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HIF-2α is essential for carotid body development and function 2
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David Macías1, Andrew S. Cowburn1,2, Hortensia Torres-Torrelo3, Patricia 6
Ortega-Sáenz3, José López-Barneo3, and Randall S. Johnson1,4,5. 7 81Department of Physiology, Development and Neuroscience. 2Department of 9
Medicine, University of Cambridge, Cambridge, UK; 3Instituto de Biomedicina 10
de Sevilla (IBiS), Seville, Spain. 4Department of Cell and Molecular Biology, 11
Karolinska Institute, Stockholm, Sweden. 12
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Keywords: hypoxia, carotid body, oxygen sensing, HIF-2α, HVR, physiological 23
responses 24
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275For correspondence: Professor Randall S. Johnson, Department of 28
Physiology, Development and Neuroscience, University of Cambridge, 29
Downing Street, Cambridge. CB2 3EG. United Kingdom. E-mail: 30
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Summary 34
Mammalian adaptation to oxygen flux occurs at many levels, from shifts in 35
cellular metabolism to physiological adaptations facilitated by the sympathetic 36
nervous system and carotid body (CB). Interactions between differing forms of 37
adaptive response to hypoxia, including transcriptional responses 38
orchestrated by the Hypoxia Inducible transcription Factors (HIFs), are 39
complex and clearly synergistic. We show here that there is an absolute 40
developmental requirement for HIF-2a, one of the HIF isoforms, for growth 41
and survival of oxygen sensitive glomus cells of the carotid body. The loss of 42
these cells renders mice incapable of ventilatory responses to hypoxia, and 43
this has striking effects on processes as diverse as arterial pressure 44
regulation, exercise performance, and glucose homeostasis. We show that 45
the expansion of the glomus cells is correlated with mTORC1 activation, and 46
is functionally inhibited by rapamycin treatment. These findings demonstrate 47
the central role played by HIF-2a in carotid body development, growth and 48
function. 49
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Introduction 59
Detecting and responding to shifts in oxygen availability is essential for animal 60
survival. Responding to changes in oxygenation occurs in cells, tissues, and 61
at the level of the whole organism. Although many of responses to oxygen flux 62
are regulated at the transcriptional level by the hypoxia inducible transcription 63
factor (HIF), at tissue and organismal levels there are even more dimensions 64
to the adaptive process. In vertebrates, the actors in adaptation are found in 65
different peripheral chemoreceptors, including the neuroepithelial cells of the 66
gills in teleosts, and in the carotid body (CB) of mammals. The CB regulates 67
many responses to changes in oxygenation, including alterations in ventilation 68
and heart rates. 69
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The carotid body is located at the carotid artery bifurcation, and contains 71
glomeruli that contain O2-sensitive, neuron-like tyrosine hydroxylase (TH)-72
positive glomus cells (type I cells). These glomus cells are responsible for a 73
rapid compensatory hypoxic ventilatory response (HVR) when a drop in 74
arterial O2 tension (PO2) occurs (Lopez-Barneo, Ortega-Saenz, et al., 2016; 75
Teppema & Dahan, 2010). In addition to this acute reflex, the CB plays an 76
important role in acclimatization to sustained hypoxia, and is commonly 77
enlarged in people living at high altitude (Arias-Stella & Valcarcel, 1976; Wang 78
& Bisgard, 2002) and in patients with chronic respiratory diseases (Heath & 79
Edwards, 1971; Heath, Smith, & Jago, 1982). This enlargement is induced 80
through a proliferative response of CB stem cells (type II cells)(Pardal, 81
Ortega-Saenz, Duran, & Lopez-Barneo, 2007; Platero-Luengo et al., 2014). 82
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During embryogenesis, the initiating event in CB organogenesis occurs when 83
multipotent neural crest cells migrate toward the dorsal aorta, to give rise to 84
the sympathoadrenal system (Le Douarin, 1986). Segregation of sympathetic 85
progenitors from the superior cervical ganglion (SCG) then creates the CB 86
parenchyma (Kameda, 2005; Kameda, Ito, Nishimaki, & Gotoh, 2008). This 87
structure is comprised of O2-sensitive glomus cells (type-I cells) (Pearse et al., 88
1973), glia-like sustentacular cells (type-II cells) (Pardal et al., 2007) and 89
endothelial cells (Annese, Navarro-Guerrero, Rodriguez-Prieto, & Pardal, 90
2017). 91
92
The cellular response to low oxygen is in part modulated by two isoforms of 93
the HIF-α transcription factor, each with differing roles in cellular and 94
organismal response to oxygen flux: HIF-1α and HIF-2α. At the organismal 95
level, mouse models hemizygous for HIFs-α (Hif-1α+/- and Hif-2α+/-) or where 96
HIF inactivation was induced in adults showed contrasting effects on CB 97
function, without in either case affecting CB morphogenesis (Hodson et al., 98
2016; Kline, Peng, Manalo, Semenza, & Prabhakar, 2002; Y. J. Peng et al., 99
2011). 100
101
Transcriptome analysis has confirmed that Hif-2α is the second most 102
abundant transcript in neonatal CB glomus cells (Tian, Hammer, Matsumoto, 103
Russell, & McKnight, 1998; Zhou, Chien, Kaleem, & Matsunami, 2016), and 104
that it is expressed at far higher levels than are found in cells of similar 105
developmental origins, including superior cervical ganglion (SCG) sympathetic 106
neurons (Gao et al., 2017). It has also been recently shown that Hif-2α, but 107
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not Hif-1α, overexpression in sympathoadrenal cells leads to enlargement of 108
the CB (Macias, Fernandez-Aguera, Bonilla-Henao, & Lopez-Barneo, 2014). 109
Here, we report that HIF-2α is required for the development of CB O2-110
sensitive glomus cells, and that mutant animals lacking CB function have 111
impaired adaptive physiological responses. 112
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Results 133
Sympathoadrenal Hif-2α loss blocks carotid body glomus cell 134
development 135
To elucidate the role of HIFα isoforms in CB development and function, we 136
generated mouse strains carrying Hif-1α or Hif-2α embryonic deletions (TH-137
HIF-1αKO and TH-HIF-2αKO) restricted to catecholaminergic tissues by 138
crossing them with a mouse strain expressing cre recombinase under the 139
control of the endogenous tyrosine hydroxylase (TH) promoter (Macias et al., 140
2014). 141
142
Histological analysis of the carotid artery bifurcation dissected from adult TH-143
HIF-2αKO mice revealed virtually no CB TH+ glomus cells (Figure 1A, bottom 144
panels) compared to HIF-2αWT littermate controls (Figure 1A, top panels). 145
Conversely, TH-HIF-1αKO mice showed a typical glomus cell organization, 146
characterized by scattered clusters of TH+ cells throughout the CB 147
parenchyma (Figure 1B, bottom panels) similar to those found in HIF-1αWT 148
littermate controls (Figure 1B, top panels). These observations were further 149
corroborated by quantification of the total CB parenchyma volume (Figure 1D-150
1F). 151
152
Other catecholaminergic organs whose embryological origins are similar to 153
those of the CB, e.g., the superior cervical ganglion (SCG), do not show 154
significant differences in structure or volume between TH-HIF-2αKO mutants 155
and control mice (Figure 1A-C). This suggests a specific role of HIF-2α in the 156
development of the CB glomus cells, and argues against a global role for the 157
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gene in development of catecholaminergic tissues. Further evidence for this 158
comes from phenotypic characterization of adrenal medulla (AM) TH+ 159
chromaffin cells. No major histological alterations were observed in adrenal 160
glands removed from TH-HIF-2αKO and TH-HIF-1αKO mutant mice compared 161
to HIF-2αWT and HIF-1αWT littermate controls (Figure 1G and H). Consistent 162
with this, the amount of catecholamine (adrenaline and noradrenaline) present 163
in the urine of TH-HIF-2αKO and TH-HIF-1αKO deficient mice was similar to 164
that found in their respective littermate controls (Figure 1I). 165
166
To determine deletion frequencies in these tissues, we crossed a loxP-flanked 167
Td-Tomato reporter strain (Madisen et al., 2010) with TH-IRES-Cre mouse 168
mice. Td-Tomato+ signal was only detected within the CB, SCG and AM of 169
mice expressing cre recombinase under the control of the Th promoter 170
(Figure 1-figure supplement 1A). Additionally, SCG and AM from HIF-2αWT, 171
HIF-1αWT, TH-HIF-2αKO and TH-HIF-1αKO were quantified for Hif-2α and Hif-172
1α deletion efficiency using genomic DNA. As expected, there is a significant 173
level of deletion of Hif-2α and Hif-1α genes detected in TH-HIF-2αKO and TH-174
HIF-1αKO mutant mice compared to HIF-2αWT and HIF-1αWT littermates 175
controls (Figure 1-figure supplement 1B and C). 176
177
To determine whether absence of CB glomus cells in adult TH-HIF-2αKO mice 178
is a result of impaired glomus cell differentiation or cell survival, we examined 179
carotid artery bifurcations dissected from TH-HIF-2αKO mice at embryonic 180
stage E18.5 (i.e., 1-2 days before birth), and postnatally (P0) (Figure 2A). 181
Differentiated TH+ glomus cells were found in the carotid bifurcation of TH-182
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HIF-2αKO mutant mice at both stages (Figure 2A). However, there is a 183
significant reduction in the total CB parenchyma volume and in the number of 184
differentiated TH+ glomus cells in TH-HIF-2αKO mice at E18.5, and this 185
reduction is even more evident in newborn mice (Figure 2B and C). Since CB 186
volume and cells number of mutant mice decreased in parallel, no significant 187
