Active repression of RAR signaling is required for head formation

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Abstract
The retinoic acid receptors (RARs) recruit coactivator and corepressor proteins to activate or repress the transcription of target genes depending on the presence of retinoic acid (RA). Despite a detailed molecular understanding of how corepressor complexes function, there is no in vivo evidence to support a necessary function for RAR-mediated repression. Signaling through RARs is required for patterning along the anteroposterior (A-P) axis, particularly in the hindbrain and posterior, although the absence of RA is required for correct anterior patterning. Because RARs and corepressors are present in regions in which RA is absent, we hypothesized that repression mediated through unliganded RARs might be important for anterior patterning. To test this hypothesis, specific reagents were used that either reduce or augment RAR-mediated repression. Derepression of RAR signaling by expressing a dominant-negative corepressor resulted in embryos that exhibited phenotypes similar to those treated by RA. Anterior structures such as forebrain and cement gland were greatly reduced, as was the expression of molecular markers. Enhancement of target gene repression using an RAR inverse agonist resulted in up-regulation of anterior neural markers and expansion of anterior structures. Morpholino antisense oligonucleotide-mediated RARα loss-of-function phenocopied the effects of RA treatment and dominant-negative corepressor expression. Microinjection of wild-type or dominant-negative RARα rescued the morpholino phenotype, confirming that RAR is functioning anteriorly as a transcriptional repressor. Lastly, increasing RAR-mediated repression potentiated head-inducing activity of the growth factor inhibitor cerberus, whereas releasing RAR-mediated repression blocked cerberus from inducing ectopic heads. We conclude that RAR-mediated repression of target genes is critical for head formation. This requirement establishes an important biological role for active repression of target genes by nuclear hormone receptors and illustrates a novel function for RARs during vertebrate development.