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HIF-2Α Is Essential for Carotid Body Development and Function 3 4 5 6 David Macías1, Andrew S

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HIF-2Α Is Essential for Carotid Body Development and Function 3 4 5 6 David Macías1, Andrew S bioRxiv preprint doi: https://doi.org/10.1101/250928; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 HIF-2α is essential for carotid body development and function 3 4 5 6 David Macías1, Andrew S. Cowburn1,2, Hortensia Torres-Torrelo3, Patricia 7 Ortega-Sáenz3, José López-Barneo3, and Randall S. Johnson1,4,5. 8 9 1Department of Physiology, Development and Neuroscience. 2Department of 10 Medicine, University of Cambridge, Cambridge, UK; 3Instituto de Biomedicina 11 de Sevilla (IBiS), Seville, Spain. 4Department of Cell and Molecular Biology, 12 Karolinska Institute, Stockholm, Sweden. 13 14 15 16 17 18 19 20 21 22 23 Keywords: hypoxia, carotid body, oxygen sensing, HIF-2α, HVR, physiological 24 responses 25 26 27 28 5For correspondence: Professor Randall S. Johnson, Department of 29 Physiology, Development and Neuroscience, University of Cambridge, 30 Downing Street, Cambridge. CB2 3EG. United Kingdom. E-mail: 31 [email protected] 32 33 1 bioRxiv preprint doi: https://doi.org/10.1101/250928; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 34 Summary 35 Mammalian adaptation to oxygen flux occurs at many levels, from shifts in 36 cellular metabolism to physiological adaptations facilitated by the sympathetic 37 nervous system and carotid body (CB). Interactions between differing forms of 38 adaptive response to hypoxia, including transcriptional responses 39 orchestrated by the Hypoxia Inducible transcription Factors (HIFs), are 40 complex and clearly synergistic. We show here that there is an absolute 41 developmental requirement for HIF-2a, one of the HIF isoforms, for growth 42 and survival of oxygen sensitive glomus cells of the carotid body. The loss of 43 these cells renders mice incapable of ventilatory responses to hypoxia, and 44 this has striking effects on processes as diverse as arterial pressure 45 regulation, exercise performance, and glucose homeostasis. We show that 46 the expansion of the glomus cells is correlated with mTORC1 activation, and 47 is functionally inhibited by rapamycin treatment. These findings demonstrate 48 the central role played by HIF-2a in carotid body development, growth and 49 function. 50 51 52 53 54 55 56 57 58 2 bioRxiv preprint doi: https://doi.org/10.1101/250928; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 59 Introduction 60 Detecting and responding to shifts in oxygen availability is essential for animal 61 survival. Responding to changes in oxygenation occurs in cells, tissues, and 62 at the level of the whole organism. Although many of responses to oxygen flux 63 are regulated at the transcriptional level by the hypoxia inducible transcription 64 factor (HIF), at tissue and organismal levels there are even more dimensions 65 to the adaptive process. In vertebrates, the actors in adaptation are found in 66 different peripheral chemoreceptors, including the neuroepithelial cells of the 67 gills in teleosts, and in the carotid body (CB) of mammals. The CB regulates 68 many responses to changes in oxygenation, including alterations in ventilation 69 and heart rates. 70 71 The carotid body is located at the carotid artery bifurcation, and contains 72 glomeruli that contain O2-sensitive, neuron-like tyrosine hydroxylase (TH)- 73 positive glomus cells (type I cells). These glomus cells are responsible for a 74 rapid compensatory hypoxic ventilatory response (HVR) when a drop in 75 arterial O2 tension (PO2) occurs (Lopez-Barneo, Ortega-Saenz, et al., 2016; 76 Teppema & Dahan, 2010). In addition to this acute reflex, the CB plays an 77 important role in acclimatization to sustained hypoxia, and is commonly 78 enlarged in people living at high altitude (Arias-Stella & Valcarcel, 1976; Wang 79 & Bisgard, 2002) and in patients with chronic respiratory diseases (Heath & 80 Edwards, 1971; Heath, Smith, & Jago, 1982). This enlargement is induced 81 through a proliferative response of CB stem cells (type II cells)(Pardal, 82 Ortega-Saenz, Duran, & Lopez-Barneo, 2007; Platero-Luengo et al., 2014). 