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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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Related in: MedlinePlus

RARαb1 and RARαb2 sequences and expression.(A) Schematic representation of RAR domains. Blue box indicates the variable A domain, which is different between RARαb1 and the previously identified RARαb2 splice variant. (B) Schematic representation of RARαb1 and RARαb2 genomic organization (adapted from Ensemble_v9). Blue bars represent the first exon, which encodes the respective A domains. White bars represent the 5′ UTRs. Black bars represent the exons that are common to the two variants. Green bars represent the target of the antisense probes used for ISH. Red arrows indicate the position of the primers used to perform RT-PCR. Purple bars indicate the position of the morpholino target sequences. (C) Alignments of the A domains of human (Hs) RARα1, mouse (Mm) RARα1, and zebrafish (Dr) RARαb1. The presence of this previously unrecognized splice variant was recently confirmed in the latest zebrafish genome assembly (Ensemble Zv9). There is no RARαa splice variant 1 ortholog in the zebrafish genome. (D) Alignments of the A domains of Hs RARα2, Mm RARα2, Dr RARαa2, and Dr RARαb2. (E) Reverse transcriptase PCR (RT-PCR) for the zebrafish rarαb isoforms. max was used as the control. -RT control did not reveal genomic contamination (data not shown). (F) Rarαb1 is expressed in the ventral anterior of the embryo and the presomitic paraxial mesoderm (arrow) at the 8 somite (s) stage. (G) Rarαb2 is expressed in rhombomeres 5 and 6, the spinal cord and the posterior lateral plate mesoderm (LPM). Arrows indicate the space between the posterior spinal cord and LPM expression domains. (H) Together, the expression patterns recapitulate a previously reported rarαb probe (referred to as rarαb1/2), which detects both isoforms [24]. In F–H, embryos are flatmounted and are dorsal views with anterior up.
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pgen-1003689-g001: RARαb1 and RARαb2 sequences and expression.(A) Schematic representation of RAR domains. Blue box indicates the variable A domain, which is different between RARαb1 and the previously identified RARαb2 splice variant. (B) Schematic representation of RARαb1 and RARαb2 genomic organization (adapted from Ensemble_v9). Blue bars represent the first exon, which encodes the respective A domains. White bars represent the 5′ UTRs. Black bars represent the exons that are common to the two variants. Green bars represent the target of the antisense probes used for ISH. Red arrows indicate the position of the primers used to perform RT-PCR. Purple bars indicate the position of the morpholino target sequences. (C) Alignments of the A domains of human (Hs) RARα1, mouse (Mm) RARα1, and zebrafish (Dr) RARαb1. The presence of this previously unrecognized splice variant was recently confirmed in the latest zebrafish genome assembly (Ensemble Zv9). There is no RARαa splice variant 1 ortholog in the zebrafish genome. (D) Alignments of the A domains of Hs RARα2, Mm RARα2, Dr RARαa2, and Dr RARαb2. (E) Reverse transcriptase PCR (RT-PCR) for the zebrafish rarαb isoforms. max was used as the control. -RT control did not reveal genomic contamination (data not shown). (F) Rarαb1 is expressed in the ventral anterior of the embryo and the presomitic paraxial mesoderm (arrow) at the 8 somite (s) stage. (G) Rarαb2 is expressed in rhombomeres 5 and 6, the spinal cord and the posterior lateral plate mesoderm (LPM). Arrows indicate the space between the posterior spinal cord and LPM expression domains. (H) Together, the expression patterns recapitulate a previously reported rarαb probe (referred to as rarαb1/2), which detects both isoforms [24]. In F–H, embryos are flatmounted and are dorsal views with anterior up.

Mentions: In contrast to the studies of RARs in mice [9]–[12], depletion of RARs has not been able to recapitulate all of the consequences of loss of RA signaling in zebrafish [8], which prompted us to determine if additional conserved RAR variants exist in zebrafish beyond what has already been reported [24]. We cloned a previously unrecognized RARα splice variant that is orthologous to human, mouse and Xenopus RARα1 termed RARαb1 (Figure 1A–1C). The previously cloned zebrafish RARα homologs RARαa and RARαb are teleost specific paralogs and both are orthologous to the splice variant 2 found in tetrapods (Figure 1B, 1D) [24]. Both rarαb1 and rarαb2 are expressed maternally and zygotically (Figure 1E), with ubiquitous expression until the tailbud stage (Figure S1A–S1I). After the tailbud stage, their expression patterns deviate (Figure 1F–1H and Figure S1J–S1O).


