RAR? is required for mesodermal gene expression prior to gastrulation

The developing vertebrate embryo is exquisitely sensitive to retinoic acid (RA) concentration, particularly during anteroposterior patterning. In contrast to Nodal and Wnt signaling, RA was not previously considered to be an instructive signal in mesoderm formation during gastrulation. Here we show that RAR? is indispensable for the expression of early mesoderm markers and is, therefore, an obligatory factor in mesodermal competence and/or maintenance. We identified several novel targets up-regulated by RAR signaling in the early gastrula that are expressed in the circumblastoporal ring and linked to mesodermal development. Despite overlapping expression patterns of the RA synthetic enzyme, Aldh1a2 and the RA- degrading enzyme, Cyp26a1, RAR?1 functions as a transcriptional activator in early mesoderm development, suggesting that RA ligand is available to the embryo earlier than previously appreciated. RAR?1 is required for cellular adhesion, as revealed by spontaneous dissociation and depletion of N-CAM mRNA in animal caps harvested from RAR?1 knockdown embryos. RAR?1 knockdown obliterates somite boundaries, and causes loss of MYOD protein in the presomitic mesoderm, but ectopic, persistent expression of MYOD protein in the trunk. Thus, RAR?1 is required for stabilizing the mesodermal fate, myogenic commitment, somite boundary formation, and terminal, skeletal muscle differentiation. Overall design: RNA-sequencing of gastrula stage embryos treated with RAR agonist TTNPB or RAR antagonist AGN193109发育中的脊椎动物胚胎对维甲酸（retinoic acid, RA）浓度极为敏感，尤其在前后轴模式建成过程中。与Nodal和Wnt信号通路不同，以往研究并未将RA视为原肠作用时期中胚层形成的指导性信号。本研究证实，视黄酸受体γ（Retinoic Acid Receptor γ, RARγ）对于早期中胚层标志物的表达不可或缺，因此是中胚层潜能获得和/或维持的必需因子。我们鉴定出多个在早期原肠胚中受RAR信号通路上调的新靶基因，这些基因在胚孔环中表达，并与中胚层发育密切相关。尽管RA合成酶Aldh1a2与RA降解酶Cyp26a1的表达模式存在重叠，但RARγ1在早期中胚层发育中作为转录激活因子发挥功能，这提示胚胎可更早接触到RA配体，早于此前的认知。通过对RARγ1敲低胚胎获取的动物帽实验结果可见，细胞发生自发解离且N-CAM mRNA表达耗竭，证实RARγ1对于细胞黏附是必需的。RARγ1敲低会消除体节边界，并导致体节前中胚层中MYOD蛋白丢失，但在躯干中引发MYOD蛋白的异位持续表达。综上，RARγ1对于稳定中胚层命运、成肌细胞定向、体节边界形成以及终末骨骼肌分化均不可或缺。总体实验设计：对经RAR激动剂TTNPB或RAR拮抗剂AGN193109处理的原肠胚阶段胚胎进行RNA测序。