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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 Cyp26a1 results in phenotypes resembling RA treatment.(A–D) Control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient embryos. A suboptimal dose of the cyp26a1 MOs was used that did not cause ostensible defects for these experiments. In D, arrow indicates loss of the MHB and line indicates shortened tail. Images are lateral views with dorsal right and anterior up. (E–H) ISH for eng2a, which marks the MHB. 100% of (E) control sibling (n = 11), (F) RARαb1 deficient (n = 7), and (G) Cyp26a1 deficient (n = 7) had eng2a expression. 85% of (H) RARαb1+Cyp26a1 deficient embryos (n = 7) had a complete absence of eng2a expression (arrow in H). Equivalent results were obtained using pax2a, which also marks the MHB (data not shown). (I–L) Hearts from control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (M–P) ISH for egfp in Tg(β-actin:GDBD-RLBD)cch1;Tg(UAS:EGFP) embryos. Lateral views with dorsal right and anterior up. (Q) Mean CM number at 48 hpf and (R) qPCR for egfp expression at 15 s in control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient embryos. Double asterisks in Q indicate a statistically significant difference relative to control and RARαb1 deficient embryos. Pound sign in Q indicates a statistically significant difference relative to RARαb1 deficient embryos.
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pgen.1003689-g005: Concurrent depletion of RARαb1 and Cyp26a1 results in phenotypes resembling RA treatment.(A–D) Control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient embryos. A suboptimal dose of the cyp26a1 MOs was used that did not cause ostensible defects for these experiments. In D, arrow indicates loss of the MHB and line indicates shortened tail. Images are lateral views with dorsal right and anterior up. (E–H) ISH for eng2a, which marks the MHB. 100% of (E) control sibling (n = 11), (F) RARαb1 deficient (n = 7), and (G) Cyp26a1 deficient (n = 7) had eng2a expression. 85% of (H) RARαb1+Cyp26a1 deficient embryos (n = 7) had a complete absence of eng2a expression (arrow in H). Equivalent results were obtained using pax2a, which also marks the MHB (data not shown). (I–L) Hearts from control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (M–P) ISH for egfp in Tg(β-actin:GDBD-RLBD)cch1;Tg(UAS:EGFP) embryos. Lateral views with dorsal right and anterior up. (Q) Mean CM number at 48 hpf and (R) qPCR for egfp expression at 15 s in control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient embryos. Double asterisks in Q indicate a statistically significant difference relative to control and RARαb1 deficient embryos. Pound sign in Q indicates a statistically significant difference relative to RARαb1 deficient embryos.

Mentions: In addition to inhibiting aldh1a2 expression, increased RA signaling promotes a negative feedback mechanism that limits RA levels by positively regulating Cyp26a1 expression 16–18. Since we observe an increase in cyp26a1 expression in RARαb1 deficient embryos (Figure 3A, 3D, 3E and Figure 4A), which was also consistent with the hypothesis that there is increased embryonic RA, we postulated that the increased Cyp26a1 may be protecting the RARαb1 deficient embryos from teratogenic increases in embryonic RA. Therefore, we concurrently depleted RARαb1 and Cyp26a1 to determine if there was a functional interaction indicative of increased embryonic RA. For these experiments, a suboptimal dose of cyp26a1 MOs (Figure S14A–S14E) was used to more easily discern a functional interaction. In either the RARαb1 or Cyp26a1 deficient embryos alone, we never observed absence of the MHB or defects in tail elongation (Figure 5A–5C, 5E–5G). However, co-depletion of RARαb1 and Cyp26a1 resulted in a loss of the MHB and truncated tails (Figure 5D, 5H), similar to increases in RA signaling 17,19,29,30. Furthermore, we found that RARαb1+Cyp26a1 deficient embryos had dismorphic hearts with a specific reduction in ventricular CM number compared to controls embryos hearts (Figure 5I–5L, 5Q), which interestingly resembles the trend we previously found in embryos with intermediate increases in RA signaling 27.