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)

RARαb1 and RARαb2 sequences and expression.(A) Schematic representation of RAR domains. Blue box indicates the variable A domain, which is different between RARαb1 and the previously identified RARαb2 splice variant. (B) Schematic representation of RARαb1 and RARαb2 genomic organization (adapted from Ensemble_v9). Blue bars represent the first exon, which encodes the respective A domains. White bars represent the 5′ UTRs. Black bars represent the exons that are common to the two variants. Green bars represent the target of the antisense probes used for ISH. Red arrows indicate the position of the primers used to perform RT-PCR. Purple bars indicate the position of the morpholino target sequences. (C) Alignments of the A domains of human (Hs) RARα1, mouse (Mm) RARα1, and zebrafish (Dr) RARαb1. The presence of this previously unrecognized splice variant was recently confirmed in the latest zebrafish genome assembly (Ensemble Zv9). There is no RARαa splice variant 1 ortholog in the zebrafish genome. (D) Alignments of the A domains of Hs RARα2, Mm RARα2, Dr RARαa2, and Dr RARαb2. (E) Reverse transcriptase PCR (RT-PCR) for the zebrafish rarαb isoforms. max was used as the control. -RT control did not reveal genomic contamination (data not shown). (F) Rarαb1 is expressed in the ventral anterior of the embryo and the presomitic paraxial mesoderm (arrow) at the 8 somite (s) stage. (G) Rarαb2 is expressed in rhombomeres 5 and 6, the spinal cord and the posterior lateral plate mesoderm (LPM). Arrows indicate the space between the posterior spinal cord and LPM expression domains. (H) Together, the expression patterns recapitulate a previously reported rarαb probe (referred to as rarαb1/2), which detects both isoforms [24]. In F–H, embryos are flatmounted and are dorsal views with anterior up.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750112&req=5

pgen-1003689-g001: RARαb1 and RARαb2 sequences and expression.(A) Schematic representation of RAR domains. Blue box indicates the variable A domain, which is different between RARαb1 and the previously identified RARαb2 splice variant. (B) Schematic representation of RARαb1 and RARαb2 genomic organization (adapted from Ensemble_v9). Blue bars represent the first exon, which encodes the respective A domains. White bars represent the 5′ UTRs. Black bars represent the exons that are common to the two variants. Green bars represent the target of the antisense probes used for ISH. Red arrows indicate the position of the primers used to perform RT-PCR. Purple bars indicate the position of the morpholino target sequences. (C) Alignments of the A domains of human (Hs) RARα1, mouse (Mm) RARα1, and zebrafish (Dr) RARαb1. The presence of this previously unrecognized splice variant was recently confirmed in the latest zebrafish genome assembly (Ensemble Zv9). There is no RARαa splice variant 1 ortholog in the zebrafish genome. (D) Alignments of the A domains of Hs RARα2, Mm RARα2, Dr RARαa2, and Dr RARαb2. (E) Reverse transcriptase PCR (RT-PCR) for the zebrafish rarαb isoforms. max was used as the control. -RT control did not reveal genomic contamination (data not shown). (F) Rarαb1 is expressed in the ventral anterior of the embryo and the presomitic paraxial mesoderm (arrow) at the 8 somite (s) stage. (G) Rarαb2 is expressed in rhombomeres 5 and 6, the spinal cord and the posterior lateral plate mesoderm (LPM). Arrows indicate the space between the posterior spinal cord and LPM expression domains. (H) Together, the expression patterns recapitulate a previously reported rarαb probe (referred to as rarαb1/2), which detects both isoforms [24]. In F–H, embryos are flatmounted and are dorsal views with anterior up.
Mentions: In contrast to the studies of RARs in mice [9]–[12], depletion of RARs has not been able to recapitulate all of the consequences of loss of RA signaling in zebrafish [8], which prompted us to determine if additional conserved RAR variants exist in zebrafish beyond what has already been reported [24]. We cloned a previously unrecognized RARα splice variant that is orthologous to human, mouse and Xenopus RARα1 termed RARαb1 (Figure 1A–1C). The previously cloned zebrafish RARα homologs RARαa and RARαb are teleost specific paralogs and both are orthologous to the splice variant 2 found in tetrapods (Figure 1B, 1D) [24]. Both rarαb1 and rarαb2 are expressed maternally and zygotically (Figure 1E), with ubiquitous expression until the tailbud stage (Figure S1A–S1I). After the tailbud stage, their expression patterns deviate (Figure 1F–1H and Figure S1J–S1O).

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