Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga 4710-057, Portugal.
Biochem Biophys Res Commun. 2012 Jul 13;423(4):889-94. doi: 10.1016/j.bbrc.2012.06.077. Epub 2012 Jun 21.
Embryo development proceeds under strict temporal control and an embryonic molecular clock (EC), evidenced by cyclic gene expression, is operating during somite formation and limb development, providing temporal information to precursor cells. In somite precursor cells, EC gene expression and periodicity depends on Retinoic acid (RA) signaling and this morphogen is also essential for limb initiation, outgrowth and patterning. Since the limb EC gene hairy2 is differentially expressed along the proximal-distal axis as growth proceeds, concomitant with changes in flank-derived RA activity in the mesenchyme, we have interrogated the role of RA signaling on limb hairy2 expression regulation. We describe RA as a positive regulator of limb hairy2 expression. Ectopic supplementation of RA induced hairy2 in a short time period, with simultaneous transient activation of Erk/MAPK, Akt/PI3K and Gli3 intracellular pathways. We further found that FGF8, an inducer of Erk/MAPK, Akt/PI3K pathways, was not sufficient for ectopic hairy2 induction. However, joint treatment with both RA and FGF8 induced hairy2, indicating that RA is creating a permissive condition for p-Erk/p-Akt action on hairy2, most likely by enhancing Gli3-A/Gli3-R levels. Finally, we observed an inhibitory action of BMP4 on hairy2 and propose a model whereby RA shapes limb hairy2 expression during limb development, by activating its expression and counteracting the inhibitory action of BMP4 on hairy2. Overall, our work reports a novel role for RA in the regulation of limb clock hairy2 gene expression and elucidates the temporal response of multiple intracellular pathways to RA signaling in limb development.
胚胎发育是在严格的时间控制下进行的，胚胎分子钟（EC）在体节形成和肢发育过程中循环基因表达，为前体细胞提供时间信息。在体节前体细胞中，EC 基因表达和周期性依赖于视黄酸（RA）信号，这种形态发生素对于肢的起始、生长和模式形成也是必不可少的。由于肢 EC 基因 hairy2 在生长过程中沿着近-远轴的差异表达，伴随着间质中侧翼衍生的 RA 活性的变化，我们研究了 RA 信号对肢 hairy2 表达调控的作用。我们描述 RA 是肢 hairy2 表达的正调节剂。RA 的异位补充在短时间内诱导 hairy2，同时瞬时激活 Erk/MAPK、Akt/PI3K 和 Gli3 细胞内途径。我们进一步发现，FGF8，一种 Erk/MAPK、Akt/PI3K 途径的诱导物，不足以诱导异位 hairy2。然而，RA 和 FGF8 的联合处理诱导了 hairy2，表明 RA 通过增强 Gli3-A/Gli3-R 水平，为 p-Erk/p-Akt 对 hairy2 的作用创造了许可条件。最后，我们观察到 BMP4 对 hairy2 的抑制作用，并提出了一个模型，即 RA 通过激活其表达并抵消 BMP4 对 hairy2 的抑制作用，在肢发育过程中塑造肢 hairy2 表达。总的来说，我们的工作报告了 RA 在调节肢时钟 hairy2 基因表达中的新作用，并阐明了多种细胞内途径对 RA 信号在肢发育中的时间反应。
