Piezo2 senses airway stretch and mediates lung inflation-induced apnoea (original) (raw)

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Acknowledgements

We thank D. Trajkovic for assistance with histology; M. Wood for assistance with electron microscopy; J. Yu for suggesting that we test the Hering–Breuer reflex; S. M. Cahalan, M. Petrus, J. Mathur, K. Marshall, S. Lee, T. Kawamura, J. Chen and P. Paolo Sanna for technical assistance; and M. Krasnow for discussions. This research was supported by NIH grants R01DE022358 to A.P. and R01HL132255, and a Giovanni Armenise-Harvard Foundation Grant to S.D.L. A.P. is an investigator of the Howard Hughes Medical Institute.

Author information

Author notes

  1. Zhaozhu Qiu & Sanjeev S. Ranade
    Present address: †Present address: Department of Physiology and Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA (Z.Q.); The Gladstone Institute, San Francisco, California 94158, USA (S.S.R.).,
  2. Keiko Nonomura, Seung-Hyun Woo and Rui B. Chang: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Molecular and Cellular Neuroscience, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, 92037, California, USA
    Keiko Nonomura, Seung-Hyun Woo, Zhaozhu Qiu, Allain G. Francisco, Sanjeev S. Ranade & Ardem Patapoutian
  2. Department of Cell Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA
    Rui B. Chang & Stephen D. Liberles
  3. Department of Biochemistry, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, 94305, California, USA
    Astrid Gillich
  4. Genomics Institute of the Novartis Research Foundation, San Diego, 92121, California, USA
    Zhaozhu Qiu

Authors

  1. Keiko Nonomura
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  2. Seung-Hyun Woo
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  3. Rui B. Chang
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  4. Astrid Gillich
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  5. Zhaozhu Qiu
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  6. Allain G. Francisco
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  7. Sanjeev S. Ranade
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  8. Stephen D. Liberles
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  9. Ardem Patapoutian
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Contributions

A.G. characterized embryonic lungs in the laboratory of M. Krasnow. Z.Q. performed the initial characterization of _Piezo2_−/− mice. A.G.F. performed plethysmograph recordings and behavioural experiments to confirm Piezo2 knockdown. S.S.R. generated Piezo2 fl/fl and Piezo2+/− mice. R.B.C. performed optogenetic experiments, whole vagus nerve electrophysiology recordings, and transducer-based Hering–Breuer reflex assessment in the S.D.L. laboratory. K.N. and S.-H.W. contributed equally to all other experiments. K.N., S.-H.W. and A.P. wrote the manuscript.

Corresponding authors

Correspondence toStephen D. Liberles or Ardem Patapoutian.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks M. Boehnke, J. M. Greally, B. Voight and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Characterization of lung development in _Piezo2_−/− mice.

a, b, Whole-mount staining for αSMA in the left lobe of lungs from E16.5 (a) and E18.5 (b) wild-type and _Piezo2_−/− mice. L1–L6, lateral branches of the left lobe. E16.5: wild-type n = 4, _Piezo2_−/− n = 3; E18.5: wild-type n = 4, _Piezo2_−/− n = 5. A normal conducting airway and vessel pattern was observed in _Piezo2_−/− lungs at late embryonic stages. c, d, Whole-mount immunostaining for E-cadherin and Muc-1 (c) or E-cadherin, T1α, and SpC (d) in left lobes from E18.5 wild-type and _Piezo2_−/− mice. Arrows indicate proximal (P) to distal (D) direction. Left panels, lower magnification; right panels, higher magnification of the distal region. Solid circles in right panels indicate alveolar progenitors in the distal region. Solid hexagons in left panels indicate nascent type I pneumocytes. Yellow dashed circles in left panels indicate nascent type II pneumocytes. A normal epithelial morphology and no defects in alveolar epithelial patterning and differentiation were observed _Piezo2_−/− lungs. e, Representative ultrastructures of wild-type and _Piezo2_−/− newborn lungs. Black arrows mark type I pneumocyte extensions. White dotted circles mark type II pneumocytes. White arrowheads within white dotted circles mark lamellar bodies. RBC, red blood cells. Samples from four mice per genotype were analysed. Normal morphology and similar abundances of type I and II pneumocytes, endothelial cells, red blood cells and surfactant proteins were observed in _Piezo2_−/− lungs compared to wild-type lungs. f, Wet-to-dry lung ratio 6 h after delivery at E18.5 to assess clearance of fetal pulmonary fluid. NS, statistically not significant. Unpaired Student’s _t_-test. Bars represent mean ± s.d. g, Per cent weight of postnatal day (P)0 lung, heart, and liver normalized to whole body weight. Bars represent mean ± s.e.m. NS, statistically not significant. Kruskal–Wallis nonparametric test (lung and liver) or one-way ANOVA (heart). Scale bars, 500 μm (a, b); 33 μm (c, d, left); 22 μm (c, d, right); 5 μm (e).

