Characterization of the role of mesenchymal Hox5 genes in alveologenesis

摘要 肺泡形成发生在肺发育的最后阶段，并负责 将肺的末端呼吸道细分为肺泡，这是有效率的关键结构 肺部的气体交换。这一过程中的缺陷导致形成简化的肺泡， 是支气管肺发育不良(BPD)的标志，BPD是一种慢性肺部疾病，表现为 早产儿接受机械通气或氧疗治疗。虽然分子 在出生后阶段调节肺泡发育的机制尚不清楚， 肺间充质中的中胚层来源的成纤维细胞已被证明是至关重要的。 肺泡形成的驱动因素。我们实验室发表的研究表明，这三个 Hox5基因(HoxA5、HOXB5和HoxC5)仅在肺间充质中表达， 这三个Hox5基因的缺失会导致严重的肺发育缺陷和围产儿 死亡。四等位基因，复合Hox5突变小鼠(Hox5 AabbCc)按孟德尔比例出生 他们的肺在出生时组织学上是正常的，然而，他们在出生时出现肺泡简化 出生后阶段。与Hox5基因在肺泡发育中的直接作用一致，表达 三种Hox5基因的表达水平均在出生后的肺泡发育阶段达到高峰 匹克。我们的实验室最近产生了Hoxa5的条件等位基因，使我们能够 绕过新生儿死亡率，评估这组婴儿的胚胎后功能 监管者。从出生时开始，肺间充质中Hoxa5的条件缺失导致 出生后肺泡简化。HOXB5和HOXC5零等位基因的添加加剧了 这个缺陷。Hox5条件突变肺表现为肌成纤维细胞异常分布、形态 和功能受损，正常肺泡形成所需的弹性蛋白网络无法形成。 无偏RNAseq分析揭示了与细胞相关的类别的基因表达变化 黏附和细胞外基质。免疫荧光和蛋白质印迹分析表明 基底膜和细胞外基质成分表达正常 在Hox5条件突变体中。然而，突变的成纤维细胞表现出显著的黏附缺陷。 培养，初步数据显示整合素5在成纤维细胞中表达缺失 Hox5条件突变体。总而言之，我们的数据表明，Hox5基因调节适当的 间充质成纤维细胞的分化和功能与控制肺基质形成的关键 用于肺泡成形术。利用遗传学，我计划阐明细胞和分子机制 Hox5在肺泡形成中对肺间充质的调节作用

ABSTRACT Alveologenesis occurs during the last stage of lung development and is responsible for subdividing the terminal airways of the lungs into alveoli, structures that are critical for efficient gas exchange in the lung. Defects in this process lead to the formation of simplified alveoli that are a hallmark of bronchopulmonary dysplasia (BPD), a chronic lung disease that presents in premature infants treated with mechanical ventilation or oxygen therapy. Although the molecular mechanisms that regulate alveolar development during postnatal stages are unknown, mesodermally-derived fibroblasts in the lung mesenchyme have been shown to be critical drivers of alveologenesis. Published work from our laboratory has demonstrated that all three Hox5 genes (HoxA5, HoxB5 and HoxC5) are exclusively expressed in the lung mesenchyme, and loss of all three Hox5 genes leads to severe developmental lung defects and perinatal death. Four-allele, compound Hox5 mutant mice (Hox5 AabbCc) are born in Mendelian ratios and their lungs are histologically normal at birth, however, they develop alveolar simplification at postnatal stages. Consistent with a direct role for Hox5 genes in alveologenesis, the expression levels of all three Hox5 genes peak during the postnatal stages when alveologenesis is at its peak. Our laboratory has recently generated a conditional allele for Hoxa5, allowing us to bypass the neonatal lethality and assess the post-embryonic functions of this group of regulators. Conditional deletion of Hoxa5 in the lung mesenchyme beginning at birth results in alveolar simplification postnatally. The addition of null alleles for Hoxb5 and Hoxc5 exacerbate this defect. Hox5 conditional mutant lungs exhibit abnormal myofibroblast distribution, shape and impaired function, and the elastin network required for proper alveologenesis fails to form. Unbiased RNAseq analyses reveal gene expression changes in categories associated with cell adhesion and extracellular matrix. Immunofluorescence and western blot analyses demonstrate that both the basement membrane and extracellular matrix components are expressed normally in Hox5 conditional mutants. However, mutant fibroblasts exhibit significant adhesion defects in culture, and preliminary data show loss of Integrin5 expression in fibroblasts derived from Hox5 conditional mutants. Collectively, our data indicate that Hox5 genes regulate the proper differentiation and function of mesenchymal fibroblasts and control lung matrix formation critical for alveologenesis. Using genetics, I plan to elucidate the cellular and molecular mechanisms of Hox5 regulation of lung mesenchyme in alveologenesis.