changes were seen in TH+ cell density at these two time points, however 188
(Figure 2D). 189
190
Consistent with the results described above (Figure 1C), the SCG volume was 191
not different in mutant newborns relative to wild type controls (Figure 2E). We 192
next assessed cell death in the developing CB, and found a progressive 193
increase in the number of TUNEL+ cells within the CB parenchyma of TH-HIF-194
2αKO mice compared to HIF-2αWT littermates (Figure 2F and G). These data 195
demonstrate that HIF-2α, but not HIF-1α, is essential for the differentiation of 196
CB glomus cells. 197
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Impaired ventilatory response and whole-body metabolic activity in TH-199
HIF-2αKO mice exposed to hypoxia 200
Although TH-HIF-2αKO deficient mice did not show an apparent phenotype in 201
the AM or SCG, it was important to determine whether their secretory or 202
electrical properties were altered (Figure 3-figure supplement 1). The 203
secretory activity of chromaffin cells under basal conditions and in response to 204
hypoxia, hypercapnia and high (20mM) extracellular K+ was not changed in 205
TH-HIF-2αKO mice (8-12 weeks old) relative to control littermates (Figure 3-206
figure supplement 1A-D). Current-voltage (I-V) relationships for Ca2+ and K+ 207
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currents of dispersed chromaffin cells (Figure 3- figure supplement 1E) and 208
SCG neurons (Figure 3-figure supplement 1F) were also similar in HIF-2αWT 209
and TH-HIF-2αKO animals. These data further support the notion that HIF-2α 210
loss in the sympathoadrenal lineages predominantly affects CB glomus cells. 211
212
To study the impact of the TH-HIF-2αKO mutation on respiratory function, we 213
examined ventilation in normoxia (21% O2) and in response to environmental 214
normobaric hypoxia (10% O2) via whole-body plethysmography. Figure 3A 215
illustrates the changes of respiratory rate during a prolonged hypoxic time-216
course in HIF-2αWT and TH-HIF-2αKO littermates. HIF-2αWT mice breathing 217
10% O2 had a biphasic response, characterized by an initial rapid increase in 218
respiratory rate followed by a slow decline. In contrast, TH-HIF-2αKO mice, 219
which have normal ventilation rates in normoxia (Figure 3A-C), respond 220
abnormally throughout the hypoxic period. Averaged respiratory rate and 221
minute volume during the initial 5 minutes of hypoxia show a substantial 222
reduction in ventilation rate in TH-HIF-2αKO mutant mice relative to HIF-2αWT 223
controls (Figure 3B and C). 224
225
Additional respiratory parameters altered by the lack of CB in response to 226
hypoxia are shown in Figure 3-supplement 2. Consistent with these data, 227
hemoglobin saturation levels dropped to 50-55% in TH-HIF-2αKO mice, as 228
compared to 73% saturation in control animals after exposure to 10% O2 for 5 229
minutes (Figure 3D). HIF-1αWT and TH-HIF-1αKO mice have similar respiratory 230
responses to 21% O2 and 10% O2, which indicates that HIF-1α is not required 231
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for the CB-mediated ventilatory response to hypoxia (Figure 3E-G; Figure 3-232
supplemental 2F-J). 233
We next determined metabolic activity in HIF-2αWT and TH-HIF-2αKO 234
littermates in a 21% O2 or 10% O2 environment. Circadian metabolic profiles 235
(VO2 and VCO2) were similar in WT and TH-HIF-2αKO mutant mice (Figure 3H 236
and I). After exposure to hypoxia, however, TH-HIF-2αKO mice showed lower 237
VO2 and VCO2 peaks and had a significantly hampered metabolic adaptation 238
to low oxygen (Figure 3J and K). 239
240
Deficient acclimatization to chronic hypoxia in mice with CBs loss 241
The CB grows and expands during sustained hypoxia, due to the proliferative 242
response of glia-like (GFAP+) neural crest-derived stem cells within the CB 243
parenchyma (Arias-Stella & Valcarcel, 1976; Pardal et al., 2007). Despite the 244
absence of adult TH+ oxygen sensing cells in TH-HIF-2αKO mice, we identified 245
a normal number of CB GFAP+ stem cells in the carotid artery bifurcation of 246
these animals (Figure 4-figure supplement 1A). We then determined the rate 247
at which CB progenitors from TH-HIF-2αKO mice were able to give rise to 248
newly formed CB TH+ glomus cells after chronic hypoxia exposition in vivo. 249
Typical CB enlargement and emergence of double BrdU+ TH+ glomus cells 250
was seen in HIF-2αWT mice after 14 days breathing 10% O2 (Figure 4-figure 251
supplement 1B-D), as previously described (Pardal et al., 2007). However, no 252
newly formed double BrdU+ TH+ glomus cells were observed in TH-HIF-2αKO 253
mice after 14 days in hypoxia (Figure 4-figure supplement 1B-D). These 254
observations are consistent with the documented role of CB glomus cells as 255
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activators of the CB progenitors proliferation in response to hypoxia (Platero-256
Luengo et al., 2014). 257
258
HIF-2αWT, HIF-1αWT and TH-HIF-1αKO mice adapted well and survived up to 259
21 days in a 10% O2 atmosphere normally. However, long-term survival of the 260
TH-HIF-2αKO deficient mice kept in a 10% O2 environment was severely 261
compromised, and only 40% reached the experimental endpoint (Figure 4A). 262
Mouse necropsies revealed internal general haemorrhages, which are most 263
likely the cause of the death. TH-HIF-2αKO mice also showed increased 264
haematocrit and haemoglobin levels relative to HIF-2αWT littermates when 265
maintained for 21 days in a 10% O2 atmosphere (Figure 4B and C). 266
Consistent with this, after 21 days in hypoxia, heart and spleen removed from 267
TH-HIF-2αKO mice were enlarged relative to HIF-2αWT littermates, likely due to 268
increased blood viscosity and extramedullary hematopoiesis, respectively 269
(Figure 4D and E). TH-HIF-1αKO mice did not show significant differences 270
compared to HIF-1αWT littermates maintained either in a 21% O2 or 10% O2 271
environment (Figure 4-figure supplement 2A-D). 272
273
Chronic exposure to hypoxia induces pulmonary arterial hypertension (PAH) 274
in mice (Cowburn et al., 2016; Stenmark, Meyrick, Galie, Mooi, & McMurtry, 275
2009). The right ventricle was found to be significantly enlarged in TH-HIF-276
2αKO mice relative to control mice after 21 days of 10% O2 exposure, 277
indicating an aggravation of pulmonary hypertension (Figure 4F). This result 278
was further confirmed by direct measurement of right ventricular systolic 279
pressure (RVSP) on anaesthetised mice (Figure 4G). Lung histology of mice 280
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chronically exposed to 10% O2 (Figure 4H) shows that the percentage of fully 281
muscularized lung peripheral vessels is significantly increased in TH-HIF-2αKO 282
mice relative to controls. The tunica media of lung peripheral vessels was also 283
significantly thicker in TH-HIF-2αKO mice compared to HIF-2αKO littermates 284
after 21 days of hypoxic exposure (Figure 4I). Taken together, these data 285
demonstrate that pulmonary adaptation to chronic hypoxia is highly reliant on 286
a normally functioning CB. 287
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Cardiovascular homeostasis is altered in mice lacking CBs 289
An important element of the CB chemoreflex is an increase in sympathetic 290
outflow (Lopez-Barneo, Ortega-Saenz, et al., 2016). To determine whether 291
this had been affected in TH-HIF-2αKO mutants, cardiovascular parameters 292
were recorded on unrestrained mice by radiotelemetry. Circadian blood 293
pressure monitoring showed a constitutive hypotensive condition that was 294
more pronounced during the diurnal period (Figure 5A and B) and did not 295
correlate with changes in heart rate, subcutaneous temperature or activity 296
(Figure 5C; Figure 5-figure supplement 1A and B). We next subjected 297
radiotelemetry-implanted HIF-2αWT and TH-HIF-2αKO littermates to a 10% O2 298
environment preceded and followed by a 24 hour period of normoxia (Figure 299
5D-F; Figure 5-figure supplement 1C-E). Normal control HIF-2αWT mice have 300
a triphasic response to this level of hypoxia, characterised by a short (10 301
minutes) initial tachycardia and hypertension, followed by a marked drop both 302
in heart rate and blood pressure; a partial to complete recovery is then seen 303
after 24-36 hours (Figure 5D and E; Figure 5-figure supplement 1C and 304
D)(Cowburn, Macias, Summers, Chilvers, & Johnson, 2017). However, while 305
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TH-HIF-2αKO mice experienced similar, but less deep, hypotension 3 hours 306
post hypoxic challenge, they failed to recover, and showed a persistent 307
hypotensive state throughout the hypoxic period (Figure 5D; Figure 5-figure 308
supplement 1C and D). Interestingly, the bradycardic effect of hypoxia on 309
heart rate was abolished in TH-HIF-2αKO animals (Figure 5E). Subcutaneous 310
temperature, an indirect indicator of vascular resistance in the skin, showed 311
an analogous trend to that seen in the blood pressure of TH-HIF-2αKO 312
mutants (Figure 5F). Activity of TH-HIF-2αKO mice during hypoxia was slightly 313
reduced relative to littermate controls (Figure 5-figure supplement 1E). 314
315
The carotid body has been proposed as a therapeutic target for neurogenic 316
hypertension (McBryde et al., 2013; Pijacka et al., 2016). To determine the 317
relationship between CB and hypertension in these mutants, we performed 318
blood pressure recordings in an angiotensin II (Ang II)-induced experimental 319
hypertension model (Figure 5G). As shown, TH-HIF-2αKO animals showed 320
lower blood pressure relative to HIF-2αWT littermate controls after 7 days of 321
Ang II infusion (Figure 5H; Figure 5-figure supplement 1F and G). Heart rate, 322
subcutaneous temperature and physical activity were unchanged throughout 323
the experiment (Figure 5K-M; Figure 5-figure supplement 1). Collectively, 324
