Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA.
Dev Biol. 2012 Apr 1;364(1):11-21. doi: 10.1016/j.ydbio.2012.01.014. Epub 2012 Jan 24.
Genetic interactions regulating intermediate stages of tubulogenesis in the developing kidney have been difficult to define. A systems biology strategy using microarray was combined with in vitro/ex vivo and genetic approaches to identify pathways regulating specific stages of tubulogenesis. Analysis of the progression of the metanephric mesenchyme (MM) through four stages of tubule induction and differentiation (i.e., epithelialization, tubular organization and elongation and early differentiation) revealed signaling pathways potentially involved at each stage and suggested key roles for a number of signaling molecules. A screen of the signaling pathways on in vitro/ex vivo nephron formation implicated a unique regulatory role for protein kinase A (PKA), through PKA-2, in a specific post-epithelialization morphogenetic step (conversion of the renal vesicle to the S-shaped body). Microarray analysis not only confirmed this stage-specificity, but also highlighted the upregulation of Wnt genes. Addition of PKA agonists to LIF-induced nephrons (previously shown to be a Wnt/beta-catenin dependent pathway) disrupted normal tubulogenesis in a manner similar to PKA-agonist treated MM/spinal-cord assays, suggesting that PKA regulates a Wnt-dependent tubulogenesis step. PKA induction of canonical Wnt signaling during tubulogenesis was confirmed genetically using MM from Batgal-reporter mice. Addition of a Wnt synthesis inhibitor to activated PKA cultures rescued tubulogenesis. By re-analysis of existing microarray data from the FGF8, Lim1 and Wnt4 knockouts, which arrest in early tubulogenesis, a network of genes involving PKA, Wnt, Lhx1, FGF8, and hyaluronic acid signaling regulating the transition of nascent epithelial cells to tubular epithelium was derived, helping to reconcile in vivo and in vitro/ex vivo data.
在发育中的肾脏中，调节小管发生的中间阶段的遗传相互作用一直难以确定。使用微阵列的系统生物学策略与体外/体外和遗传方法相结合，以鉴定调节小管发生特定阶段的途径。对后肾间充质（MM）通过小管诱导和分化的四个阶段（即上皮化、管状组织和伸长以及早期分化）的进展进行分析，揭示了每个阶段可能涉及的信号通路，并暗示了许多信号分子的关键作用。对体外/体外肾单位形成的信号通路进行筛选，表明蛋白激酶 A（PKA）通过 PKA-2 在特定的上皮后形态发生步骤（将肾小泡转化为 S 形体）中具有独特的调节作用。微阵列分析不仅证实了这种阶段特异性，而且还强调了 Wnt 基因的上调。PKA 激动剂添加到 LIF 诱导的肾单位（先前显示为 Wnt/beta-catenin 依赖性途径）中，以类似于 PKA-激动剂处理的 MM/脊髓板测定的方式破坏了正常的小管发生，表明 PKA 调节 Wnt 依赖性小管发生步骤。使用来自 Batgal 报告小鼠的 MM 进行遗传验证，证实了 PKA 在小管发生过程中诱导经典 Wnt 信号。向激活的 PKA 培养物中添加 Wnt 合成抑制剂可挽救小管发生。通过重新分析 FGF8、Lim1 和 Wnt4 敲除（在早期小管发生中停滞）的现有微阵列数据，涉及 PKA、Wnt、Lhx1、FGF8 和透明质酸信号的基因网络，调节初生上皮细胞向管状上皮的转变，有助于协调体内和体外/体外数据。
