Phox2b mutation mediated by Atoh1 expression impaired respiratory rhythm and ventilatory responses to hypoxia and hypercapnia
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Evaluation Summary:
This study provides a novel mouse model for the study of the central respiratory chemoreceptor circuit and, therefore, of interest for the respiratory physiology community. Nonetheless, in its present form, this work still lacks more physiological, developmental, and anatomical characterizations to place this study in a broader context and gain new insights into the physiology of respiratory chemoreflexes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Mutations in the transcription factor Phox2b cause congenital central hypoventilation syndrome (CCHS). The syndrome is characterized by hypoventilation and inability to regulate breathing to maintain adequate O 2 and CO 2 levels. The mechanism by which CCHS impact respiratory control is incompletely understood, and even less is known about the impact of the non-polyalanine repeat expansion mutations (NPARM) form. Our goal was to investigate the extent by which NPARM Phox2b mutation affect (a) respiratory rhythm; (b) ventilatory responses to hypercapnia (HCVR) and hypoxia (HVR); and (c) number of chemosensitive neurons in mice. We used a transgenic mouse line carrying a conditional Phox2b Δ8 mutation (same found in humans with NPARM CCHS). We crossed them with Atoh1 cre mice to introduce mutation in regions involved with respiratory function and central chemoreflex control. Ventilation was measured by plethysmograph during neonatal and adult life. In room air, mutation in neonates and adult did not greatly impact basal ventilation. However, Phox2b Δ8 , Atoh1 cre increased breath irregularity in adults. The HVR and HCVR were impaired in neonates. The HVR, but not HCVR, was still partially compromised in adults. The mutation reduced the number of Phox2b + /TH - -expressing neurons as well as the number of fos-activated cells within the ventral parafacial region (also named retrotrapezoid nucleus [RTN] region) induced by hypercapnia. Our data indicates that Phox2b Δ8 mutation in Atoh1 -expressing cells impaired RTN neurons, as well as chemoreflex under hypoxia and hypercapnia specially early in life. This study provided new evidence for mechanisms related to NPARM form of CCHS neuropathology.
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Author Response
Reviewer #1 (Public Review):
Authors introduced new strategy of genetic manipulation in mice to reveal functional development of the retrotrapezoid nucleus (RTN) neurons that is known as an important brainstem region for central chemoreception and the dysfunction is relate to congenital central hypoventilation syndrome (CCHS) neuropathology. They used a conditional mutation of Phox2b within Atoh1derived cells (Atoh1Cre/Phox2bΔ8 mice) and examined a) respiratory rhythm; b) ventilatory responses to hypercapnia and hypoxia and c) number of RTN-chemosensitive neurons. They found that 1) mice with mutant Phox2b expression showed a suppressed breath activity to hypoxia and hypercapnia in neonates; 2) adult mutant mice presented irregular breathing pattern, partial recovery of the ventilatory response to hypoxia and complete …
Author Response
Reviewer #1 (Public Review):
Authors introduced new strategy of genetic manipulation in mice to reveal functional development of the retrotrapezoid nucleus (RTN) neurons that is known as an important brainstem region for central chemoreception and the dysfunction is relate to congenital central hypoventilation syndrome (CCHS) neuropathology. They used a conditional mutation of Phox2b within Atoh1derived cells (Atoh1Cre/Phox2bΔ8 mice) and examined a) respiratory rhythm; b) ventilatory responses to hypercapnia and hypoxia and c) number of RTN-chemosensitive neurons. They found that 1) mice with mutant Phox2b expression showed a suppressed breath activity to hypoxia and hypercapnia in neonates; 2) adult mutant mice presented irregular breathing pattern, partial recovery of the ventilatory response to hypoxia and complete recovery of response to hypercapnia; 3) anatomical data showed reduced number of activated neurons by hypercapnia and Phox2b immunoreactivity in the RTN. They concluded that conditionally expression of Phox2b mutation by Atoh1 affected development of the RTN neurons and suggested that Atoh1/Phox2b system in the RTN was essential for the activation of breathing under hypoxic and hypercapnia condition. They thought that their findings provided new evidence for mechanisms related to CCHS neuropathology. The conclusions of this paper are well supported by data, but careful discussion seems to be required for comparison with results of various previous studies performed by different genetic strategies for the RTN development.