these data highlight the CB as a systemic cardiovascular regulator which, 325
contributes to baseline blood pressure modulation, is critical for 326
cardiovascular adaptations to hypoxia and delays the development of Ang II-327
induced experimental hypertension. 328
329
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Adaptive responses to exercise and high glucose are affected in mice 330
with CB dysfunction 331
We next questioned whether this mutant, which lacks virtually all CB function, 332
can inform questions about adaptation to other physiological stressors. In the 333
first instance, we assessed metabolic activity during a maximal exertion 334
exercise test. Oxygen consumption and carbon dioxide production (VO2 and 335
VCO2, respectively) were comparable in HIF-2αWT and TH-HIF-2αKO mice at 336
the beginning of the test. However, TH-HIF-2αKO mutants reached VO2max 337
much earlier than wild type controls, with significantly decreased heat 338
generation (Figure 6A, B and D). There was no shift in energetic substrate 339
usage, as evidenced by the lack of difference in respiratory exchange ratio 340
(RER) (Figure 6C). The time to exhaustion trended lower in mutants, and 341
there was a significant reduction in both aerobic capacity and power output 342
relative to littermate controls (Figure 6E-G). Interestingly, although no 343
significant changes were found in glucose blood content across the 344
experimental groups and conditions (Figure 6H), lactate levels in TH-HIF-2αKO 345
mice were significantly lower than those observed in HIF-2αWT mice after 346
running (Figure 6I). 347
348
We next examined the response of TH-HIF-2αKO mice to hyperglycemic and 349
hypoglycemic challenge. Baseline glucose levels of HIF-2αWT and TH-HIF-350
2αKO animals were not statistically different under fed and fasting conditions 351
(Figure 6J and K), and metabolic activity during fasting was unaffected (Figure 352
6-figure supplement 1A-D). However, TH-HIF-2αKO mutant mice showed 353
significant changes in glucose tolerance, with no change in insulin tolerance 354
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when compared to HIF-2αWT littermates (Figure 6L and M). TH-HIF-2αKO mice 355
also showed an initially hampered lactate clearance relative to control mice 356
(Figure 6N). Together, these data demonstrate that the mutants demonstrate 357
intriguing roles for the CB in metabolic adaptations triggered by both exercise 358
and hyperglycemia. 359
360
CB development and proliferation in response to hypoxia require 361
mTORC1 activation 362
Hypoxia is an mTORC1 activator that can induce cell proliferation and growth 363
in a number of organs, e.g., the lung parenchyma (Brusselmans et al., 2003; 364
Cowburn et al., 2016; Elorza et al., 2012; Hubbi & Semenza, 2015; Torres-365
Capelli et al., 2016). Physiological proliferation and differentiation of the CB 366
induced by hypoxia closely resembles CB developmental expansion (Annese 367
et al., 2017; Pardal et al., 2007; Platero-Luengo et al., 2014); therefore a 368
potential explanation for the developmental defects in CB expansion observed 369
in above could be defects in mTORC1 activation. 370
371
To test this hypothesis, we first determined mTORC1 activation levels in the 372
CB of mice maintained at 21% O2 or 10% O2 for 14 days. This was done via 373
immunofluorescent detection of the phosphorylated form of the ribosomal 374
protein S6 (p-S6) (Elorza et al., 2012; Ma & Blenis, 2009). In normoxia, there 375
was a faint p-S6 expression within the TH+ glomus cells of the CB in wild type 376
mice; this contrasts with the strong signal detected in SCG sympathetic 377
neurons (Figure 7A, left panels; Figure 7-figure supplement 1A, left panel). 378
However, after 14 days in a 10% O2 environment, we saw a significantly 379
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increased p-S6 signal within the CB TH+ cells while the SCG sympathetic 380
neurons remained at levels similar to those seen in normoxia (Figure 7A and 381
B; Figure 7-figure supplement 1A and B). TH expression, which is induced by 382
hypoxia (Schnell et al., 2003), was also significantly elevated in CB TH+ cells, 383
and trended higher in SCG sympathetic neurons (Figure 7B; Figure 7-figure 384
supplement 1B). 385
386
We next assessed the role of HIF-2α in this hypoxia-induced mTORC1 387
activation. We began with evaluating expression of p-S6 in late stage 388
embryos. In E18.5 embryos there was a striking accumulation of p-S6; this is 389
significantly reduced in HIF-2α deficient CB TH+ cells in embryos from this 390
stage of development (Figure 7C and D). p-S6 levels in SCG TH+ sympathetic 391
neurons of HIF-2αWT and TH-HIF-2αKO E18.5 embryos were unaffected, 392
however (Figure 7-figure supplement 1C and D). 393
394
TH expression trended lower in both CB and SCG TH+ cells from TH-HIF-395
2αKO E18.5 embryos relative to HIF-2αWT littermate controls (Figure 7D; 396
Figure 7-figure supplement 1D). These data indicate that HIF-2α regulates 397
mTORC1 activity in the CB glomus cells during development. To determine 398
whether this pathway could be pharmacologically manipulated, we then 399
studied the effect of mTORC1 inhibition by rapamycin on CB proliferation and 400
growth following exposure to environmental hypoxia. mTORC1 activity, as 401
determined by p-S6 staining on SCG and CB TH+ cells after 14 days of 402
hypoxic treatment, was inhibited after in vivo rapamycin administration (Figure 403
7-figure supplement 1E). Interestingly, histological analysis of rapamycin 404
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injected wild type mice maintained for 14 days at 10% O2 showed decreased 405
CB parenchyma volume, TH+ cells number, and showed a dramatic reduction 406
in the number of proliferating double BrdU+ TH+ cells when compared to 407
vehicle-injected mice (Figure 7E-H). 408
409
In addition, we assessed the ventilatory acclimatization to hypoxia (VAH), as a 410
progressive increase in ventilation rates is associated with CB growth during 411
sustained exposure to hypoxia (Hodson et al., 2016). Consistent with the 412
histological analysis above, rapamycin treated mice had a decreased VAH 413
compared to vehicle-injected control mice following 7 days at 10% O2 (Figure 414
7K). Haematocrit levels were comparable between vehicle control and 415
rapamycing treated wild type mice after 7 days in hypoxia (Figure 7-figure 416
supplement 1F). 417
418
Collectively, these data indicate that mTORC1 activation is required for CB 419
expansion in a HIF-2α-dependent manner, and this activation regulates CB 420
growth and subsequent VAH under prolonged low pO2 conditions. 421
422
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Discussion 423
Metabolic homeostasis depends on a complex system of O2-sensing cells and 424
organs. The carotid body is central to this, via its regulation of metabolic and 425
ventilatory responses to oxygen (Lopez-Barneo, Macias, Platero-Luengo, 426
Ortega-Saenz, & Pardal, 2016). At the cellular level, the HIF transcription 427
factor is equally central for responses to oxygen flux. However, how HIF 428
contributes to the development and function of CB O2-sensitivity is still not 429
completely understood. Initial studies performed on global Hif-1α-/- or Hif-2α-/- 430
mutant mice revealed that HIFs are critical for development (Compernolle et 431
al., 2002; Iyer et al., 1998; J. Peng, Zhang, Drysdale, & Fong, 2000; Ryan, Lo, 432
& Johnson, 1998; Scortegagna et al., 2003; Tian et al., 1998), but only HIF-433
2α-/- embryos showed defects in catecholamine homeostasis, which indicated 434
a potential role in sympathoadrenal development (Tian et al., 1998). 435
436
Investigations using hemizygous Hif-1α+/- and Hif-2α+/- mice showed no 437
changes in the CB histology or overall morphology (Kline et al., 2002; Y. J. 438
Peng et al., 2011). However, our findings demonstrate that embryological 439
deletion of Hif-2α, but not Hif-1α, specifically in sympathoadrenal tissues 440
(TH+) prevents O2-sensitive glomus cell development, without affecting SCG 441
sympathetic neurons or AM chromaffin cells of the same embryological origin. 442
It was previously reported that HIF-2α, but not HIF-1α overactivation in 443
catecholaminergic tissues led to marked enlargement of the CB (Macias et al., 444
2014). Mouse models of Chuvash polycythemia, which have a global increase 445
in HIF-2α levels, also show CB hypertrophy and enhanced acute hypoxic 446
ventilatory response (Slingo, Turner, Christian, Buckler, & Robbins, 2014). 447
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Thus either over-expression or deficiency of HIF-2α clearly perturbs CB 448
development and, by extension, function. 449
450
Sustained hypoxia is generally thought to attenuate cell proliferation in a HIF-451
1α-dependent fashion (Carmeliet et al., 1998; Goda et al., 2003; Koshiji et al., 452
2004). However, environmental hypoxia can also trigger growth of a number 453
of specialized tissues, including the CB (Arias-Stella & Valcarcel, 1976; Pardal 454
et al., 2007) and the pulmonary arteries (Madden, Vadula, & Kurup, 1992). In 455
this context, the HIF-2α isoform seems to play the more prominent role, and 456
has been associated with pulmonary vascular remodeling as well as with 457
bronchial epithelial proliferation (Brusselmans et al., 2003; Bryant et al., 2016; 458
Cowburn et al., 2016; Kapitsinou et al., 2016; Tan et al., 2013; Torres-Capelli 459
et al., 2016). 460
461
CB enlargement induced by hypoxia to a large extent recapitulates the cellular 462
events that give rise to mature O2-sensitive glomus cells in development. In a 463
recent study, global HIF-2α, but not HIF-1α, inactivation in adult mice was 464
shown to impair CB growth and associated VAH caused by exposure to 465
chronic hypoxia (Hodson et al., 2016). Therefore, we used this physiological 466
