Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 ​kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 ​kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.

非洲爪蟾的发育依赖于肾前体细胞在神经胚阶段分组到背外侧中胚层的特定区域，称为肾区。早期神经胚阶段肾场的形成取决于作用于肾脏主转录调节因子（如 pax8 或 lhx1）上游的视黄酸 (RA) 信号。虽然lhx1可能是肾区 RA 介导的转录激活的直接目标，但 RA 如何控制肾区的出现仍知之甚少。为了更好地了解 RA 对肾场肾脏规范的控制，我们对在肾场出现前分离的外侧中胚层外植体中受 RA 破坏影响的基因进行了转录组学分析，并在不同时间点培养直至早期神经胚阶段. 除了直接参与前肾发育的基因（pax8、lhx1、osr2、mecom）、hox（hoxa1、a3、b3、b4、c5和d1) 和 hox 辅因子meis3是 RA 下游编码转录因子 (TF) 的一组突出基因。支持 meis3 在肾场中的作用的想法，我们观察到 meis3 耗尽导致pax8在肾场中的表达受到严重抑制。Meis3 耗尽仅轻微影响lhx1和aldh1a2的表达，表明 meis3 主要作用于pax8的上游。进一步论证 meis3 和 hox 在控制 pax8 中的作用，meis3、hoxb4 和 pbx1 的组合在动物帽中的表达诱导pax8表达，但不诱导lhx1的表达. 同样的 TF 组合也能够反式激活先前鉴定的pax8增强子 Pax8-CNS1。Pax8-CNS1 中存在的潜在 PBX-Hox 结合基序的诱变进一步允许识别其中两个转录激活所必需的基序。最后，我们在报告分析中测试了调控序列的缺失，该转基因包含 36.5 kb 的热带假丝酵母 pax8 基因，该基因允许表达截短的 pax8-GFP 融合蛋白，重现内源性pax8表达。该转基因包括三个保守的pax8增强子，Pax8-CNS1，Pax8-CNS2 和 Pax8-CNS3。单独删除 Pax8-CNS1 不会影响报告基因表达，但删除包含 Pax8-CNS1 和 Pax8-CNS2 的 3.5 kb 区域会导致严重抑制报告基因在耳基板和肾区的表达。