA, and employment of a novel transgenic RA sensor line support the hypothesis that the increases in RA signaling in RAR deficient embryos are the result of increased embryonic RA coupled with compensatory RAR expression.

View Article: PubMed Central - PubMed

Affiliation: The Heart Institute, Molecular Cardiovascular Biology and Developmental Biology Divisions, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.

ABSTRACT
Normal embryonic development and tissue homeostasis require precise levels of retinoic acid (RA) signaling. Despite the importance of appropriate embryonic RA signaling levels, the mechanisms underlying congenital defects due to perturbations of RA signaling are not completely understood. Here, we report that zebrafish embryos deficient for RA receptor αb1 (RARαb1), a conserved RAR splice variant, have enlarged hearts with increased cardiomyocyte (CM) specification, which are surprisingly the consequence of increased RA signaling. Importantly, depletion of RARαb2 or concurrent depletion of RARαb1 and RARαb2 also results in increased RA signaling, suggesting this effect is a broader consequence of RAR depletion. Concurrent depletion of RARαb1 and Cyp26a1, an enzyme that facilitates degradation of RA, and employment of a novel transgenic RA sensor line support the hypothesis that the increases in RA signaling in RAR deficient embryos are the result of increased embryonic RA coupled with compensatory RAR expression. Our results support an intriguing novel mechanism by which depletion of RARs elicits a previously unrecognized positive feedback loop that can result in developmental defects due to teratogenic increases in embryonic RA.

Show MeSH
Related in: MedlinePlus