proliferative response as a model to understand the developmental 467
mechanisms underpinning of HIF-2α deletion in O2-sensitive glomus cells. 468
469
Contrasting effects of HIF-α isoforms on the mammalian target of rapamycin 470
complex 1 (mTORC1) activity can be found in the literature that mirror the 471
differential regulation of cellular proliferation induced by hypoxia in different 472
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tissues (see above) (Brugarolas et al., 2004; Elorza et al., 2012; Liu et al., 473
2006). Our findings reveal that mTORC1, a central regulator of cellular 474
metabolism, proliferation and survival (Laplante & Sabatini, 2012), is 475
necessary for CB O2-sensitive glomus cell proliferation; and further, that 476
impact in the subsequent enhancement of the hypoxic ventilatory response 477
induced by chronic hypoxia. Importantly, we found that mTORC1 activity is 478
reduced in HIF-2α-deficient embryonic glomus cells, but not in SCG 479
sympathetic neurons, which likely explains the absence of a functional adult 480
CB in these mutants. 481
482
As HIF-2α regulates mTORC1 activity to promote proliferation, this 483
connection could prove relevant in pathological situations; e.g., both somatic 484
and germline Hif-2α gain-of-function mutations have been found in patients 485
with paraganglioma, a catecholamine-secreting tumor of neural crest origin 486
(Lorenzo et al., 2013; Zhuang et al., 2012). Indeed, CB tumors are 10 times 487
more frequent in people living at high altitude (Saldana, Salem, & Travezan, 488
1973). As further evidence of this link, it has been shown that the mTOR 489
pathway is commonly activated in both paraganglioma and 490
pheochromocytoma tumors (Du et al., 2016; Favier, Amar, & Gimenez-491
Roqueplo, 2015). 492
493
CB chemoreception plays a crucial role in homeostasis, particularly in 494
regulating cardiovascular and respiratory responses to hypoxia. Our data 495
show that mice lacking CB O2-sensitive cells (TH-HIF-2αKO mice) have 496
impaired respiratory, cardiovascular and metabolic adaptive responses. 497
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Although previous studies have linked HIF-2α and CB function (Hodson et al., 498
2016; Y. J. Peng et al., 2011), none of them exhibited a complete CB loss of 499
function. This might explain the differing effects observed in hemizygous Hif-500
2α+/- mice (Hodson et al., 2016; Y. J. Peng et al., 2011) compared to our TH-501
HIF-2αKO mice. 502
Mice with a specific HIF-1α ablation in CB O2-sensitive glomus cells (TH-HIF-503
1αKO mice) did not show altered hypoxic responses in our experimental 504
conditions. Consistent with this, hemizygous Hif-1α+/- mice exhibited a normal 505
acute HVR (Hodson et al., 2016; Kline et al., 2002) albeit some otherwise 506
aberrant CB activities in response to hypoxia (Kline et al., 2002; Ortega-507
Saenz, Pascual, Piruat, & Lopez-Barneo, 2007). 508
509
Alterations in CB development and/or function have recently acquired 510
increasing potential clinical significance and have been associated with 511
diseases such as heart failure, obstructive sleep apnea, chronic obstructive 512
pulmonary disease, sudden death infant syndrome and hypertension 513
(Iturriaga, Del Rio, Idiaquez, & Somers, 2016; Lopez-Barneo, Ortega-Saenz, 514
et al., 2016). 515
516
We describe here the first mouse model with specific loss of carotid body O2-517
sensitive cells: a model that is viable in normoxia but shows impaired 518
physiological adaptations to both acute and chronic hypoxia. In addition, we 519
describe new and likely important roles for the CB in systemic blood pressure 520
regulation, exercise performance and glucose management. Further 521
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investigation of this mouse model could aid in revealing roles of the carotid 522
body in both physiology and disease. 523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
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Material and Methods 547
Mice husbandry 548
This research has been regulated under the Animals (Scientific Procedures) 549
Act 1986 Amendment Regulations 2012 following ethical review by the 550
University of Cambridge Animal Welfare and Ethical Review Body (AWERB). 551
Targeted deletion of Hif-1α, Hif-2α and Td-Tomato in sympathoadrenal 552
tissues was achieved by crossing homozygous mice carrying loxP-flanked 553
alleles Hif1atm3Rsjo (Ryan et al., 1998), Epas1tm1Mcs (Gruber et al., 2007) or 554
Gt(ROSA)26Sortm9(CAG-tdTomato)Hze (Madisen et al., 2010) into a mouse strain of 555
cre recombinase expression driven by the endogenous tyrosine hydroxylase 556
promoter (Thtm1(cre)Te)(Lindeberg et al., 2004), which is specific of 557
catecholaminergic cells. LoxP-flanked littermate mice without cre 558
recombinase (HIF-1αflox/flox, HIF-2αflox/flox or Td-Tomatoflox/flox) were used as 559
control group (HIF-1αWT, HIF-2αWT and Td-Tomato, respectively) for all the 560
experiments. C57BL/6J wild type mice were purchased from The Jackson 561
laboratory (Bar Harbor, Maine, US). All the experiments were performed with 562
age and sex matched control and experimental mice. 563
564
Tissue preparation, immunohistochemistry and morphometry 565
Newborn (P0) and adult mice (8-12 weeks old) were killed by an overdose of 566
sodium pentobarbital injected intraperitoneally. E18.5 embryos were collected 567
and submerged into a cold formalin solution (Sigma-Aldrich, Dorset, UK). The 568
carotid bifurcation and adrenal gland, were dissected, fixed for 2 hours with 569
4% paraformaldehyde (Santa Cruz Biotechnology) and cryopreserved (30% 570
sucrose in PBS) for cryosectioning (10 µm thick, Bright Instruments, Luton, 571
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24
UK). Tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), 572
bromodeoxyuridine (BrdU) and phospho-S6 ribosomal protein (p-S6) were 573
detected using the following polyclonal antibodies: rabbit anti-TH (1:5000, 574
Novus Biologicals, Abindong, UK; NB300-109) for single immunofluorescence 575
or chicken anti-TH (1:1000, abcam, Cambridge, UK; ab76442) for double 576
immunofluorescence, rabbit anti-GFAP (1:500, Dako, Cambridge, UK; 577
Z0334), rat anti-BrdU (1:100, abcam, ab6326), and rabbit anti-phospho-S6 578
ribosomal protein (1:200, Cell Signaling, 2211), respectively. Fluorescence-579
based detection was visualized using, Alexa-Fluor 488- and 568-conjugated 580
anti-rabbit IgG (1:500, ThermoFisher, Waltham, MA US), Alexa-Fluor 488-581
conjugated anti-chicken IgG (1:500, ThermoFisher) and Alexa-Fluor 488-582
conjudaged anti-rat IgG (1:500, ThermoFisher) secondary antibodies. 583
Chromogen-based detection was performed with Dako REAL Detection 584
Systems, Peroxidase/DAB+, Rabbit/Mouse (Dako, K5001) and counterstained 585
with Carrazzi hematoxylin. For the Td-Tomato detection, carotid bifurcation 586
and adrenal gland were dissected, embedded with OCT, flash-frozen into 587
liquid N2 and sectioned (10 µm thick, Bright Instruments). Direct Td-Tomato 588
fluorescence was imaged after nuclear counterstain with 4',6-diamidino-2-589
phenylindole (DAPI). 590
CB glomus cell counting, area measurements and pixel mean intensity were 591
calculated on micrographs (Leica DM-RB, Wetzlar, Gernany) taken from 592
sections spaced 40 µm apart across the entire CB using Fiji software 593
(Schindelin et al., 2012) as previously reported (Macias et al., 2014). CB and 594
SCG volume was estimated on the same micrographs according to Cavalieri’s 595
principle. Lung tissue was removed in the distended state by infusion into the 596
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25
trachea of 0.8% agarose and then fixed in 4% paraformaldehyde for paraffin 597
embedding. Lung sections (6 µm thick) were stained with H&E and EVG stain 598
to assess morphology (Merck, Darmstadt, Germany). Vessel medial thickness 599
was measured on micrographs using Fiji software. To determine the degree of 600
muscularization of small pulmonary arteries, serial lung tissue sections were 601
stained with anti-α smooth muscle actin (1:200, Sigma-Aldrich, A2547). 602
Tissue sections were independently coded and quantified by a blinded 603
investigator. 604
605
Gene deletion 606
Genomic DNA was isolated from dissected SCG and AM using DNeasy Blood 607
& Tissue Kit (Qiagen, Hilden, Germany). Relative gene deletion was assayed 608
by qPCR (One-Step Plus Real-Time PCR System; ThermoFisher Scientific) 609
using TaqMan™ Universal PCR Master Mix (ThermoFisher Scientific) and 610
PrimeTime Std qPCR Assay (IDT, Coralville, IA US). 611
For Hif-1α, 612
Hif1_probe: 5’-/56-FAM/CCTGTTGGTTGCGCAGCAAGCATT/3BHQ_1/-3’ 613
Hif1_F: 5’-GGTGCTGGTGTCCAAAATGTAG-3’ 614
Hif1_R: 5’-ATGGGTCTAGAGAGATAGCTCCACA-3’ 615
For Hif-2α, 616
Hif2_probe: 5’-/56-FAM/CCACCTGGACAAAGCCTCCATCAT/3IABkFQ/-3’ 617
Hif2_F: 5’-TCTATGAGTTGGCTCATGAGTTG-3’ 618
Hif2_R: 5’-GTCCGAAGGAAGCTGATGG-3’ 619
Relative gene deletion levels were calculated using the 2ΔΔCT method. 620
621
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26
Tissue preparation for amperometry and patch clamp experiments 622
Adrenal glands and SCG were quickly removed and transferred to ice-cooled 623
PBS. Dissected AM was enzymatically dispersed in two steps. First, tissue 624
was placed in 3 ml of extraction solution (in mM: 154 NaCl, 5.6 KCl, 10 625
Hepes, 11 glucose; pH 7.4) containing 425 U/ml collagenase type IA and 4-5 626
mg of bovine serum albumin (all from Sigma-Aldrich), and incubated for 30 627
min at 37°C. Tissue was mechanically dissociated every 10 minutes by 628
pipetting. Then, cell suspension was centrifuged (165 g and 15-20ºC for 4 629
min), and the pellet suspended in 3 ml of PBS containing 9550 U/ml of trypsin 630
and 425 U/ml of collagenase type IA (all from Sigma-Aldrich). Cell suspension 631
was further incubated at 37°C for 5 min and mechanically dissociated by 632
