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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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Concurrent depletion of RARαb1 and RARαb2 promotes increased RA signaling and atrial CM number.qPCR for (A) RA signaling responsive gene, (B) RA metabolizing gene, and (C) zebrafish rar expression in control sibling, RARαb1 deficient, RARαb2 deficient, RARαb1+RARαb2 (suboptimal doses) deficient, RA treated, and DEAB treated embryos at the 8 s stage. (D–G) ISH for egfp expression in Tg(12XRARE-ef1a:EGFP)sk72 embryos. Brackets indicate the length of egfp expression in the spinal cord. (H) Measurements of the length in arbitrary units (AU) of egfp expression in the spinal cord of Tg(12XRARE-ef1a:EGFP)sk72 embryos. (I–L) Hearts from control and RARαb depleted Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (M) Mean CM number from Tg(-5.1myl7:DsRed-NLS)f2 hearts at 48 hpf. (N) qPCR for CM marker gene expression at 48 hpf. While modest increases in vmhc expression in RARαb1+RARαb2 deficient embryos were observed relative to RARαb1 (suboptimal dose) deficient embryos, corresponding increases in ventricular CM number were not observed.
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pgen.1003689-g004: Concurrent depletion of RARαb1 and RARαb2 promotes increased RA signaling and atrial CM number.qPCR for (A) RA signaling responsive gene, (B) RA metabolizing gene, and (C) zebrafish rar expression in control sibling, RARαb1 deficient, RARαb2 deficient, RARαb1+RARαb2 (suboptimal doses) deficient, RA treated, and DEAB treated embryos at the 8 s stage. (D–G) ISH for egfp expression in Tg(12XRARE-ef1a:EGFP)sk72 embryos. Brackets indicate the length of egfp expression in the spinal cord. (H) Measurements of the length in arbitrary units (AU) of egfp expression in the spinal cord of Tg(12XRARE-ef1a:EGFP)sk72 embryos. (I–L) Hearts from control and RARαb depleted Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (M) Mean CM number from Tg(-5.1myl7:DsRed-NLS)f2 hearts at 48 hpf. (N) qPCR for CM marker gene expression at 48 hpf. While modest increases in vmhc expression in RARαb1+RARαb2 deficient embryos were observed relative to RARαb1 (suboptimal dose) deficient embryos, corresponding increases in ventricular CM number were not observed.

Mentions: We next wanted to determine if increases in RA signaling responsive genes were specific to RARαb1 depletion, so we examined RA responsive gene expression in RARαb2 deficient embryos. Previous studies found that RARαb2 deficient embryos lack forelimbs (pectoral fins) and tbx5a expression [8], [28], which we confirmed (Figure S7A, S7C, S7D, S7F, S7H, S7I). However, similar to RARαb1 depletion (Figure 3A and Figure 4A), RARαb2 deficient embryos had increased expression of RA signaling responsive genes (Figure 4A). While the previous studies found a loss of forelimbs, defects in heart development were not reported. Despite the loss of forelimbs and increase in RA signaling responsive genes, we did not observe an increase in heart size, CM number or CM gene expression (Figure S8A–S8D). Therefore, although eliciting similar increases in RA signaling responsive gene expression, individual depletion of RARαb1 and RARαb2 results in distinct defects.


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)

Concurrent depletion of RARαb1 and RARαb2 promotes increased RA signaling and atrial CM number.qPCR for (A) RA signaling responsive gene, (B) RA metabolizing gene, and (C) zebrafish rar expression in control sibling, RARαb1 deficient, RARαb2 deficient, RARαb1+RARαb2 (suboptimal doses) deficient, RA treated, and DEAB treated embryos at the 8 s stage. (D–G) ISH for egfp expression in Tg(12XRARE-ef1a:EGFP)sk72 embryos. Brackets indicate the length of egfp expression in the spinal cord. (H) Measurements of the length in arbitrary units (AU) of egfp expression in the spinal cord of Tg(12XRARE-ef1a:EGFP)sk72 embryos. (I–L) Hearts from control and RARαb depleted Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (M) Mean CM number from Tg(-5.1myl7:DsRed-NLS)f2 hearts at 48 hpf. (N) qPCR for CM marker gene expression at 48 hpf. While modest increases in vmhc expression in RARαb1+RARαb2 deficient embryos were observed relative to RARαb1 (suboptimal dose) deficient embryos, corresponding increases in ventricular CM number were not observed.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750112&req=5

pgen.1003689-g004: Concurrent depletion of RARαb1 and RARαb2 promotes increased RA signaling and atrial CM number.qPCR for (A) RA signaling responsive gene, (B) RA metabolizing gene, and (C) zebrafish rar expression in control sibling, RARαb1 deficient, RARαb2 deficient, RARαb1+RARαb2 (suboptimal doses) deficient, RA treated, and DEAB treated embryos at the 8 s stage. (D–G) ISH for egfp expression in Tg(12XRARE-ef1a:EGFP)sk72 embryos. Brackets indicate the length of egfp expression in the spinal cord. (H) Measurements of the length in arbitrary units (AU) of egfp expression in the spinal cord of Tg(12XRARE-ef1a:EGFP)sk72 embryos. (I–L) Hearts from control and RARαb depleted Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (M) Mean CM number from Tg(-5.1myl7:DsRed-NLS)f2 hearts at 48 hpf. (N) qPCR for CM marker gene expression at 48 hpf. While modest increases in vmhc expression in RARαb1+RARαb2 deficient embryos were observed relative to RARαb1 (suboptimal dose) deficient embryos, corresponding increases in ventricular CM number were not observed.
Mentions: We next wanted to determine if increases in RA signaling responsive genes were specific to RARαb1 depletion, so we examined RA responsive gene expression in RARαb2 deficient embryos. Previous studies found that RARαb2 deficient embryos lack forelimbs (pectoral fins) and tbx5a expression [8], [28], which we confirmed (Figure S7A, S7C, S7D, S7F, S7H, S7I). However, similar to RARαb1 depletion (Figure 3A and Figure 4A), RARαb2 deficient embryos had increased expression of RA signaling responsive genes (Figure 4A). While the previous studies found a loss of forelimbs, defects in heart development were not reported. Despite the loss of forelimbs and increase in RA signaling responsive genes, we did not observe an increase in heart size, CM number or CM gene expression (Figure S8A–S8D). Therefore, although eliciting similar increases in RA signaling responsive gene expression, individual depletion of RARαb1 and RARαb2 results in distinct defects.

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