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 viv...