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， 将肺的末端气道细分为肺泡，肺泡是有效呼吸的关键结构。 肺部的气体交换。这个过程中的缺陷导致了简化肺泡的形成， 是支气管肺发育不良（BPD）的标志，BPD是一种慢性肺部疾病， 接受机械通气或氧疗的早产儿。虽然分子 在出生后阶段调节肺泡发育的机制是未知的， 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， Hox5基因（HoxA5、HoxB5和HoxC5）仅在肺间充质中表达， 所有三个Hox5基因的缺失导致严重的发育性肺缺陷和围产期 死亡四等位基因复合Hox 5突变小鼠（Hox 5 AabbCc）以孟德尔比率出生 他们的肺在出生时组织学上是正常的，然而，他们在出生时发展为肺泡简化。 产后阶段。与Hox 5基因在肺泡发生中的直接作用一致， 所有三种Hox 5基因的水平在出生后阶段达到峰值，此时肺泡形成处于其最低水平。 峰我们的实验室最近产生了Hoxa5的条件等位基因，使我们能够 绕过新生儿的致命性，并评估这组婴儿的胚胎后功能。 监管部门从出生开始肺间质中Hoxa5的条件性缺失导致 出生后肺泡简化。Hoxb5和Hoxc5的无效等位基因的添加加剧了 这个缺陷。Hox 5条件突变肺表现出异常的肌成纤维细胞分布、形状 和功能受损，并且适当的肺泡形成所需的弹性蛋白网络不能形成。 无偏RNAseq分析揭示了与细胞凋亡相关的基因表达变化 粘附和细胞外基质。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都正常表达 在Hox5条件突变体中。然而，突变的成纤维细胞表现出明显的粘附缺陷， 培养，初步数据显示在来源于人成纤维细胞的成纤维细胞中整合素β 5表达丧失。 Hox5条件突变体。总的来说，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以及控制肺基质形成至关重要 用于肺泡形成。利用遗传学，我计划阐明细胞和分子机制， 肺泡形成中肺间充质的Hox 5调节。

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.