pipetting. Enzymatic reaction was stopped by adding ≈10 ml of cold complete 633
culture medium (DMEM/F-12 (21331-020 GIBCO, ThermoFisher Scientific) 634
supplemented with penicillin (100 U/ml)/streptomycin (10 µg/ml), 2 mM L-635
glutamine and 10% fetal bovine serum. The suspension was centrifuged (165 636
g and 15-20ºC for 4 min), the pellet gently suspended in 200 µl of complete 637
culture medium. Next, cells were plated on small coverslips coated with 1 638
mg/ml poly-L-lysine and kept at 37ºC in a 5% CO2 incubator for settlement. 639
Afterwards, the petri dish was carefully filled with 2 ml of culture medium and 640
returned to the incubator for 2 hours. Under these conditions, cells could be 641
maintained up to 12-14 hours to perform experiments. 642
To obtain SCG dispersed cells, the tissue was placed in a 1.5 ml tube 643
containing enzymatic solution (2.5 mg/ml collagenase type IA in PBS) and 644
incubated for 15 min in a thermoblock at 37°C and 300 rpm. The suspension 645
was mechanically dissociated by pipetting and centrifuged for 3 min at 200 g. 646
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The pellet was suspended in the second enzymatic dispersion (1mg/ml of 647
trypsin in PBS), incubated at 37°C, 300 rpm for 25 min and dissociated again 648
with a pipette. Enzymatic reaction was stopped adding a similar solution than 649
used for chromaffin cells (changing fetal bovine serum for newborn calf 650
serum). Finally, dispersed SCG cells were plated onto poly-L-lysine coated 651
coverslips and kept at the 37ºC incubator. Experiments with SCG cells were 652
done between 24-48h after finishing the culture. 653
654
To prepare AM slices, the capsule and adrenal gland cortex was removed and 655
the AM was embedded into low melting point agarose at when reached 47 ºC. 656
The agarose block is sectioned (200 µm thick) in an O2-saturated Tyrode's 657
solution using a vibratome (VT1000S, Leica). AM slices were washed twice 658
with the recording solution (in mM: 117 NaCl, 4.5 KCl, 23 NaHCO3, 1 MgCl2, 1 659
CaCl2, 5 glucose and 5 sucrose) and bubbled with carbogen (5% CO2 and 660
95% O2) at 37 ºC for 30 min in a water bath. Fresh slices are used for the 661
experiments during 4-5h after preparation. 662
663
Patch-clamp recordings 664
The macroscopic currents were recorded from dispersed mouse AM and SCG 665
cells using the whole cell configurations of the patch clamp technique. 666
Micropipettes (≈3 MΩ) were pulled from capillary glass tubes (Kimax, Kimble 667
Products, Rockwood, TN US) with a horizontal pipette puller (Sutter 668
instruments model P-1000, Novato, CA US) and fire polished with a 669
microforge MF-830 (Narishige, Amityville, NY US). Voltage-clamp recordings 670
were obtained with an EPC-7 amplifier (HEKA Electronik, Lambrecht/Pfalz, 671
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28
Germany). The signal was filtered (3 kHz), subsequently digitized with an 672
analog/digital converter (ITC-16 Instrutech Corporation, HEKA Electronik) and 673
finally sent to the computer. Data acquisition and storage was done using the 674
Pulse/pulsefit software (Heka Electronik) at a sampling interval of 10 µsec. 675
Data analysis is performed with the Igor Pro Carbon (Wavemetrics, Portland, 676
OR US) and Pulse/pulsefit (HEKA Electronik) programs. All experiments were 677
conducted at 30-33°C. Macroscopic Ca2+, Na+, and K+ currents were recorded 678
in dialyzed cells. The solutions used for the recording of Na+ and Ca2+ 679
currents contained (in mM): external: 140 NaCl, 10 BaCl2, 2.5 KCl, 10 680
HEPES, and 10 glucose; pH 7.4 and osmolality 300 mOsm/kg; and internal: 681
110 CsCl, 30 CsF, 10 EGTA, 10 HEPES, and 4 ATP-Mg; pH 7.2 and 682
osmolality 285 mOsm/kg. The external solution used for the recording of 683
whole cell K+ currents was (in mM): 117 NaCl, 4.5 KCl, 23 NaHCO3, 1 MgCl2, 684
2.5 CaCl2, 5 glucose and 5 sucrose. The internal solution to record K+ 685
currents was (in mM): 80 potassium glutamate, 50 KCl, 1 MgCl2, 10 HEPES, 686
4 MgATP, and 5 EGTA, pH 7.2. Depolarizing steps of variable duration 687
(normally 10 or 50 msec) from a holding potential of -70 mV to different 688
voltages ranging from -60 up to +50 mV (with voltage increments of 10 mV) 689
were used to record macroscopic ionic currents in AM and SCG cells. 690
691
Amperometric recording 692
To perform the experiments, single slices were transferred to the chamber 693
placed on the stage of an upright microscope (Axioscope, Zeiss, Oberkochen, 694
Germany) and continuously perfused by gravity (flow 1–2 ml min-1) with a 695
solution containing (in mM): 117 NaCl, 4.5 KCl, 23 NaHCO3, 1 MgCl2, 2.5 696
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29
CaCl2, 5 glucose and 5 sucrose. The ‘normoxic’ solution was bubbled with 5% 697
CO2, 20% O2 and 75 % N2 (PO2 ≈ 150 mmHg). The ‘hypoxic’ solution was 698
bubbled with 5% CO2 and 95% N2 (PO2 ≈ 10–20 mmHg). The ‘hypercapnic’ 699
solution was bubbled with 20% CO2, 20% O2 and 60 % N2 (PO2 ≈ 150 700
mmHg). The osmolality of solutions was ≈ 300 mosmol kg−1 and pH = 7.4. In 701
the 20 mM K+ solution, NaCl was replaced by KCl equimolarly (20 mM). All 702
experiments were carried out at a chamber temperature of 36°C. 703
Amperometric currents were recorded with an EPC-8 patch-clamp amplifier 704
(HEKA Electronics), filtered at 100 Hz and digitized at 250 Hz for storage in a 705
computer. Data acquisition and analysis were carried out with an ITC-16 706
interface and PULSE/PULSEFIT software (HEKA Electronics). The secretion 707
rate (fC min−1) was calculated as the amount of charge transferred to the 708
recording electrode during a given period of time. 709
710
Catecholamine measurements 711
Catecholamine levels were determined by ELISA (Abnova, Taipei, Taiwan) 712
following manufacture’s instructions. Spontaneous urination was collected 713
from control and experimental mice (8-12 weeks old) at similar daytime 714
(9:00am). HIF-2αWT and TH-HIF-2αKO or HIF-1αWT, TH-HIF-1αKO samples 715
were collected and assayed in different days. 716
717
Tunel assay 718
Cell death was determined on CB sections using Click-iT™ TUNEL Alexa 719
Fluor™ 488 Imaging Assay (ThermoFisher Scientific), following manufacture’s 720
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indications. TUNEL+ cells across the whole CB parenchyma were counted 721
and normalized by area. 722
723
Plethysmography 724
Respiratory parameters were measured by unrestrained whole-body 725
plethysmography (Data Sciences International, St. Paul, MN US). Mice were 726
maintained in a hermetic chamber with controlled normoxic airflow (1.1 l/min) 727
of 21% O2 (N2 balanced, 5000ppm CO2, BOC Healthcare, Manchester, UK) 728
until they were calm (30-40 minutes). Mice were then exposed to pre-mixed 729
hypoxic air (1.1 l/min) of 10% O2 (N2 balanced, 5000ppm CO2, BOC 730
Healthcare) for 30 min (time-course experiments) or 10 min (VAH 731
experiments). Real-time data were recorded and analyzed using Ponemah 732
software (Data Sciences International). Each mouse was monitored 733
throughout the experiment and periods of movements and/or grooming were 734
noted and subsequently removed from the analysis. The transition periods 735
between normoxia and hypoxia (2-3 min) were also removed from the 736
analysis. 737
738
Oxygen saturations 739
Oxygen saturations were measured using MouseOx Pulse Oximeter (Starr 740
Life Sciences Corp,Oakmont, PA US) on anaesthetised (isofluorane) mice. 741
Anaesthetic was vaporized with 2 l/min of 100% O2 gas flow (BOC 742
Healthcare) and then shift to 2 l/min of pre-mixed 10% O2 flow (N2 balanced, 743
5000ppm CO2, BOC Healthcare) for 5 min. Subsequently, gas flow was 744
switched back to 100% O2. 745
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31
746
Whole-body metabolic assessments 747
Energy expenditure was measured and recorded using the Columbus 748
Instruments Oxymax system (Columbus, OH US) according to the 749
manufacturer’s instructions. Mice were randomly allocated to the chambers 750
and they had free access to food and water throughout the experiment with 751
the exception of fasting experiments. An initial 18-24h acclimation period was 752
disregarded for all the experiments. Once mice were acclimated to the 753
chamber, the composition of the influx gas was switched from 21% O2 to 10% 754
O2 using a PEGAS mixer (Columbus Instruments). For maximum exertion 755
testing, mice were allowed to acclimatize to the enclosed treadmill 756
environment for 15 min before running. The treadmill was initiated at 5 757
meters/min and the mice warmed up for 5 minutes. The speed was increased 758
up to 27 meters/min or exhaustion at 2.5 min intervals on a 15° incline. 759
Exhaustion was defined as the third time the mouse refused to run for more 760
than 3-5 seconds or when was unable to stay on the treadmill. Blood glucose 761
and lactate levels were measured before and after exertion using a StatStrip 762
Xpress (nova biomedical, Street-Waltham, MA US) glucose and lactate 763
meters. 764
765
Chronic hypoxia exposure and in vivo CB proliferation 766
Mice were exposed to a normobaric 10% O2 atmosphere in an enclosed 767
chamber with controlled O2 and CO2 levels for up to 21 days. Humidity and 768
CO2 levels were quenched throughout the experiments by using silica gel 769
(Sigma-Aldrich) and spherasorb soda lime (Intersurgical, Wokingham, UK), 770
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32
respectively. Haematocrit was measured in a bench-top haematocrit 771
centrifuge. Haemoglobin levels were analyzed with a Vet abc analyzer 772
(Horiba, Kyoto, Japan) according to the manufacture’s instructions. Heart, 773
spleen tissues were removed and their wet weights measured. Right 774
ventricular systolic pressures were assessed using a pressure-volume loop 775
system (Millar, Houston, TX US) as reported before (Cowburn et al., 2016). 776
