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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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RARαb1 deficient embryos have enlarged hearts with increased CM number.(A, B) Hearts from control sibling and RARαb1 deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (C–H) ISH for CM differentiation marker genes. (I–L) ISH for CM progenitor marker genes. Brackets in I and J indicate length of nkx2.5 expression. Arrows in K indicate posterior and anterior limits of the hand2 expression domains in the LPM. In C–L, views are dorsal with anterior up. (M) Mean CM number at 48 hpf. (N) qPCR for CM differentiation marker gene expression at 48 hpf. (O) Areas of the amount of cells expressing the CM differentiation marker genes at the 20 s and 22 s stages. (P) qPCR for CM differentiation marker gene and nkx2.5 expression at 24 hpf. (Q) qPCR for CM progenitor gene expression at the 8 s stage. Asterisk in all graphs indicate a statistically significant difference compared to controls (p<0.05). Error bars in all graphs indicate standard deviation.
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pgen-1003689-g002: RARαb1 deficient embryos have enlarged hearts with increased CM number.(A, B) Hearts from control sibling and RARαb1 deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (C–H) ISH for CM differentiation marker genes. (I–L) ISH for CM progenitor marker genes. Brackets in I and J indicate length of nkx2.5 expression. Arrows in K indicate posterior and anterior limits of the hand2 expression domains in the LPM. In C–L, views are dorsal with anterior up. (M) Mean CM number at 48 hpf. (N) qPCR for CM differentiation marker gene expression at 48 hpf. (O) Areas of the amount of cells expressing the CM differentiation marker genes at the 20 s and 22 s stages. (P) qPCR for CM differentiation marker gene and nkx2.5 expression at 24 hpf. (Q) qPCR for CM progenitor gene expression at the 8 s stage. Asterisk in all graphs indicate a statistically significant difference compared to controls (p<0.05). Error bars in all graphs indicate standard deviation.

Mentions: We used a translation blocking morpholino (MO) to examine the function of RARαb1 (Figure 1B). By 48 hours post-fertilization (hpf), RARαb1 deficient embryos had enlarged hearts with increased CM number and expression of CM marker genes myl7, vmhc and amhc (Figure 2A, 2B, 2M, 2N and Figure S2A–S2D). Similar increases in CM number were also found at 55 hpf (Figure S3A–S3C), suggesting the major addition of surplus CMs occurs during earlier stages of development. Consistent with this idea, we observed an expansion of CM differentiation (myl7, vmhc, and amhc) and progenitor (nkx2.5 and hand2) marker expression in RARαb1 deficient embryos at earlier stages via in situ hybridization (ISH) and quantitative real-time PCR (qPCR; Figure 2C–2L, 2O–2Q). Injecting the RARαb1 MO along with rarαb1 mRNA that lacks the 5′UTR MO binding sequence is able to rescue the increased heart size, supporting the specificity of the phenotype (Figure S4A–S4D). Together, these results suggest that RARαb1 deficient embryos have increased CM specification, number and heart size.


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 enlarged hearts with increased CM number.(A, B) Hearts from control sibling and RARαb1 deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (C–H) ISH for CM differentiation marker genes. (I–L) ISH for CM progenitor marker genes. Brackets in I and J indicate length of nkx2.5 expression. Arrows in K indicate posterior and anterior limits of the hand2 expression domains in the LPM. In C–L, views are dorsal with anterior up. (M) Mean CM number at 48 hpf. (N) qPCR for CM differentiation marker gene expression at 48 hpf. (O) Areas of the amount of cells expressing the CM differentiation marker genes at the 20 s and 22 s stages. (P) qPCR for CM differentiation marker gene and nkx2.5 expression at 24 hpf. (Q) qPCR for CM progenitor gene expression at the 8 s stage. Asterisk in all graphs indicate a statistically significant difference compared to controls (p<0.05). Error bars in all graphs indicate standard deviation.
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pgen-1003689-g002: RARαb1 deficient embryos have enlarged hearts with increased CM number.(A, B) Hearts from control sibling and RARαb1 deficient Tg(-5.1myl7:DsRed-NLS)f2 embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (C–H) ISH for CM differentiation marker genes. (I–L) ISH for CM progenitor marker genes. Brackets in I and J indicate length of nkx2.5 expression. Arrows in K indicate posterior and anterior limits of the hand2 expression domains in the LPM. In C–L, views are dorsal with anterior up. (M) Mean CM number at 48 hpf. (N) qPCR for CM differentiation marker gene expression at 48 hpf. (O) Areas of the amount of cells expressing the CM differentiation marker genes at the 20 s and 22 s stages. (P) qPCR for CM differentiation marker gene and nkx2.5 expression at 24 hpf. (Q) qPCR for CM progenitor gene expression at the 8 s stage. Asterisk in all graphs indicate a statistically significant difference compared to controls (p<0.05). Error bars in all graphs indicate standard deviation.
Mentions: We used a translation blocking morpholino (MO) to examine the function of RARαb1 (Figure 1B). By 48 hours post-fertilization (hpf), RARαb1 deficient embryos had enlarged hearts with increased CM number and expression of CM marker genes myl7, vmhc and amhc (Figure 2A, 2B, 2M, 2N and Figure S2A–S2D). Similar increases in CM number were also found at 55 hpf (Figure S3A–S3C), suggesting the major addition of surplus CMs occurs during earlier stages of development. Consistent with this idea, we observed an expansion of CM differentiation (myl7, vmhc, and amhc) and progenitor (nkx2.5 and hand2) marker expression in RARαb1 deficient embryos at earlier stages via in situ hybridization (ISH) and quantitative real-time PCR (qPCR; Figure 2C–2L, 2O–2Q). Injecting the RARαb1 MO along with rarαb1 mRNA that lacks the 5′UTR MO binding sequence is able to rescue the increased heart size, supporting the specificity of the phenotype (Figure S4A–S4D). Together, these results suggest that RARαb1 deficient embryos have increased CM specification, number and heart size.

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