Hypoxia and mitochondria in spine development and congenital scoliosis

摘要 脊椎骨在胚胎发育过程中起源于体节。体节是从 体节前中胚层（PSM），并且该过程被称为体节发生。体细胞发生是由钥匙控制的 PSM中的Notch信号以与体节形成相匹配的周期性振荡。缺氧发生 在循环系统建立之前，胚胎发育中自然会出现这种情况。然而，缺氧的加剧 因为它可能发生在怀孕期间，破坏了PSM中的振荡Notch信号，并导致异常的 体节发生和改变的脊柱发育。脊椎肋骨发育不全（SCDO）的特征是严重的 脊椎畸形，由Notch组成部分的纯合性功能丧失突变引起 信号通路携带类似纯合突变的小鼠表现出与人类疾病相似的表型。杂合 Notch LOF突变导致的先天性脊柱侧凸的人类缺陷虽然更常见，但程度更轻 (CS)，其也在杂合小鼠突变体中表型复制。这些小鼠的表型恶化， 妊娠缺氧缺氧反应的介质是缺氧诱导的转录因子 因子-1 α（HIF 1）和HIF 2。HIF 1和HIF 2在体节发生中的作用尚未得到解决。填补 基于这一知识空白，我们使用TCre转基因小鼠（HIF 1 突变体）。PSM中HIF 1的缺失导致异常的体节发生和脊柱畸形， SCDO/CS和妊娠缺氧。相反，初步数据显示，HIF 2对于脊柱不稳定性是不必要的。 但是伴随的HIF 1和HIF 2的缺失改善了在HIF 1中观察到的脊柱异常。 变种人缺氧增加了HIF的稳定性和转录活性;因此，我们对HIF的表达很感兴趣。 观察到妊娠缺氧和PSM中HIF 1的缺失以类似的方式改变体节发生。 值得注意的是，HIF 1的丢失和妊娠缺氧都导致PSM中细胞内缺氧的增加。 此外，初步研究结果表明，线粒体呼吸的损害，这是作为一个 减少细胞内缺氧的工具，部分纠正了在HIF 1突变体中观察到的棘缺陷。考虑 我们的初步数据，我们的工作假设是，在PSM和妊娠缺氧中， 增加细胞内缺氧，这反过来又使Notch信号通路失调， 体节发生我们还假设PSM中HIF 1的缺失通过增加细胞内HIF 2的表达而稳定HIF 2。 缺氧，并且增强的HIF 2转录活性介导了由于HIF 1缺失引起的一些效应。我们 假设将在两个目标中进行测试。完成拟议的实验将建立，为第一 据我们所知，HIF和线粒体呼吸在体节发生中的作用。它将定位HIF 1 在PSM中Notch信号通路的上游。它将为我们目前对宇宙的理解提供新的线索。 HIF 1和HIF 2之间的体内交叉对话。最后，它将为致病机制提供新的见解 导致先天性脊柱侧凸

ABSTRACT Vertebrae originate from somites during embryonic development. Somites are segmented from the presomitic mesoderm (PSM) and the process is known as somitogenesis. Somitogenesis is controlled by key Notch signals in the PSM that oscillate with a periodicity matching that of somite formation. Hypoxia occurs naturally in developing embryos before the circulatory system is established. However, exacerbation of hypoxia as it may take place during gestation disrupts the oscillatory Notch signals in the PSM and leads to abnormal somitogenesis and altered spine development. Spondylocostal Dysostosis (SCDO) is characterized by severe vertebral malformations and is caused by homozygous loss-of-function mutations of components of the Notch signaling pathway. Mice carrying similar homozygous mutations phenocopy the human disease. Heterozygous Notch LOF mutations cause the more modest, although more frequent, human defect of congenital scoliosis (CS), which is also phenocopied in heterozygous mouse mutants. These mice phenotypes are worsened by gestational hypoxia. The mediators of the hypoxic response are the transcription factors Hypoxia-Inducible Factor-1alpha (HIF1) and HIF2. The role of HIF1 and HIF2 in somitogenesis has not been addressed. To fill this gap of knowledge, we conditionally inactivated HIF1 in the PSM using TCre transgenic mice (HIF1 mutants). Loss of HIF1 in the PSM causes abnormal somitogenesis and spine malformations reminiscent of SCDO/CS and gestational hypoxia. Conversely, preliminary data showed that HIF2 is not necessary for spine development, but the concomitant loss of HIF1 and HIF2 ameliorates the spine abnormalities observed in HIF1 mutants. Hypoxia increases stability and transcriptional activity of the HIFs; therefore, we were intrigued by the observation that gestational hypoxia and loss of HIF1 in the PSM alter somitogenesis in a similar manner. Notably, both loss of HIF1 and gestational hypoxia led to an increase in intracellular hypoxia in the PSM. Furthermore, preliminary findings revealed that the impairing of mitochondrial respiration, which was used as a tool to reduce intracellular hypoxia, partially corrected the spine defects observed in HIF1 mutants. Considering our preliminary data, ourworking hypothesis is thatboth loss of HIF1 in the PSM and gestational hypoxia increase intracellular hypoxia, which in turn dysregulates the Notch signaling pathway and alters somitogenesis. We also hypothesize that loss of HIF1 in the PSM stabilizes HIF2 by increasing intracellular hypoxia, and the augmented HIF2 transcriptional activity mediates some of the effects due to loss of HIF1. Our hypotheses will be tested in two Aims. Accomplishment of the proposed experiments will establish, for the first time to our knowledge, the role of HIFs and mitochondria respiration in somitogenesis. It will position HIF1 upstream of the Notch signaling pathway in PSM. It will shed new light into our current understanding of the in vivo cross-talks between HIF1 and HIF2. Lastly, it will provide novel insights into the pathogenetic mechanisms leading to congenital scoliosis because of gestational hypoxia.