We would like to thank the reviewer for the comments on our manuscript. In the present version, we made several corrections as suggested by the reviewers to facilitate interpretation and strength the manuscript.
Reviewer #2 (Public Review):
Mutations in the Phox2B gene can lead to congenital central hypoventilation syndrome with variable presentations. Two distinct classes of causative mutations have been found in the human population. The first group consists of mutations that result in trinucleotide, polyalanine repeat expansions, referred to as PARM. The second group are non- polyalanine repeat expansion mutations (NPARM) that includes missense, nonsense, and frameshift mutations. Each group (and even specific mutations) present with differing clinical phenotype severity, with NPARM mutations typically being more severe. As Phox2B is expressed across a multitude of cell types across the life an individual, there remains much to be understood as to the cell specific effects of various Phox2B mutations on phenotype. To add to our understanding, the authors utilized a conditional Phox2bΔ8 allele that, upon recombination, replaces Exon 3 and UTR with a mutated exon and IRES GFP reporter. This approach allows for an inducible NPARM mutation and reporter expression in a targeted cell type. The authors focused on Atoh1 expressing cells using an Atoh1 expressing Cre recombinase line (Atoh1_Cre). Atoh1 has been shown to also be coexpressed in the RTN and in the para and inter-trigeminal regions of the Pons. After inducing the Phox2B mutations in one allele, the authors examined respiratory features in both adults and neonate mice under room air, hypercapnia (7%) and Hypoxia (8%). The Atoh1_Cre; Phox2bΔ8 adult mice showed a significant body weight difference. Under their plethysmography approach neonate mice breathing room air showed few differences with a potential difference in tidal volume. Notably adult mice show irregularity in their breathing. Both adult and neonate mice may show compromised chemosensory deficits. A potential hypercapnic deficit likely resolves in the adult but there may remain a compromised hypoxic reflex in the adult. Notably, Atoh1_Cre; Phox2bΔ8 mice showed reduced cfos expression in the RTN after hypercapnic stimulation and reduced Phox2B immuno-reactivity.
The premise of the paper is to examine how a distinct mutation in a specific cellular context may contribute to clinical outcomes. The potential phenotypes are interesting and illuminate how differing mutations may drive different phenotypes or phenotype severity. While the RTN is likely a key mediator of the reported phenotypes, the conclusions drawn by the authors cannot be fully supported with the data presented.
We would like to thank the reviewer for the comments. In the present version, we have made all changes suggested and we performed new sets of additional experiments to strengthen the work. We are very enthusiastic about the new version of the manuscript, and we believe it opened new questions that could be addressed in the future.
The authors assign all phenotypes to RTN function. However, there are other documented and potential undocumented areas of Atoh1 and Phox2b overlap that could either impact breathing directly or indirectly through metabolism and stress responses (PMID 8184995). As noted above, para trigeminal neurons including those in the ITR also co-express Atoh1 and Phox2B and are captured in the Atoh1_Cre; Phox2bΔ8 mouse model. The inter-trigeminal region is associated with apneic reflexes and jaw opening (PMID: 19914183). Thus, perturbations to this center may underlie the increased irregularity seen in adult life. A potential role in chemosensory function cannot be entirely ruled out either. While Rose et al. assert that the RTN and para- and inter- trigeminal neurons are the only ones co-expressing Atoh1 and Phox2B (using antibodies), the persistent cumulative GFP labeled fate map offered by the Atoh1_Cre; Phox2bΔ8 model would allow the authors to rule in or rule out any other uncharacterized overlapping populations. Such a fate map may also help to inform as to why the adult mice are significantly underweight. The weight phenotype may stem from metabolic dysregulation, changes in behavior, or feeding. Changes in metabolism may drive secondary changes in breathing and chemosensory reflexes that play a role in the reported phenotypes. Ultimately, the relative roles of para-trigeminal and RTN neurons in these phenotypes should be dissected out.
Yes, we ran a new series of experiments and noticed that Phox2b+ neurons in the pons as well as the number of TH cells in the A1, A2, A6, and C1 were not affected by the mutation. Unfortunately, we were unable to quantify the number of Phox2b-expressing neurons in the paratrigeminal region.
