Retinoic acid control of pax8 during renal specification of Xenopus pronephros involves hox and meis3
Retinoic acid control of pax8 during renal specification of Xenopus pronephros involves hox and meis3
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原爪蟾肾脏特化过程中视黄酸对 pax8 的控制涉及 hox 和 meis3
DOI：
10.1016/j.ydbio.2022.10.009
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发表时间：
2023
影响因子：
2.7
通讯作者：
Riou Jean-Fran?ois
中科院分区：
文献类型：
--
作者：
Durant-Vesga Jennifer;Suzuki Nanoka;Ochi Haruki;Le Bouffant Ronan;Eschstruth Alexis;Ogino Hajime;Umbhauer Muriel;Riou Jean-Fran?ois
原爪蟾的发育依赖于神经期的肾前体，这些前体聚集在背外侧中胚层的一个特定区域，称为肾野。早期神经期肾野的形成依赖于肾主转录调控因子（如pax8或lhx1）上游的视黄酸（RA）信号。虽然hx1可能是RA介导的肾野转录激活的直接靶点，但RA如何控制肾野的出现仍然知之甚少。为了更好地理解RA对肾野的肾脏规范的控制，我们在肾野出现之前分离的侧中胚层外植体和在不同时间点培养到早期神经期的外植体中进行了受RA破坏影响的基因转录组学分析。除了直接参与肾原发育的基因（pax8,lhx1,osr2,mecom）外，hox （hoxa1,a3,b3,b4，c5和d1）和hox辅助因子3是RA下游编码转录因子（tf）的重要基因群。为了支持meis3在肾区发挥作用的观点，我们观察到meis3的缺失导致肾区pax8表达的严重抑制。Meis3缺失仅轻微影响flhx1和aldh1a2的表达，表明Meis3主要作用于pax8的上游。进一步论证了meis3和hox在pax8调控中的作用，在动物帽中，meis3、hoxb4和pbx1的组合表达可诱导pax8的表达，但不能诱导flhx1的表达。同样的tf组合也能够反激活先前鉴定的pax8增强子Pax8-CNS1。Pax8-CNS1中存在的潜在PBX-Hox结合基序的突变进一步确定了其中两个必需的转激活基序。最后，我们在报告基因检测中检测了包含36.5 kb theX的先前表征的转基因基因的调控序列缺失。该基因允许表达截断的pax8-GFP融合蛋白，再现内源性pax8的表达。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不会影响报告基因的表达，但删除包含Pax8-CNS1和Pax8-CNS2的3.5 kb区域会导致严重抑制肾区和肾区报告基因的表达。
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. Althoughlhx1might 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,c5andd1) and the hox co-factormeis3appear 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 ofpax8expression in the kidney field. Meis3 depletion only marginally affects expression oflhx1andaldh1a2suggesting that meis3 principally acts upstream ofpax8. 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 inducespax8expression, but not that oflhx1. The same combination of TFs is also able to transactivate a previously identifiedpax8enhancer, 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 theX. tropicalis pax8gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenouspax8expression. This transgene includes three conservedpax8enhancers, 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.