CPLANE protein INTU regulates growth and patterning of the mouse lungs through cilia-dependent Hh signaling.

Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of Inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increased Hh target gene expression and tubulogenesis in explanted lungs in wild type, but not Intu mutants, suggesting impaired Hh signaling response in the absence of Intu. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find a diminishing difference in gene expression between the left and right lungs in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs. Wild type and Intu mutant lungs were isolated at embryonic day 12.5, and left and right lobes were separated. Total RNAs were prepared from each sample for sequencing, such that comparisons can be made between wild and mutant, as well as between left and right.重症型先天性肺畸形可在出生时致命。对这类出生缺陷的预防与早期干预，有赖于对肺发育分子机制的全面认知。本研究发现，内翻蛋白（Inturned，Intu）——一种纤毛与平面极性效应基因——的缺失，会严重破坏小鼠胚胎肺的生长与分支形态发生。与我们既往研究中Intu在纤毛发生与刺猬（Hedgehog，Hh）信号通路中发挥重要作用的结果一致，我们发现肺上皮与间质组织中的初级纤毛数量均大幅减少。同时，Hh信号通路的直接靶基因Gli1与Ptch1的表达量显著降低，这表明Intu突变肺组织中依赖纤毛的Hh信号通路遭到破坏。Hh通路激活因子平滑蛋白（smoothened）的激动剂，可提升野生型小鼠离体肺组织的Hh靶基因表达与管腔发生，但对Intu突变体无此效果，这提示缺失Intu会导致Hh信号通路应答受损。进一步实验显示，同时敲除Gli2与Intu可完全阻断肺的分支形态发生，有力支持了Intu通过依赖纤毛的Hh信号通路调控肺生长与模式建成的机制。转录组学分析（transcriptomics analysis）发现，Intu突变肺组织中存在约200个差异表达基因（differentially expressed genes，DEGs），其中包括已知的Hh靶基因Gli1、Ptch1/2与Hhip。差异表达基因中显著富集了与肌肉分化及功能相关的基因，这与Hh信号通路在气道平滑肌分化中的重要作用相符。此外，我们发现Intu突变体中左右肺之间的基因表达差异逐渐消失，这表明Intu在小鼠肺的不对称生长与模式建成中发挥重要作用。本研究于胚胎第12.5天分离野生型与Intu突变小鼠的肺组织，分离其左右肺叶，随后从每份样本中提取总RNA进行测序，以便开展野生型与突变型、左侧与右侧肺组织之间的比较分析。