Both the adult and neonate plethysmography was not collected in line with current best practices. Adult whole body plethysmography is best carried out in a temperature controlled chamber held at thermo-neutrality. This minimizes any thermo-regulatory and metabolic effects on respiratory drive. Concurrent measurement of one or more metabolic parameters such as VO2 or VCO2 is required to determine if baseline breathing, and chemosensory reflex phenotypes may be affected by changes metabolism or persistent metabolic imbalances (acidosis or alkalosis). Whole body measurements in neonates are do not allow for accurate assessment of tidal volume. Rather head out or facemark pneumotachography are more accurate, (PMID: 25017785).
We totally agree with the reviewer. In fact, some information and misconception were noticed in the previous version. We added the correct way in which the respiratory parameters were measured in both neonate and adult mice. Additionally, we performed head-out plethysmograph in a subset of neonates (control and mutant) and added it in the result section. We also measure VO2 and VE/VO2 in neonates and adults.
Reviewer #3 (Public Review):
The work by Ferreira and colleagues set to define the functional consequences of a PHOX2B (Phox2bdelta8) mutation, belonging to the group of non-polyalanine repeat expansions, when restricted to Atoh1 expressing cells. In doing so, the authors generated a new mouse model (Atoh1Cre,Phox2bdelta8 mice) for the study of the central respiratory chemoreceptor circuit. Ferreira et al., found that these conditional mutants present with largely unaffected breathing parameters in postnatal life. However, neonatal breathing irregularities, normally observable in control neonates, are not corrected with the maturation of the conditional mutants. Furthermore, the authors found that conditional Atoh1Cre,Phox2bdelta8 neonates fail to display ventilatory responses to hypoxic (low O2 content in air) and hypercapnic (high CO2 content in air) challenges. The authors show that Atoh1Cre,Phox2bdelta8 adult mice appear to "recover" the capacity to response to hypercapnic, but not hypoxic, challenges. Lastly, the authors found reduced numbers of Phox2b+ cells in an "area" where the retrotrapezoid nucleus, a key center in the respiratory chemoreceptor circuit, normally locates.
Strengths:
The most exciting aspect of this work is the modelling of the Phox2bdelta8 mutation in one element of the central neuronal circuit mediating respiratory reflexes, that is in the retrotrapezoid nucleus. To date, mutations in the PHOX2B gene are commonly associated with most patients diagnosed with central congenital hypoventilation syndrome (CCHS), a disease characterized by hypoventilation and absence of chemoreflexes, in the neonatal period, which in severe cases can lead to respiratory arrest during sleep. Two distinct types of PHOX2B mutations have been identified in CCHS patients: i) polyalanine repeat expansions, and ii) non-polyalanine repeat expansions. Non-polyalanine repeat expansions tend to be more prevalent in severe cases of CCHS. Thus, the characterization of the Phox2bdelta8 mutation could allow for a better understanding of the etiology behind CCHS.
Weaknesses:
Whereas the most exciting part of this work is the modelling of the Phox2bdelta8 mutation in retrotrapezoid neurons using conditional mutagenesis driven by Atoh1 (i.e. Atoh1Cre,Phox2bdelta8 mice), the weakness of this study is the lack of a clear physiological, developmental, and anatomical distinction between this approach and similar studies already reported elsewhere, for instance the use of Atoh1Cre,Phox2bflox/flox and P2b::CreBAC1;Atoh1lox/lox mice (Ruffault et al., 2015, DOI: 10.7554/eLife.07051), Egr2cre;P2b27Alacki (Ramanantsoa et al., 2011, DOI: 10.1523/JNEUROSCI.1721-11.2011), Atoh1Phox2bCKO mice (Huang et al., 2017, DOI: 10.1016/j.neuron.2012.06.027) and Egr2cre;Lbx1FS (Hernandez-Miranda et al., 2018, DOI: 10.1073/pnas.1813520115).
Several conclusions presented in this work are not directly supported by the provided data. For instance, the reduction in the number of retrotrapezoid neurons in Atoh1Cre,Phox2bdelta8 mice or the reduction of fos+ activated retrotrapezoid neurons after CO2 exposure, as the identity of retrotrapezoid neurons was not thoroughly determined. Furthermore, the authors conclude from their plethysmograph (respiratory recordings) data that Atoh1Cre,Phox2bdelta8 neonatal mice display an impaired ventilatory responses to hypoxia (low O2 in air) and hypercapnia (high CO2 in air), but that these mutant animals recover the capacity to respond to hypercapnia, but not to hypoxia, in the adult life. This is a bit of an overstatement, as their plethysmograph recordings show that adult Atoh1Cre,Phox2bdelta8 mice do respond to low O2 in air, as these mice accelerate respiration, increase tidal volumes and minute ventilation in the same fashion as control mice. However, what the presented data show is that adult Atoh1Cre,Phox2bdelta8 mice do not sustain the ventilatory response as efficient as control mice.
