Together, our study supports a novel paradigm whereby RARαb depletion elicits a positive feedback mechanism that can result in teratogenic increases in RA signaling. Importantly, our work highlights that loss and gain of RA signaling can cause similar developmental defects. RA signaling is required to restrict CM specification 25
, while high increases in RA signaling can eliminate CM specification () 27
. However, our present findings suggest that low increases in RA signaling, achieved when treating embryos with µM concentrations of RA or through RARαb depletion, can also promote increases in both atrial and ventricular CM specification (). As we found previously, modest, but slightly higher increases of RA signaling can promote atrial CM specification without significantly affecting ventricular CM specification 27
, which is strikingly similar to what we found with concurrent depletion of the RARαb variants here (). Moreover, intermediate increases in RA signaling can inhibit ventricular CM specification, which is similar what we observed when concurrently depleting RARαb1 and Cyp26a1 (). It also appears that modulation of Hox activity downstream of both gain and loss RA signaling is at least partially responsible for the increases in CM specification, suggesting the hypothesis that the similar effects on CM number are actually due to opposite perturbations of anterior-posterior patterning within the ALPM. Therefore, our analysis corroborates and extends previous observations that there are differential effects on atrial and ventricular CM populations as there is a progressive increase from low to intermediate levels of RA signaling in the early embryo.
Models of the effects of RA signaling on heart patterning and the RA feedback mechanism.
It is interesting that depletion of RARα homologs using MOs in zebrafish, presented in this study, and Xenopus
elicit similar phenotypic responses. In Xenopus
embryos, RARα depletion alone results in loss of the MHB 6
. While depletion of RARαb1 alone does not result in MHB defects in zebrafish embryos, we have found that RARαb1+Cyp26a1 deficient embryos completely lack the MHB. Taken together, these results suggest that the underlying consequences of increased RA signaling due to depletion of RARα homologs are likely conserved at least in Xenopus
and zebrafish embryos, but that in Xenopus
perhaps the role of Cyp26 enzymes in protecting the brain has been lost. Despite similarities in the phenotypes that both point to an increase in RA signaling in RARα and RARαb deficient Xenopus
and zebrafish embryos, our results contrast with the model proposed by Koide et al. 6
, which concluded that RARs are required to function as transcriptional repressors. Importantly, the tools used in the previous study, including dominant-negative RARs, transcriptional co-repressors, and inverse agonists, would not have allowed them to distinguish between a transcriptional de-repressive model and the positive feedback mechanism involving the production of excess RA supported here.
In addition to the phenotypic similarities when depleting RARα homologs in Xenopus
and zebrafish, depletion of zebrafish RARαbs results in compensatory RAR expression similar to RARα depletion in mice 23
, supporting the hypothesis that this feedback response to RARα deficiency is conserved in vertebrates. Importantly, the response to RAR depletion is likely different than complete ablation of RARs. RAR KO mice have not been reported to have compensatory increases in other RARs 11
, suggesting that a complete loss of RAR expression may cause a breakdown of this feedback loop. However, when considering the probability that RAR expression would be completely lost vs. depleted, we postulate that insults resulting in depletion of RAR expression would be much more likely. Consistent with this idea, variable levels of RAR expression deficiency, which in the case of RARβ can be due to epigenetic silencing, is commonly observed in a variety of cancers 13
Given the conserved feedback mechanisms already recognized that limit fluctuations in RA signaling in vertebrates 16
, it seems logical that a conserved mechanism that senses RAR deficiency would also exist to prevent loss of RA signaling. We propose that this newly recognized positive feedback mechanism would be more suitable to prevent transient deficiency in RARs. As demonstrated here, persistent RARαb depletion can result in a hypervigilant response of RA signaling and RA-induced teratogenic defects. Overall, these data provide insight into a previously unappreciated RAR-dependent positive feedback mechanism (), which is active during development. Further elucidation of this RA signaling feedback mechanism may illuminate the etiology of poorly understood RA-insensitive cancers 13
and congenital defects 1