Mice were anaesthetized (isoflurane) and right-sided heart catheterization 777
was performed through the right jugular vein. Lungs were processed for 778
histology as describe above. Analogous procedures were followed for mice 779
maintained in normoxia. 780
For hypoxia-induced in vivo CB proliferation studies, mice were injected 781
intraperitoneally once a week with 50 mg/kg of BrdU (Sigma-Aldrich) and 782
constantly supplied in their drinking water (1mg/ml) as previously shown 783
(Pardal et al., 2007). In vivo mTORC inhibition was achieved by 784
intraperitoneal injection of rapamycin (6 mg kg-1 day-1). Tissues were collected 785
for histology after 14 days at 10% O2 as described above. VAH was recorded 786
in vehicle control and rapamycin-injected C57BL/6J mice after 7 days in 787
chronic hypoxia by plethysmography as explained above. 788
789
Blood pressure measurement by radio-telemetry 790
All radio-telemetry hardware and software was purchased from Data Science 791
International. Surgical implantation of radio-telemetry device was performed 792
following University of Cambridge Animal Welfare and Ethical Review Body’s 793
(AWERB) recommendations and according to manufacturer’s instructions. 794
After surgery, mice were allowed to recover for at least 10 days. All baseline 795
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33
telemetry data were collected over a 72h period in a designated quiet room to 796
ensure accurate and repeatable results. Blood pressure monitoring during 797
hypoxia challenge was conducted in combination with Columbus Instruments 798
Oxymax system and PEGAS mixer. Mice were placed in metabolic chambers 799
and allowed to acclimate for 24h before the oxygen content of the flow gas 800
was reduced to 10%. Mice were kept for 48h at 10% O2 before being returned 801
to normal atmospheric oxygen for a further 24h. For hypertension studies, 802
mice were subsequently implanted with a micro osmotic pump (Alzet, 803
Cupertino, CA; 1002 model) for Ang II (Sigma-Aldrich) infusion (490 ng min-1 804
kg-1) for 2 weeks. Blood pressure, heart rate, temperature and activity were 805
monitored for a 48h period after 7, 14 and 21 days after osmotic pump 806
implantation. 807
808
Glucose, insulin and lactate tolerant tests 809
All the blood glucose and lactate measurements were carried out with a 810
StatStrip Xpress (nova biomedical) glucose and lactate meters. Blood was 811
sampled from the tail vein. For glucose tolerance test (GTT), mice were 812
placed in clean cages and fasted for 14-15 hours (overnight) with free access 813
to water. Baseline glucose level (0 time point) was measured and then a 814
glucose bolus (Sigma-Aldrich) of 2 g/kg in PBS was intraperitoneally injected. 815
Glucose was monitored after 15, 30, 60, 90, 120 and 150 minutes. For insulin 816
tolerance test, mice were fasted for 4 hours and baseline glucose was 817
measured (0 time point). Next, insulin (Roche, Penzberg, Germany) was 818
intraperitoneally injected (0.75 U/kg) and blood glucose content recorded after 819
15, 30, 60, 90 and 120 minutes. For lactate tolerance test, mice were injected 820
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34
with a bolus of 2 g/kg of sodium lactate (Sigma-Aldrich) in PBS and blood 821
lactate measured after 15, 30, 60, 90 and 120 minutes. 822
823
Statistical analysis 824
Particular statistic tests and sample size can be found in the figures and figure 825
legends. Data are presented as the mean ± standard error of the mean (SEM). 826
Shapiro-Wilk normality test was used to determine Gaussian data distribution. 827
Statistical significance between two groups was assessed by un-paired t-test in 828
cases of normal distribution with a Welch’s correction if standard deviations 829
were different. In cases of non-normal distribution (or low sample size for 830
normality test) the non-parametric Mann-Whitney U test was used. For multiple 831
comparisons we used two-way ANOVA followed by Fisher’s LSD test. Non-832
linear regression (one-phase association) curve fitting, using a least squares 833
method, was used to model the time course data in Figs. 3, 5 and 834
Supplemental Fig. S6. Extra sum-of-squares F test was used to compare 835
fittings, and determine whether the data were best represented by a single 836
curve, or by separate ones. Individual area under the curve (AUC) was 837
calculated for the circadian time-courses in Figs. 3, 5; Supplemental Figs. S6, 838
S7, followed by un-paired t-test or non-parametric Mann-Whitney U test as 839
explained above. Statistical significance was considered if p ≤ 0.05. Analysis 840
was carried out using Prism6.0f (GraphPad software, La Jolla, CA US) for Mac 841
OS X. 842
843
Ackowledgements 844
This work is funded by the Wellcome Trust (grant WT092738MA). 845
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35
846
Competing interests: 847
No competing interest declared. 848
849
850
Author Contributions 851
Conceptualization, D.M. and R.S.J.; Investigation, D.M., A.C., H.T.T., and 852
P.O.S.; Writing-Original Draft, D.M. and R.S.J.; Writing-Review & Editing, 853
D.M., A.C., H.T.T., P.O.S., J.L.B., and R.S.J.; Supervision, D.M., J.L.B. and 854
R.S.J.; Funding Acquisition, R.S.J. 855
856
857
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36
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Figure Legends 1243
Figure 1. Selective loss of carotid body glomus cells in sympathoadrenal-1244
specific Hif-2α, but not Hif-1α, deficient mice. (A and B) Tyrosine hydroxylase 1245
(TH) immunostaining on carotid bifurcation sections from HIF-2αWT (A, top 1246
panels), TH-HIF-2αKO (A, bottom panels), HIF-1αWT (B, top panels) and TH-1247
HIF-1αKO (B, bottom panels) mice. Micrographs showed in A (bottom) were 1248
selected to illustrate the rare presence of TH+ glomus cells in TH-HIF-2αKO 1249
mice (white arrowheads). Dashed rectangles (left panels) are shown at higher 1250
magnification on the right panels. SCG, superior cervical ganglion; CB, carotid 1251
body; ICA, internal carotid artery; ECA, external carotid artery. Scale bars: 1252
200 µm (left panels), 50 µm (right panels). (C-F) Quantification of total SCG 1253
volume (C), total CB volume (D), TH+ cell number (E) and TH+ cell density (F) 1254
on micrograph from HIF-2αWT (black dots, n = 4 for SCG and n = 6 for CB), 1255
TH-HIF-2αKO (red squares, n = 4), HIF-1αWT (black triangles, n = 4) and TH-1256
HIF-1αKO (blue triangles, n = 3) mice. Data are expressed as mean ± SEM. 1257
Mann-Whitney test, **p < 0.001; ns, non-significant. (G and H) TH-1258
immunostained adrenal gland sections from HIF-2αWT (G, top panel), TH-HIF-1259
2αKO (G, bottom panel), HIF-1αWT (H, top panel) and TH-HIF-1αKO (H, bottom 1260
panel) littermates. AM, adrenal medulla. Scale bars: 200 µm. (I) Normalized 1261
adrenaline (left) and noradrenaline (right) urine content measured by ELISA. 1262
HIF-2αWT (black dots, n = 8), TH-HIF-2αKO (red squares, n = 7), HIF-1αWT 1263
(black triangles, n = 7) and TH-HIF-1αKO (blue triangles, n = 4). Mann-Whitney 1264
test, ns, non-significant. 1265
1266
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59
Figure 1-figure supplement 1. Sympathoadrenal gene deletion by TH-IRES-1267
Cre mouse strain. (A) TH-Cre-mediated recombination in carotid bifurcation 1268
(left panels) and adrenal gland (right panels) sections dissected from the TH-1269
Td-tomato reporter mice (Cre+) compared to Td-Tomato (Cre-) mice. Notice 1270
the presence of Td-tomato fluorescence into the CB glomus cells, SCG 1271
sympathetic neurons and adrenal medulla due to TH-Cre activity (lower 1272
panels). Dashed lines delineate the location of SCG and AM within Td-tomato 1273
(Cre-) sections. SCG, superior cervical ganglion; CB, carotid body; AM, 1274
adrenal medulla. Scale bars: 100 µm for carotid bifurcation and 200 µm for 1275
adrenal gland sections. (B-C) Relative Hif-2α (B) and Hif-1α (C) gene deletion 1276
of dissected SCG (left graphs) and AM (right graphs) from TH-HIF-2αKO (red, 1277
n = 4) and TH-HIF-1αKO (blue, n = 4) compared to their respective littermate 1278
controls (HIF-2αWT, n = 4; HIF-1αWT, n = 2). Data are expressed as mean 1279
±SEM. Unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. 1280
1281
Figure 2. Progressive CB glomus cells death during development of TH-1282
HIF2αKO mouse. (A) Representative micrographs illustrating CB TH+ glomus 1283
cells appearance at embryo stage E18.5 (left) and postnatal stage P0 (right) 1284
in HIF-2αWT (top panels) and TH-HIF-2αKO (bottom panels) mice. Dashed 1285
rectangles (left panels) are shown at higher magnification on the right panels. 1286
SCG, superior cervical ganglion; CB, carotid body; ICA, internal carotid artery; 1287
ECA, external carotid artery. Scale bars: 100 µm. (B-E) Quantitative 1288
histological analysis of CB volume (B), TH+ cells number (C), TH+ cell density 1289
(D) and SCG volume (E) from TH-HIF-2αKO compared to HIF-2αWT mice. HIF-1290
2αWT (E18.5, n = 8; P0, n = 4; SCG, n = 5), TH-HIF-2αKO (E18.5, n = 7; P0, n 1291
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60
= 5; SCG, n = 5). SCG, superior cervical ganglion. Data are expressed as 1292
mean ±SEM. Two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 1293
0.0001, ns, non significant. (F) Representative pictures of double TH+ and 1294
TUNEL+ (white arrows) stained carotid bifurcation sections from HIF-2αWT (top 1295
panels) and TH-HIF-2αKO (bottom panels) mice at E18.5 (left) and P0 (right) 1296
stages. (G) Number of TUNEL+ cells within the CB (TH+ area) of HIF-2αWT 1297
(E18.5, n = 9; P0, n = 5) and TH-HIF-2αKO (E18.5, n = 5; P0, n = 4) mice at 1298
E18.5 and P0 stages. Data are expressed as mean ±SEM. Two-way ANOVA, 1299
*p < 0.05, **p < 0.01, ****p < 0.0001. Scale bars: 50 µm 1300
1301
Figure 3. Effects of HIF-2α and HIF-1α sympathoadrenal loss on the HVR 1302
and whole-body metabolic activity in response to hypoxia. (A and E) HVR in 1303