We would like to thank the reviewer for the comments, strengths, and weakness of our study. In the present version, we have made a significant change throughout the manuscript as suggested by the editor and reviewers. In addition, we performed new sets of experiments to strengthen our work. We are very enthusiastic about the current version, and we believe it will open new questions that need to be addressed in future studies
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Evaluation Summary:
This study provides a novel mouse model for the study of the central respiratory chemoreceptor circuit and, therefore, of interest for the respiratory physiology community. Nonetheless, in its present form, this work still lacks more physiological, developmental, and anatomical characterizations to place this study in a broader context and gain new insights into the physiology of respiratory chemoreflexes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
-
Reviewer #1 (Public Review):
Authors introduced new strategy of genetic manipulation in mice to reveal functional development of the retrotrapezoid nucleus (RTN) neurons that is known as an important brainstem region for central chemoreception and the dysfunction is relate to congenital central hypoventilation syndrome (CCHS) neuropathology. They used a conditional mutation of Phox2b within Atoh1-derived cells (Atoh1Cre/Phox2bΔ8 mice) and examined a) respiratory rhythm; b) ventilatory responses to hypercapnia and hypoxia and c) number of RTN-chemosensitive neurons. They found that 1) mice with mutant Phox2b expression showed a suppressed breath activity to hypoxia and hypercapnia in neonates; 2) adult mutant mice presented irregular breathing pattern, partial recovery of the ventilatory response to hypoxia and complete recovery of …
Reviewer #1 (Public Review):
Authors introduced new strategy of genetic manipulation in mice to reveal functional development of the retrotrapezoid nucleus (RTN) neurons that is known as an important brainstem region for central chemoreception and the dysfunction is relate to congenital central hypoventilation syndrome (CCHS) neuropathology. They used a conditional mutation of Phox2b within Atoh1-derived cells (Atoh1Cre/Phox2bΔ8 mice) and examined a) respiratory rhythm; b) ventilatory responses to hypercapnia and hypoxia and c) number of RTN-chemosensitive neurons. They found that 1) mice with mutant Phox2b expression showed a suppressed breath activity to hypoxia and hypercapnia in neonates; 2) adult mutant mice presented irregular breathing pattern, partial recovery of the ventilatory response to hypoxia and complete recovery of response to hypercapnia; 3) anatomical data showed reduced number of activated neurons by hypercapnia and Phox2b immunoreactivity in the RTN. They concluded that conditionally expression of Phox2b mutation by Atoh1 affected development of the RTN neurons and suggested that Atoh1/Phox2b system in the RTN was essential for the activation of breathing under hypoxic and hypercapnia condition. They thought that their findings provided new evidence for mechanisms related to CCHS neuropathology.
The conclusions of this paper are well supported by data, but careful discussion seems to be required for comparison with results of various previous studies performed by different genetic strategies for the RTN development.