3 bioRxiv preprint doi: https://doi.org/10.1101/250928; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 83 During embryogenesis, the initiating event in CB organogenesis occurs when 84 multipotent neural crest cells migrate toward the dorsal aorta, to give rise to 85 the sympathoadrenal system (Le Douarin, 1986). Segregation of sympathetic 86 progenitors from the superior cervical ganglion (SCG) then creates the CB 87 parenchyma (Kameda, 2005; Kameda, Ito, Nishimaki, & Gotoh, 2008). This 88 structure is comprised of O2-sensitive glomus cells (type-I cells) (Pearse et al., 89 1973), glia-like sustentacular cells (type-II cells) (Pardal et al., 2007) and 90 endothelial cells (Annese, Navarro-Guerrero, Rodriguez-Prieto, & Pardal, 91 2017). 92 93 The cellular response to low oxygen is in part modulated by two isoforms of 94 the HIF-α transcription factor, each with differing roles in cellular and 95 organismal response to oxygen flux: HIF-1α and HIF-2α. At the organismal 96 level, mouse models hemizygous for HIFs-α (Hif-1α+/- and Hif-2α+/-) or where 97 HIF inactivation was induced in adults showed contrasting effects on CB 98 function, without in either case affecting CB morphogenesis (Hodson et al., 99 2016; Kline, Peng, Manalo, Semenza, & Prabhakar, 2002; Y. J. Peng et al., 100 2011). 101 102 Transcriptome analysis has confirmed that Hif-2α is the second most 103 abundant transcript in neonatal CB glomus cells (Tian, Hammer, Matsumoto, 104 Russell, & McKnight, 1998; Zhou, Chien, Kaleem, & Matsunami, 2016), and 105 that it is expressed at far higher levels than are found in cells of similar 106 developmental origins, including superior cervical ganglion (SCG) sympathetic 107 neurons (Gao et al., 2017). It has also been recently shown that Hif-2α, but 4 bioRxiv preprint doi: https://doi.org/10.1101/250928; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 108 not Hif-1α, overexpression in sympathoadrenal cells leads to enlargement of 109 the CB (Macias, Fernandez-Aguera, Bonilla-Henao, & Lopez-Barneo, 2014). 110 Here, we report that HIF-2α is required for the development of CB O2- 111 sensitive glomus cells, and that mutant animals lacking CB function have 112 impaired adaptive physiological responses. 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 5 bioRxiv preprint doi: https://doi.org/10.1101/250928; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 133 Results 134 Sympathoadrenal Hif-2α loss blocks carotid body glomus cell 135 development 136 To elucidate the role of HIFα isoforms in CB development and function, we 137 generated mouse strains carrying Hif-1α or Hif-2α embryonic deletions (TH- 138 HIF-1αKO and TH-HIF-2αKO) restricted to catecholaminergic tissues by 139 crossing them with a mouse strain expressing cre recombinase under the 140 control of the endogenous tyrosine hydroxylase (TH) promoter (Macias et al., 141 2014). 142 143 Histological analysis of the carotid artery bifurcation dissected from adult TH- 144 HIF-2αKO mice revealed virtually no CB TH+ glomus cells (Figure 1A, bottom 145 panels) compared to HIF-2αWT littermate controls (Figure 1A, top panels). 146 Conversely, TH-HIF-1αKO mice showed a typical glomus cell organization, 147 characterized by scattered clusters of TH+ cells throughout the CB 148 parenchyma (Figure 1B, bottom panels) similar to those found in HIF-1αWT 149 littermate controls (Figure 1B, top panels). These observations were further 150 corroborated by quantification of the total CB parenchyma volume (Figure 1D- 151 1F). 152 153 Other catecholaminergic organs whose embryological origins are similar to 154 those of the CB, e.g., the superior cervical ganglion (SCG), do not show 155 significant differences in structure or volume between TH-HIF-2αKO mutants 156 and control mice (Figure 1A-C). This suggests a specific role of HIF-2α in the 157 development of the CB glomus cells, and argues against a global role for the 6 bioRxiv preprint doi: https://doi.org/10.1101/250928; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 158 gene in development of catecholaminergic tissues. Further evidence for this 159 comes from phenotypic characterization of adrenal medulla (AM) TH+ 160 chromaffin cells. No major histological alterations were observed in adrenal 161 glands removed from TH-HIF-2αKO and TH-HIF-1αKO mutant mice compared 162 to HIF-2αWT and HIF-1αWT littermate controls (Figure 1G and H).
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