TH-HIF-2αKO (A, red line, n = 4) and TH-HIF-1αKO (E, blue line, n = 3) 1304
deficient mice compared to control HIF-2αWT (A, black line, n = 4) and HIF-1305
1αWT (E, black line, n = 3) mice. Shift and duration of 10% O2 stimulus (30 1306
minutes) are indicated. Shaded areas represent the period of time analysed in 1307
B and C or F and G, respectively. Data are presented as mean breaths/min 1308
every minute ±SEM. Two-way ANOVA, **p < 0.01, ns, non-significant. (B and 1309
C) Averaged respiratory rate (B) and minute volume (C) of HIF-2αWT (black, n 1310
= 4) and TH-HIF-2αKO (red, n = 4) mice before and during the first 5 minutes 1311
of 10% O2 exposure (shaded rectangle in A). Data are expressed as mean 1312
±SEM. Two-way ANOVA, *p < 0.05, ***p < 0.001, ****p < 0.0001, ns, non-1313
significant. (D) Peripheral O2 saturation before, during and after hypoxia in 1314
HIF-2αWT (black, n = 8) and TH-HIF-2αKO (red, n = 9) mice. Data are 1315
expressed as mean percentage every 10 seconds ±SEM. Two-way ANOVA, 1316
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****p < 0.0001. (F and G) Averaged respiratory rate (F) and minute volume (F) 1317
of HIF-1αWT (black, n = 3) and TH-HIF-1αKO (blue, n = 3) mice before and 1318
during the first 5 minutes of 10% O2 exposure (shaded rectangle in E). Data 1319
are expressed as mean ±SEM. Two-way ANOVA, *p < 0.05, **p < 0.01. (H 1320
and I) Baseline metabolic activity of HIF-2αWT (black, n = 3) and TH-HIF-2αKO 1321
(red, n = 3) mice. Data are presented as mean oxygen consumption (VO2, H) 1322
and carbon dioxide generation (VCO2, I) every hour ± SEM. White and black 1323
boxes depict diurnal and nocturnal periods, respectively. Area under the curve 1324
followed by Mann-Whitney test, ns, non-significant. (J and K) Whole-body 1325
metabolic analysis of HIF-2αWT (black, n = 4) and TH-HIF-2αKO (red, n = 6) 1326
mice in response to 10% O2. Shift and duration of 10% O2 stimulus are 1327
indicated. Data are presented as mean oxygen consumption (VO2, J) and 1328
carbon dioxide generation (VCO2, K) every hour ± SEM. White and black 1329
boxes depict diurnal and nocturnal periods, respectively. Recovery across the 1330
hypoxia period was analysed by one-phase association curve fitting. ****p < 1331
0.0001. 1332
1333
Figure 3-figure supplement 1.Secretory and electrophysiological properties 1334
in AM and SCG from TH-HIF-2αKO mice. (A and B) Representative 1335
amperometric recordings of catecholamine release by chromaffin cells from 1336
HIF-2αWT and TH-HIF-2αKO mice (8-12 weeks old) in response to low PO2 1337
(PO2 ≈ 10-15 mmHg), hypercapnia (20% CO2) and 20 mM KCl. (C) 1338
Percentage of AM slices with response to hypoxia, 20% CO2 and 20 mM KCl, 1339
respectively. HIF-2αWT n = 8; TH-HIF-2αKO, n = 7. (D) Averaged secretion rate 1340
of chromaffin cells from HIF-2αWT and TH-HIF-2αKO mice exposed to hypoxia 1341
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62
(PO2 ≈ 10-15 mmHg), hypercapnia (20% CO2) and 20 mM KCl. Each data 1342
point represents the mean of 2-3 recordings per mouse. HIF-2αWT, n = 8; TH-1343
HIF-2αKO, n = 7. Data are expressed as mean ±SEM. One-way ANOVA, non-1344
significant. (E and F) Ca2+ and K+ current/voltage (I/V) curves of AM 1345
chromaffin cells (E) and SCG sympathetic neurons (F) dissociated from HIF-1346
2αWT (n = 9 cells from 3 mice) and TH-HIF-2αKO (n = 10 cells from 3 mice) 1347
mice. Data are expressed as mean ±SEM. Two-way ANOVA, ns, non-1348
significant. 1349
1350
Figure 3-figure supplement 2. Respiratory parameters of TH-HIF-2αKO and 1351
TH-HIF-1αKO mice exposed to acute hypoxia. (A-J) Averaged tidal volume (A 1352
and F), inspiration time (B and G), expiration time (C and H), peak inspiratory 1353
flow (D and I) and peak expiratory flow (E and J) of TH-HIF-2αKO (n = 4) and 1354
TH-HIF-1αKO (n = 3) respect to control mice (HIF-2αWT, n = 4; HIF-1αWT, n = 1355
3) before and during the first 5 minutes breathing 10% O2. Data are 1356
expressed as mean ±SEM. Two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 1357
0.001. 1358
1359
Figure 4. Impaired acclimatisation to chronic hypoxia and severe pulmonary 1360
hypertension in mice lacking CBs. (A) Kaplan-Meier survival curve of the 1361
indicated mice housed at 10% O2 up to 21 days. HIF-2αWT (n = 12), TH-HIF-1362
2αKO (n = 11), HIF-1αWT (n = 8) and TH-HIF-1αKO (n = 8). ***p < 0.001. (B and 1363
C) Haematocrit (B) and haemoglobin (C) levels of HIF-2αWT (black) and TH-1364
HIF-2αKO (red) littermates kept in normoxia (21% O2) and after 21 days at 1365
10% O2. In B, HIF-2αWT (21% O2, n = 6; 10% O2, n = 21), TH-HIF-2αKO (21% 1366
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63
O2, n = 7; 10% O2, n = 8). In C, HIF-2αWT (21% O2, n = 5; 10% O2, n = 8), TH-1367
HIF-2αKO (21% O2, n = 5; 10% O2, n = 7). Data are expressed as mean 1368
±SEM. Two-way ANOVA, *p < 0.05, ***p < 0.001, ****p < 0.0001, ns, non-1369
significant. (D and E) Normalized heart (D) and spleen (E) weight of HIF-2αWT 1370
(black) and TH-HIF-2αKO (red) before and after 21 days in hypoxia (10% O2). 1371
In D, HIF-2αWT (21% O2, n = 16; 10% O2, n = 23), TH-HIF-2αKO (21% O2, n = 1372
15; 10% O2, n = 13). In E, HIF-2αWT (21% O2, n = 5; 10% O2, n = 23), TH-HIF-1373
2αKO (21% O2, n = 5; 10% O2, n = 13). Data are expressed as mean ±SEM. 1374
Two-way ANOVA, ****p < 0.0001, ns, non-significant. (F) Fulton index on 1375
hearts dissected from HIF-2αWT (black, 21% O2, n = 17; 10% O2, n = 23) and 1376
TH-HIF-2αKO (red, 21% O2, n = 15; 10% O2, n = 17) mice before and after 21 1377
days in hypoxia (10% O2). Data are expressed as mean ±SEM. Two-way 1378
ANOVA, ****p < 0.0001, ns, non-significant. RV, right ventricle. LV, left 1379
ventricle. (G) Right ventricular systolic pressure (RVSP) recorded on HIF-1380
2αWT (black, 21% O2, n = 6; 10% O2, n = 7) and TH-HIF-2αKO (red, 21% O2, n 1381
= 5; 10% O2, n = 5) mice maintained in normoxia (21% O2) or hypoxia (10% 1382
O2) for 21 days. Data are expressed as mean ±SEM. Two-way ANOVA, **p < 1383
0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant. (H and I) Lung 1384
peripheral vessels muscularization (H) and arterial medial thickness (I) in HIF-1385
2αWT and TH-HIF-2αKO mice exposed to 10% O2 for 14 and/or 21 days. In H, 1386
HIF-2αWT (10% O2 14d, n = 4; 10% O2 21d, n = 3), TH-HIF-2αKO (10% O2 1387
14d, n = 6; 10% O2 21d, n = 3). In I, HIF-2αWT (21% O2, n = 5; 10% O2 21d, n 1388
= 8), TH-HIF-2αKO (21% O2, n = 5; 10% O2 21d, n = 8). Data are expressed 1389
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64
as mean ±SEM. Two-way ANOVA, *p < 0.05, **p < 0.01, ****p < 0.0001, ns, 1390
non-significant. 1391
1392
Figure 4-figure supplement 1. Impaired hypoxia-induced CB proliferation in 1393
TH-HIF-2αKO mice. (A) Glial fibrillary acidic protein (GFAP) immunostaining to 1394
illustrate the presence of CB stem cells in carotid bifurcation of both HIF-2αWT 1395
and TH-HIF-2αKO mice. Scale bars: 50 µm. (B) Double TH+ BrdU+ 1396
immunofluorescence performed on carotid bifurcation sections from HIF-2αWT 1397
(top) and TH-HIF-2αKO (bottom) mice exposed to 10% O2 for 14 days. Dashed 1398
rectangles are shown at higher magnification on the right panels. Newly 1399
formed TH+ cells are indicated with white arrows. Scale bars: 200 µm (left 1400
pictures), 50 µm (right pictures). (C and D) Quantification of total CB volume 1401
(C, n = 4 per genotype and condition) and double BrdU+ TH+ cells number (D, 1402
n = 3 per genotype and condition) in HIF-2αWT (black) and TH-HIF-2αKO (red) 1403
mice maintained for 14 days in hypoxia (10% O2). Data are presented as 1404
mean ±SEM. Unpaired t-test, **p < 0.01, ***p < 0.001. 1405
1406
Figure 4-figure supplement 2. Adaptation to hypoxia in TH-HIF-1αKO 1407
animals. (A) Haematocrit levels of HIF-1αWT (black, 21% O2, n = 4; 10% O2, n 1408
= 8) and TH-HIF-1αKO (blue, 21% O2, n = 4; 10% O2, n = 8) mice housed at 1409
21% O2 or after 21 days at 10% O2. Data are expressed as mean ±SEM. 1410
Two-way ANOVA, ****p < 0.001, ns, non-significant. (B and C) Normalized 1411
heart (B) and spleen (C) weight of HIF-1αWT (black) and TH-HIF-1αKO (blue) 1412
mice before and after 21 days of hypoxia (10% O2) exposure. In B, HIF-1αWT, 1413
21% O2, n = 9; 10% O2, n = 8; TH-HIF-1αKO, n = 9, per condition. In C, 21% 1414
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65
O2, n = 4 per genotype; 10% O2, n = 5 per genotype. Data are expressed as 1415
mean ±SEM. Two-way ANOVA, *p < 0.05, ns, non-significant. (D) Fulton 1416
index on hearts dissected from HIF-1αWT (black) and TH-HIF-1αKO (blue) mice 1417
before and after 21 days in hypoxia (10% O2). HIF-1αWT, 21% O2, n = 7; 10% 1418
O2, n = 8; TH-HIF-1αKO, n = 8, per condition. Data are expressed as mean 1419
±SEM. Two-way ANOVA, ****p < 0.0001, ns, non-significant. RV, right 1420
ventricle. LV, left ventricle. 1421
1422
Figure 5. Effects of CB dysfunction on blood pressure regulation. (A-C) 1423
Circadian variations in baseline systolic blood pressure (A, continued line), 1424
diastolic blood pressure (A, broken line), mean blood pressure (B) and heart 1425
rate (C) of HIF-2αWT (black, n = 10) and TH-HIF-2αKO (red, n = 8) littermates 1426
recorded by radiotelemetry. White and black boxes depict diurnal and 1427
nocturnal periods, respectively. Data are presented as averaged values every 1428
hour ±SEM. Area under the curve followed by unpaired t-test are shown. BP, 1429
blood pressure. HR, heart rate in beats per minute. (D-F) Mean blood 1430
pressure (D), heart rate (E) and subcutaneous temperature (F) alteration in 1431
response to hypoxia in radiotelemetry-implanted TH-HIF-2αKO (red, n = 5) 1432
compared to HIF-2αWT (black, n = 5). Shift and duration of 10% O2 are 1433
indicated in the graph. White and black boxes represent diurnal and nocturnal 1434
periods, respectively. Data are presented as averaged values every hour 1435
±SEM. Recovery across the hypoxia period was analysed by one-phase 1436