-
Reviewer #2 (Public Review):
Mutations in the Phox2B gene can lead to congenital central hypoventilation syndrome with variable presentations. Two distinct classes of causative mutations have been found in the human population. The first group consists of mutations that result in trinucleotide, polyalanine repeat expansions, referred to as PARM. The second group are non- polyalanine repeat expansion mutations (NPARM) that includes missense, nonsense, and frameshift mutations. Each group (and even specific mutations) present with differing clinical phenotype severity, with NPARM mutations typically being more severe. As Phox2B is expressed across a multitude of cell types across the life an individual, there remains much to be understood as to the cell specific effects of various Phox2B mutations on phenotype. To add to our …
Reviewer #2 (Public Review):
Mutations in the Phox2B gene can lead to congenital central hypoventilation syndrome with variable presentations. Two distinct classes of causative mutations have been found in the human population. The first group consists of mutations that result in trinucleotide, polyalanine repeat expansions, referred to as PARM. The second group are non- polyalanine repeat expansion mutations (NPARM) that includes missense, nonsense, and frameshift mutations. Each group (and even specific mutations) present with differing clinical phenotype severity, with NPARM mutations typically being more severe. As Phox2B is expressed across a multitude of cell types across the life an individual, there remains much to be understood as to the cell specific effects of various Phox2B mutations on phenotype. To add to our understanding, the authors utilized a conditional Phox2bΔ8 allele that, upon recombination, replaces Exon 3 and UTR with a mutated exon and IRES GFP reporter. This approach allows for an inducible NPARM mutation and reporter expression in a targeted cell type. The authors focused on Atoh1 expressing cells using an Atoh1 expressing Cre recombinase line (Atoh1_Cre). Atoh1 has been shown to also be co-expressed in the RTN and in the para and inter-trigeminal regions of the Pons. After inducing the Phox2B mutations in one allele, the authors examined respiratory features in both adults and neonate mice under room air, hypercapnia (7%) and Hypoxia (8%). The Atoh1_Cre; Phox2bΔ8 adult mice showed a significant body weight difference. Under their plethysmography approach neonate mice breathing room air showed few differences with a potential difference in tidal volume. Notably adult mice show irregularity in their breathing. Both adult and neonate mice may show compromised chemosensory deficits. A potential hypercapnic deficit likely resolves in the adult but there may remain a compromised hypoxic reflex in the adult. Notably, Atoh1_Cre; Phox2bΔ8 mice showed reduced cfos expression in the RTN after hypercapnic stimulation and reduced Phox2B immuno-reactivity.
The premise of the paper is to examine how a distinct mutation in a specific cellular context may contribute to clinical outcomes. The potential phenotypes are interesting and illuminate how differing mutations may drive different phenotypes or phenotype severity. While the RTN is likely a key mediator of the reported phenotypes, the conclusions drawn by the authors cannot be fully supported with the data presented.
The authors assign all phenotypes to RTN function. However, there are other documented and potential undocumented areas of Atoh1 and Phox2b overlap that could either impact breathing directly or indirectly through metabolism and stress responses (PMID 8184995). As noted above, para trigeminal neurons including those in the ITR also co-express Atoh1 and Phox2B and are captured in the Atoh1_Cre; Phox2bΔ8 mouse model. The inter-trigeminal region is associated with apneic reflexes and jaw opening(PMID: 19914183). Thus, perturbations to this center may underlie the increased irregularity seen in adult life. A potential role in chemosensory function cannot be entirely ruled out either. While Rose et al. assert that the RTN and para- and inter- trigeminal neurons are the only ones co-expressing Atoh1 and Phox2B (using antibodies), the persistent cumulative GFP labeled fate map offered by the Atoh1_Cre; Phox2bΔ8 model would allow the authors to rule in or rule out any other uncharacterized overlapping populations. Such a fate map may also help to inform as to why the adult mice are significantly underweight. The weight phenotype may stem from metabolic dysregulation, changes in behavior, or feeding. Changes in metabolism may drive secondary changes in breathing and chemosensory reflexes that play a role in the reported phenotypes. Ultimately, the relative roles of para-trigeminal and RTN neurons in these phenotypes should be dissected out.
Both the adult and neonate plethysmography was not collected in line with current best practices. Adult whole body plethysmography is best carried out in a temperature controlled chamber held at thermo-neutrality. This minimizes any thermo-regulatory and metabolic effects on respiratory drive. Concurrent measurement of one or more metabolic parameters such as VO2 or VCO2 is required to determine if baseline breathing and chemosensory reflex phenotypes may be affected by changes metabolism or persistent metabolic imbalances (acidosis or alkalosis). Whole body measurements in neonates are do not allow for accurate assessment of tidal volume. Rather head out or facemark pneumotachography are more accurate, (PMID: 25017785).