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 Cyp26a1 results in phenotypes resembling RA treatment.(A–D) Control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient embryos. A suboptimal dose of the cyp26a1 MOs was used that did not cause ostensible defects for these experiments. In D, arrow indicates loss of the MHB and line indicates shortened tail. Images are lateral views with dorsal right and anterior up. (E–H) ISH for eng2a, which marks the MHB. 100% of (E) control sibling (n = 11), (F) RARαb1 deficient (n = 7), and (G) Cyp26a1 deficient (n = 7) had eng2a expression. 85% of (H) RARαb1+Cyp26a1 deficient embryos (n = 7) had a complete absence of eng2a expression (arrow in H). Equivalent results were obtained using pax2a, which also marks the MHB (data not shown). (I–L) Hearts from control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (M–P) ISH for egfp in Tg(β-actin:GDBD-RLBD)cch1;Tg(UAS:EGFP) embryos. Lateral views with dorsal right and anterior up. (Q) Mean CM number at 48 hpf and (R) qPCR for egfp expression at 15 s in control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient embryos. Double asterisks in Q indicate a statistically significant difference relative to control and RARαb1 deficient embryos. Pound sign in Q indicates a statistically significant difference relative to RARαb1 deficient embryos.
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pgen.1003689-g005: Concurrent depletion of RARαb1 and Cyp26a1 results in phenotypes resembling RA treatment.(A–D) Control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient embryos. A suboptimal dose of the cyp26a1 MOs was used that did not cause ostensible defects for these experiments. In D, arrow indicates loss of the MHB and line indicates shortened tail. Images are lateral views with dorsal right and anterior up. (E–H) ISH for eng2a, which marks the MHB. 100% of (E) control sibling (n = 11), (F) RARαb1 deficient (n = 7), and (G) Cyp26a1 deficient (n = 7) had eng2a expression. 85% of (H) RARαb1+Cyp26a1 deficient embryos (n = 7) had a complete absence of eng2a expression (arrow in H). Equivalent results were obtained using pax2a, which also marks the MHB (data not shown). (I–L) Hearts from control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (M–P) ISH for egfp in Tg(β-actin:GDBD-RLBD)cch1;Tg(UAS:EGFP) embryos. Lateral views with dorsal right and anterior up. (Q) Mean CM number at 48 hpf and (R) qPCR for egfp expression at 15 s in control sibling, RARαb1 deficient, Cyp26a1 deficient, and RARαb1+Cyp26a1 deficient embryos. Double asterisks in Q indicate a statistically significant difference relative to control and RARαb1 deficient embryos. Pound sign in Q indicates a statistically significant difference relative to RARαb1 deficient embryos.
Mentions: In addition to inhibiting aldh1a2 expression, increased RA signaling promotes a negative feedback mechanism that limits RA levels by positively regulating Cyp26a1 expression 16–18. Since we observe an increase in cyp26a1 expression in RARαb1 deficient embryos (Figure 3A, 3D, 3E and Figure 4A), which was also consistent with the hypothesis that there is increased embryonic RA, we postulated that the increased Cyp26a1 may be protecting the RARαb1 deficient embryos from teratogenic increases in embryonic RA. Therefore, we concurrently depleted RARαb1 and Cyp26a1 to determine if there was a functional interaction indicative of increased embryonic RA. For these experiments, a suboptimal dose of cyp26a1 MOs (Figure S14A–S14E) was used to more easily discern a functional interaction. In either the RARαb1 or Cyp26a1 deficient embryos alone, we never observed absence of the MHB or defects in tail elongation (Figure 5A–5C, 5E–5G). However, co-depletion of RARαb1 and Cyp26a1 resulted in a loss of the MHB and truncated tails (Figure 5D, 5H), similar to increases in RA signaling 17,19,29,30. Furthermore, we found that RARαb1+Cyp26a1 deficient embryos had dismorphic hearts with a specific reduction in ventricular CM number compared to controls embryos hearts (Figure 5I–5L, 5Q), which interestingly resembles the trend we previously found in embryos with intermediate increases in RA signaling 27.

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