association curve fitting. BP, blood pressure. HR, heart rate in beats per 1437
minute. (G) Schematic representation of the protocol followed in the Ang II-1438
induced experimental hypertension model. (H-M) Mean blood pressure and 1439
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66
heart rate recorded from HIF-2αWT (black, n = 8) and TH-HIF-2αKO (red, n = 8) 1440
mice after 7 days (H and K), 14 days (I and L) and 21 days (J and M) of Ang II 1441
infusion. White and black boxes depict diurnal and nocturnal periods, 1442
respectively. Data are presented as averaged values every hour ±SEM. Area 1443
under the curve followed by unpaired t-test are shown. BP, blood pressure. 1444
HR, heart rate in beats per minute. 1445
1446
Figure 5-figure supplement 1. Cardiovascular homeostasis in response to 1447
hypoxia and Ang II-induced experimental hypertension. (A and B) Circadian 1448
oscillations in subcutaneous temperature (A) and physical activity (B) of 1449
radiotelemetry-implanted HIF-2αWT (black, n = 10) and TH-HIF-2αKO (red, n = 1450
8). White and black boxes depict diurnal and nocturnal periods, respectively. 1451
Data are presented as averaged values every hour ±SEM. Area under the 1452
curve followed by unpaired t-test are shown. (C and D) Systolic blood 1453
pressure (C) and diastolic blood pressure (D) alteration in response to 1454
hypoxia in TH-HIF-2αKO (red, n = 5) compared to HIF-2αWT (black, n = 5) 1455
measured by radiotelemetry. Shift and duration of 10% O2 are indicated in the 1456
graph. White and black boxes depict diurnal and nocturnal periods, 1457
respectively. Data are presented as averaged blood pressure values every 1458
hour ±SEM. Recovery across the hypoxia period was analysed by one-phase 1459
association curve fitting. (E) Physical activity recorded by radiotelemetry of 1460
HIF-2αWT (black, n = 5) and TH-HIF-2αKO (red, n = 5) littermates exposed to 1461
10% O2 environment. Shift and duration of 10% O2 are indicated in the graph. 1462
White and black boxes depict diurnal and nocturnal periods, respectively. 1463
Data are presented as averaged blood pressure values every hour ±SEM. 1464
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Area under the curve followed by unpaired t-test is shown. (F-Q) Circadian 1465
fluctuations in systolic blood pressure, diastolic blood pressure, subcutaneous 1466
temperature and physical activity of HIF-2αWT (black, n = 5) and TH-HIF-2αKO 1467
(red, n = 5) after 7 days (F-I), 14 days (J-M) and 21 days (N-Q) of Ang II 1468
infusion recorded by radiotelemetry. White and black boxes represent diurnal 1469
and nocturnal periods, respectively. Data are presented as averaged values 1470
every hour ±SEM. Area under the curve followed by unpaired t-test are 1471
shown. ns, non-significant. 1472
1473
Figure 6. Exercise performance and glucose management in mice lacking 1474
CBs. (A-D). Time course showing the increment in O2 consumption (A), 1475
increment in CO2 generation (B), respiratory exchange ratio (C) and 1476
increment in heat production (D) from HIF-2αWT (black, n = 8) and TH-HIF-1477
2αKO (red, n = 10) mice during exertion on a treadmill. Mice were warmed up 1478
for 5 minutes (5m/min) and then the speed was accelerated 2m/min at 2.5 1479
minutes intervals until exhaustion. Averaged measurements every 2.5 1480
minutes intervals ±SEM are charted. Two-way ANOVA, *p < 0.05, **p < 0.01, 1481
***p < 0.001, ****p < 0.0001. RER, respiratory exchange ratio. (E) Running 1482
total time spent by HIF-2αWT (black, n = 8) and TH-HIF-2αKO (red, n = 10) 1483
mice to become exhausted. Data are expressed as mean ±SEM. Unpaired t-1484
test. (F and G) Aerobic capacity (F) and power output (G) measured in HIF-1485
2αWT (black, n = 8) mice compared to TH-HIF-2αKO (red, n = 10) mice when 1486
performing at VO2max. Data are expressed as mean ±SEM. Unpaired t-test. 1487
*p < 0.05. (H and I) Plasma glucose (H) and lactate (I) levels of HIF-2αWT 1488
(black, before, n = 7; after, n = 12) and TH-HIF-2αKO (black, before, n = 6; 1489
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after, n = 12) littermates before and after running to exhaustion. Data are 1490
expressed as mean ±SEM. Two-way ANOVA, *p < 0.05. ***p < 0.001. ns, 1491
non-significant. (J and K) Plasma glucose levels of HIF-2αWT (black, n = 6) 1492
and TH-HIF-2αKO (red, n = 6) mice fed (J, n = 6 per genotype) or fasted over 1493
night (K, HIF-2αWT (n = 8) and TH-HIF-2αKO (n = 10). Data are expressed as 1494
mean ±SEM. Unpaired t-test. (L-M) Time courses showing the tolerance of 1495
HIF-2αWT and TH-HIF-2αKO mice to glucose (L, n = 7 per genotype) insulin 1496
(M, n = 11 per genotype) and lactate (N, n = 7 per genotype) injected bolus. 1497
Data are presented as mean ±SEM. Two-way ANOVA, *p < 0.05, **p < 0.01, 1498
ns, non-significant. 1499
1500
Figure 6-figure supplement 1. Related to Figure 6. Whole-body metabolic 1501
activity of TH-HIF-2αKO in response to fasting-induced hypoglycemia. (A-D) 1502
Oxygen consumption (A, VO2), carbon dioxide generation (B, VCO2), 1503
respiratory exchange rate (C) and heat production (D) of HIF-2αWT (black, n = 1504
7) and TH-HIF-2αKO (red, n = 7) littermates under feed and over night fasting 1505
conditions. White and black boxes represent diurnal and nocturnal periods, 1506
respectively. Data are presented as averaged values every hour ±SEM. Area 1507
under the curve followed by unpaired t-test are shown. ns, non-significant. 1508
1509
Figure 7. mTORC1 activation in CB development and hypoxia-induced CB 1510
neurogenesis. (A) Double TH and p-S6 (mTORC1 activation marker) 1511
immunofluorescence on adult wild type CB slices to illustrate the increased p-1512
S6 expression after 14 days in hypoxia (10% O2). For clarity, bottom panels 1513
only show p-S6 staining. TH+ outlined area (top panels) indicates the region 1514
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used for pixels quantification. Scale bars: 50 µm. (B) Quantification of p-S6 1515
(top) and TH (bottom) mean pixels intensity within the CB TH+ cells of wild 1516
type mice breathing 21% O2 or 10% O2 for 14 days. Each data point depicts 1517
the average of 3-4 pictures. 21% O2, n = 5; 10% O2 14d, n = 9 per genotype. 1518
Data are presented as mean ±SEM. Unpaired t-test, *p < 0.05. (C) 1519
Representative micrographs of TH and p-S6 double immunostaining of HIF-1520
2αWT and TH-HIF-2αKO embryonic (E18.5) carotid bifurcations. TH+ outlined 1521
area indicates the region used for pixels quantification. Scale bars: 50µm. (D) 1522
Quantitative analysis performed on HIF-2αWT (black, n = 12) and TH-HIF-2αKO 1523
(red, n = 10) micrographs previously immuno-stained for p-S6 and TH. Each 1524
data point depicts the average of 3-4 pictures. Data are presented as mean 1525
±SEM. Unpaired t-test, **p < 0.01. (E) Double BrdU and TH 1526
immunofluorescence on carotid bifurcation sections from wild type mice 1527
exposed to 10% O2 and injected with rapamycin or vehicle control for 14 days. 1528
Notice the striking decrease in double BrdU+ TH+ cells (white arrowheads) in 1529
the rapamycin treated mice compared to vehicle control animals. Scale bars: 1530
100µm. (F-H) Quantification of total CB volume (F), TH+ cells number (G) and 1531
percentage of double BrdU+ TH+ over the total TH+ cells (H) in wild type mice 1532
breathing 10% O2 and injected with rapamycin or vehicle control for 14 days. 1533
n = 5 mice per treatment. (K) Averaged breaths per minute during the first 2 1534
minutes of acute hypoxia (10% O2) exposure from vehicle or rapamycin 1535
injected mice before and after 7 days of chronic hypoxia (10% O2) treatment. 1536
n = 14 mice per condition and treatment. Data are presented as mean ±SEM. 1537
B, D, F, G and H, unpaired t-test, *p < 0.05, ****p < 0.0001. K, two-way 1538
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ANOVA followed by Tukey’s multiple comparison test. ****p < 0.0001, ns, 1539
non-significant. 1540
1541
Figure 7-figure supplement 1. mTORC1 activity in SCG TH+ sympathetic 1542
neurons and effect of rapamycin treatment. (A) Double TH and p-S6 1543
(mTORC1 activation marker) immunofluorescence on adult wild type SCG 1544
slices after 14 days in hypoxia (10% O2). TH+ outlined area indicates the 1545
region used for pixels quantification. Scale bars: 100 µm. SCG, superior 1546
cervical ganglion. (B) Quantification of p-S6 (red) and TH (green) mean pixels 1547
intensity within the SCG TH+ area of wild type mice breathing 21% O2 or 10% 1548
O2 for 14 days. Each data point depicts the average of 3-4 pictures. 21% O2, 1549
n = 3; 10% O2, n = 4. Data are presented as mean ±SEM. Unpaired t-test, ns, 1550
non-significant. (C) Representative micrographs of TH and p-S6 double 1551
immunostaining of HIF-2αWT and TH-HIF-2αKO embryonic (E18.5) carotid 1552
bifurcations. SCG TH+ outlined area indicates the region used for pixels 1553
quantification. SCG, superior cervical ganglion. Scale bars: 100 µm. (D) 1554
Quantitative analysis performed on HIF-2αWT (black) and TH-HIF-2αKO (red) 1555
micrographs previously immune-stained for TH (green) and p-S6 (red). Each 1556
data point depicts the average of 3-4 pictures. 21% O2, n = 12; 10% O2, n = 1557
10. Data are presented as mean ±SEM. Unpaired t-test, ns, non-significant. 1558
(E) Double TH and p-S6 immunofluorescence on vehicle control or rapamycin 1559
injected adult wild type SCG slices after 14 days breathing in a 10% O2 1560
atmosphere. Notice the absence of p-S6 signal as a consequence of mTORC 1561
inhibition by rapamycin. SCG, superior cervical ganglion. CB, carotid body. 1562
Scale bars: 100 µm. (F) Haematocrit levels of vehicle control (n = 14) and 1563
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rapamycin (n = 14) injected mice after 7 days at 10% O2. Un-paired t-test, ns, 1564
non-significant. 1565
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