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Reviewer #3 (Public Review):
The work by Ferreira and colleagues set to define the functional consequences of a PHOX2B (Phox2bdelta8) mutation, belonging to the group of non-polyalanine repeat expansions, when restricted to Atoh1 expressing cells. In doing so, the authors generated a new mouse model (Atoh1Cre,Phox2bdelta8 mice) for the study of the central respiratory chemoreceptor circuit. Ferreira et al., found that these conditional mutants present with largely unaffected breathing parameters in postnatal life. However, neonatal breathing irregularities, normally observable in control neonates, are not corrected with the maturation of the conditional mutants. Furthermore, the authors found that conditional Atoh1Cre,Phox2bdelta8 neonates fail to display ventilatory responses to hypoxic (low O2 content in air) and hypercapnic (high CO2 …
Reviewer #3 (Public Review):
The work by Ferreira and colleagues set to define the functional consequences of a PHOX2B (Phox2bdelta8) mutation, belonging to the group of non-polyalanine repeat expansions, when restricted to Atoh1 expressing cells. In doing so, the authors generated a new mouse model (Atoh1Cre,Phox2bdelta8 mice) for the study of the central respiratory chemoreceptor circuit. Ferreira et al., found that these conditional mutants present with largely unaffected breathing parameters in postnatal life. However, neonatal breathing irregularities, normally observable in control neonates, are not corrected with the maturation of the conditional mutants. Furthermore, the authors found that conditional Atoh1Cre,Phox2bdelta8 neonates fail to display ventilatory responses to hypoxic (low O2 content in air) and hypercapnic (high CO2 content in air) challenges. The authors show that Atoh1Cre,Phox2bdelta8 adult mice appear to "recover" the capacity to response to hypercapnic, but not hypoxic, challenges. Lastly, the authors found reduced numbers of Phox2b+ cells in an "area" where the retrotrapezoid nucleus, a key center in the respiratory chemoreceptor circuit, normally locates.
Strengths:
The most exciting aspect of this work is the modelling of the Phox2bdelta8 mutation in one element of the central neuronal circuit mediating respiratory reflexes, that is in the retrotrapezoid nucleus. To date, mutations in the PHOX2B gene are commonly associated with most patients diagnosed with central congenital hypoventilation syndrome (CCHS), a disease characterized by hypoventilation and absence of chemoreflexes, in the neonatal period, which in severe cases can lead to respiratory arrest during sleep. Two distinct types of PHOX2B mutations have been identified in CCHS patients: i) polyalanine repeat expansions, and ii) non-polyalanine repeat expansions. Non-polyalanine repeat expansions tend to be more prevalent in severe cases of CCHS. Thus, the characterization of the Phox2bdelta8 mutation could allow for a better understanding of the etiology behind CCHS.
Weaknesses:
Whereas the most exciting part of this work is the modelling of the Phox2bdelta8 mutation in retrotrapezoid neurons using conditional mutagenesis driven by Atoh1 (i.e. Atoh1Cre,Phox2bdelta8 mice), the weakness of this study is the lack of a clear physiological, developmental, and anatomical distinction between this approach and similar studies already reported elsewhere, for instance the use of Atoh1Cre,Phox2bflox/flox and P2b::CreBAC1;Atoh1lox/lox mice (Ruffault et al., 2015, DOI: 10.7554/eLife.07051), Egr2cre;P2b27Alacki (Ramanantsoa et al., 2011, DOI: 10.1523/JNEUROSCI.1721-11.2011), Atoh1Phox2bCKO mice (Huang et al., 2017, DOI: 10.1016/j.neuron.2012.06.027) and Egr2cre;Lbx1FS (Hernandez-Miranda et al., 2018, DOI: 10.1073/pnas.1813520115).
Several conclusions presented in this work are not directly supported by the provided data. For instance, the reduction in the number of retrotrapezoid neurons in Atoh1Cre,Phox2bdelta8 mice or the reduction of fos+ activated retrotrapezoid neurons after CO2 exposure, as the identity of retrotrapezoid neurons was not thoroughly determined. Furthermore, the authors conclude from their plethysmograph (respiratory recordings) data that Atoh1Cre,Phox2bdelta8 neonatal mice display an impaired ventilatory responses to hypoxia (low O2 in air) and hypercapnia (high CO2 in air), but that these mutant animals recover the capacity to respond to hypercapnia, but not to hypoxia, in the adult life. This is a bit of an overstatement, as their plethysmograph recordings show that adult Atoh1Cre,Phox2bdelta8 mice do respond to low O2 in air, as these mice accelerate respiration, increase tidal volumes and minute ventilation in the same fashion as control mice. However, what the presented data show is that adult Atoh1Cre,Phox2bdelta8 mice do not sustain the ventilatory response as efficient as control mice.
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