Limits...
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.

Show MeSH

Related in: MedlinePlus

RARαb1 deficient embryos have increased expression of RA signaling responsive genes.(A) qPCR for RA signaling responsive gene expression at the 8 s stage. (B, C) ISH for hoxb5b expression at the 8 s stage. Bracket indicates length of expression in the LPM. Views are dorsal with anterior up. (D, E) ISH for cyp26a1 expression at the 8 s stage. Arrows in E indicate increased expression in the tailbud and spinal cord. Views are lateral with anterior up and dorsal right. (F–H) Fronto-lateral views of Tg(-5.1myl7:GFP)f2 embryos at 48 hpf of control sibling, RARαb1 deficient embryos, and RARαb1+Hoxb5b deficient embryos. (I–K) Hearts from control sibling, RARαb1 deficient embryos and RARαb1+Hoxb5b deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (L) Percentage of control+rarαb1 MOs (n = 60), hoxb5b+rarαb1 MOs (n = 68) showing enlarged and normal hearts. (M) qPCR for CM differentiation gene expression at 48 hpf. (N) Mean CM number at 48 hpf.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750112&req=5

pgen-1003689-g003: RARαb1 deficient embryos have increased expression of RA signaling responsive genes.(A) qPCR for RA signaling responsive gene expression at the 8 s stage. (B, C) ISH for hoxb5b expression at the 8 s stage. Bracket indicates length of expression in the LPM. Views are dorsal with anterior up. (D, E) ISH for cyp26a1 expression at the 8 s stage. Arrows in E indicate increased expression in the tailbud and spinal cord. Views are lateral with anterior up and dorsal right. (F–H) Fronto-lateral views of Tg(-5.1myl7:GFP)f2 embryos at 48 hpf of control sibling, RARαb1 deficient embryos, and RARαb1+Hoxb5b deficient embryos. (I–K) Hearts from control sibling, RARαb1 deficient embryos and RARαb1+Hoxb5b deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (L) Percentage of control+rarαb1 MOs (n = 60), hoxb5b+rarαb1 MOs (n = 68) showing enlarged and normal hearts. (M) qPCR for CM differentiation gene expression at 48 hpf. (N) Mean CM number at 48 hpf.

Mentions: The increased atrial and ventricular CM number in RARαb1 deficient embryos are reminiscent of RA signaling deficient embryos [25], [26]. Therefore, we examined hoxb5b expression, which functions downstream of RA signaling to restrict atrial CM number [26] and is likely a direct target of RARs (Figure S5A–S5D). Unexpectedly, we found that hoxb5b expression was increased in RARαb1 deficient embryos (Figure 3A–3C). While this was initially perplexing, our recent studies showed that Hoxb5b overexpression is able to mimic many of the teratogenic effects of RA treatment [27]. Therefore, we asked if the increases in hoxb5b expression in RARαb1 deficient embryos could be a cause of the enlarged hearts. While depletion of hoxb5b alone using a low concentration of hoxb5b MO does not affect CM number (Figure S6A–S6C), we found that concurrent depletion of RARαb1 and Hoxb5b largely restored heart morphology, CM differentiation marker expression, and CM number relative to the RARαb1 deficient embryos (Figure 3F–3N), suggesting that the increased CM number in RARαb1 deficient embryos is in part a consequence of the increased hoxb5b expression.


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 deficient embryos have increased expression of RA signaling responsive genes.(A) qPCR for RA signaling responsive gene expression at the 8 s stage. (B, C) ISH for hoxb5b expression at the 8 s stage. Bracket indicates length of expression in the LPM. Views are dorsal with anterior up. (D, E) ISH for cyp26a1 expression at the 8 s stage. Arrows in E indicate increased expression in the tailbud and spinal cord. Views are lateral with anterior up and dorsal right. (F–H) Fronto-lateral views of Tg(-5.1myl7:GFP)f2 embryos at 48 hpf of control sibling, RARαb1 deficient embryos, and RARαb1+Hoxb5b deficient embryos. (I–K) Hearts from control sibling, RARαb1 deficient embryos and RARαb1+Hoxb5b deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (L) Percentage of control+rarαb1 MOs (n = 60), hoxb5b+rarαb1 MOs (n = 68) showing enlarged and normal hearts. (M) qPCR for CM differentiation gene expression at 48 hpf. (N) Mean CM number at 48 hpf.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003689-g003: RARαb1 deficient embryos have increased expression of RA signaling responsive genes.(A) qPCR for RA signaling responsive gene expression at the 8 s stage. (B, C) ISH for hoxb5b expression at the 8 s stage. Bracket indicates length of expression in the LPM. Views are dorsal with anterior up. (D, E) ISH for cyp26a1 expression at the 8 s stage. Arrows in E indicate increased expression in the tailbud and spinal cord. Views are lateral with anterior up and dorsal right. (F–H) Fronto-lateral views of Tg(-5.1myl7:GFP)f2 embryos at 48 hpf of control sibling, RARαb1 deficient embryos, and RARαb1+Hoxb5b deficient embryos. (I–K) Hearts from control sibling, RARαb1 deficient embryos and RARαb1+Hoxb5b deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (L) Percentage of control+rarαb1 MOs (n = 60), hoxb5b+rarαb1 MOs (n = 68) showing enlarged and normal hearts. (M) qPCR for CM differentiation gene expression at 48 hpf. (N) Mean CM number at 48 hpf.
Mentions: The increased atrial and ventricular CM number in RARαb1 deficient embryos are reminiscent of RA signaling deficient embryos [25], [26]. Therefore, we examined hoxb5b expression, which functions downstream of RA signaling to restrict atrial CM number [26] and is likely a direct target of RARs (Figure S5A–S5D). Unexpectedly, we found that hoxb5b expression was increased in RARαb1 deficient embryos (Figure 3A–3C). While this was initially perplexing, our recent studies showed that Hoxb5b overexpression is able to mimic many of the teratogenic effects of RA treatment [27]. Therefore, we asked if the increases in hoxb5b expression in RARαb1 deficient embryos could be a cause of the enlarged hearts. While depletion of hoxb5b alone using a low concentration of hoxb5b MO does not affect CM number (Figure S6A–S6C), we found that concurrent depletion of RARαb1 and Hoxb5b largely restored heart morphology, CM differentiation marker expression, and CM number relative to the RARαb1 deficient embryos (Figure 3F–3N), suggesting that the increased CM number in RARαb1 deficient embryos is in part a consequence of the increased hoxb5b expression.

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