Depletion of Retinoic Acid Receptors Initiates a Novel Positive Feedback Mechanism that Promotes Teratogenic Increases in Retinoic Acid

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.

Cyp26 enzymes are required to balance the cardiac and vascular lineages within the anterior lateral plate mesoderm

“…In contrast to the previous hypothesis, our complementary analysis of ISH, lineage tracing and cardiomyocyte counting indicate that Cyp26 loss results in increased atrial progenitor specification and atrial cardiomyocytes; this is independent of effects on ventricular progenitor specification and cell number, which were unchanged. Importantly, the independent effects on chamber specification in Cyp26-deficient embryos corroborate recent studies examining increases and decreases of RA signaling in vertebrates using other methods (Waxman et al, 2008;Waxman and Yelon, 2009;D'Aniello et al, 2013). Specifically, loss of Cyp26 results in chamber defects that are equivalent to treatment with moderate RA concentrations (∼0.1 μM), although treatment with higher concentrations of RA is able to eliminate both atrial and ventricular progenitors (Waxman and Yelon, 2009).…”

“…2O), whereas singular knockdown of cyp26a1 or cyp26c1 did not affect cardiomyocyte number. Interestingly, the specific increase in atrial cells in Cyp26-deficient embryos is remarkably similar to the effects of modest increases in RA signaling (Waxman et al, 2008;Waxman and Yelon, 2009;D'Aniello et al, 2013). Therefore, our data demonstrate that depletion of both Cyp26 enzymes is required to produce a specific increase in atrial differentiation that is reminiscent of modest increases in RA signaling resulting from RA treatment.…”

“…To determine the role that Cyp26 enzymes play in early cardiovascular development, we used previously characterized morpholino oligonucleotides (MOs) to knockdown the Cyp26 enzymes (Hernandez et al, 2007;D'Aniello et al, 2013). We also used the giraffe (gir) mutant, which contains a point mutation that causes a truncation in Cyp26a1 (Emoto et al, 2005).…”

“…S2A,C) (Hernandez et al, 2007;D'Aniello et al, 2013). Double knockdown of cyp26a1 and cyp26c1 (referred to as Cyp26-deficient embryos) or injection of cyp26c1 MO into gir mutant embryos (referred to as gir+C) both caused more severe phenotypes, with larger pericardial edema and smaller heads compared with cyp26a1-deficient and gir embryos alone (supplementary material Fig.…”

Retinoic Acid Signaling Coordinates Macrophage-Dependent Injury and Repair after AKI

“…This may result from a compensatory increase in RA synthesis that in turn suppresses M1 spectrum macrophages in PTEC-DNRAR mice. Depletion of RARa variants in zebrafish embryos also initiates a compensatory increase in RA synthesis, 56 suggesting that inhibiting RAR signaling activates a similar positive feedback mechanism. On this basis, we propose that inhibition of PTEC RA signaling decreases M2 spectrum macrophage markers, but at the same time a compensatory increase in local RA synthesis acts through a different mechanism to repress inflammatory renal macrophages after IR-AKI.…”