Retinoic Acid Receptor Gamma Activity Plays a Critical Role in Regulating Early Mouse Gastruloid Development

Regulation of all-trans retinoic acid (ATRA) signalling is crucial to early embryonic de-velopment. We show here that in embryonic stem (ES) cell-derived gastruloids, which mimic normal development in response to the Wnt/beta-catenin agonist CHIR9901, expression of retinoic acid receptor (RAR) gamma was spatially restricted to primitive cells that co-expressed ES cell and early progenitor cell markers, i.e., Nanog, Sox2, and Oct4. In contrast, RAR alpha expression was ubiquitous. mRNAs for the key enzymes involved in ATRA synthesis (Aldh1a2) and degradation (Cyp26a1) were not seen in cells that ex-pressed RAR gamma. Treatment of ES cell-derived gastruloids with physiologically relevant (10nM) levels of ATRA or with a highly selective RAR gamma agonist blocked normal developmental processes, preventing symmetry-breaking and axial elongation. This was not seen following treatments with an RAR alpha agonist, where there was a tendency for enhanced axial elongation. Brachyury (TBXT) immuno-positive cells localised in the posterior end of elongated gastruloids in control- and RAR alpha agonist-treated cultures, with Sox2 immuno-positive cells seen more widely, whilst both TBXT and Sox2 immu-no-positive cells were randomly distributed throughout ATRA- and RAR gamma agonist-treated gastruloids. Concurrent treatment of gastruloids with 10nM ATRA and 100nM of an RAR gamma antagonist partially abrogated the ATRA-mediated block to axial elongation. Conversely, 10nM RAR gamma antagonist treatments were associated with the formation of multi-axis gastruloid elongations, with comparatively little effect seen after treatments with an RAR alpha antagonist. These findings reveal that RAR gamma plays a crucial role in the development of embryonic tissues.