Extended Data Figure 2 Piezo2 expression in the respiratory system.

a, Co-immunostaining for GFP and Nefh in P0 Piezo2 GFP reporter lung. Arrow, NEB. Smaller panels on right show a magnified view of NEB stained for GFP and CGRP (a marker of NEBs). b, c, GFP immunostaining in the jugular–nodose complex from P0 (b) and adult (c) Piezo2 GFP reporter mice. Dotted line demarcates the boundary of the jugular–nodose complex. d, e, GFP immunostaining in P0 trigeminal ganglion (d) or adult thoracic DRG (e) from Piezo2 GFP reporter mice. Dotted line in (d) demarcates the boundary of the trigeminal ganglion. f, Co-immunostaining for GFP and NeuN in a sagittal section of P0 Piezo2 GFP brainstem. g, h, GFP and αBTX (a marker of neuromuscular junction) co-staining in P0 Piezo2 GFP diaphragm (g) and intercostal muscles (h). i, Co-immunostaining for GFP and NeuN in adult Piezo2 GFP lumbar spinal cord. Dotted circle indicates motor neuron localization. j, Co-immunostaining for GFP and NeuN in adult Piezo2 GFP brainstem. Dotted circle marks dorsal nucleus of the vagus nerve, X. k, Co-immunostaining for GFP and Tuj1 in thoracic sympathetic ganglia from adult Piezo2 GFP reporter mice. l, Schematic summary of Piezo2–GFP expression in the respiratory system. VII, facial nucleus; BötC, the Bötzinger complex; VRG, ventral respiratory group; RB, rib bone; Vt, ventricle. Scale bars, 20 μm (smaller panels on right in a), 100 μm (all other panels).

Extended Data Figure 3 Characterization of tdTomato expression in the respiratory system of Piezo2-GFP-IRES-Cre (Piezo2 GFP);Ai9 reporter mice.

a, b, Immunostaining for PECAM1 (a) and CGRP (b) with tdTomato epifluorescence in postnatal lungs. Arrow in (b) indicates tdTomato+ nerve fibre innervating NEB. Dotted line in (b) demarcates the lung epithelium. c, tdTomato epifluorescence in P0 jugular–nodose complex. Dotted line in (c) demarcates the boundary of the jugular–nodose complex. d, tdTomato epifluorescence in adult thoracic DRG. e, f, tdTomato epifluorescence in adult nucleus of the solitary tract (NTS) (e) and in adult spinal trigeminal nucleus (Sp5) (f), where axons of nodose and jugular/trigeminal sensory neurons project, respectively52,53. Smaller panels on right in f show a magnified view of Sp5 with Nefh staining. g–i, PECAM1 immunostaining with tdTomato epifluorescence in P0 brainstem (g), adult diaphragm (h), and P0 intercostal muscle (i). j, PV immunostaining with tdTomato epifluorescence in postnatal lumbar spinal cord. Dotted circle marks motor neuron localization. k, Tuj1 immunostaining with tdTomato epifluorescence in adult thoracic sympathetic ganglia. l, ChAT immunostaining and tdTomato epifluorescence in tracheal parasympathetic ganglia from adult reporter mice. Scale bars, 25 μm (a, l, smaller panels on right in f), 20 μm (b), 100 μm (c–k).

Extended Data Figure 4 Characterization of tissue-specific Cre activities via Ai9 reporters.

a, b, tdTomato epifluorescence in P0 Tie2Cre;Ai9 lung with PECAM1 staining (a) or CGRP staining (b). c, tdTomato epifluorescence in P0 Tie2Cre;Ai9 jugular–nodose complex with PECAM1 staining. Dotted line demarcates the boundary of the jugular–nodose complex. d, tdTomato epifluorescence in adult Tie2Cre;Ai9 thoracic DRG. Dotted line demarcates the boundary of the DRG. e, tdTomato epifluorescence in P0 Tie2Cre;Ai9 trigeminal ganglion with Advillin staining. TdTomato signal co-localizes with PECAM1+ endothelial cells in a and c. f–i, tdTomato epifluorescence in P0 Phox2bCre;Ai9 lung with CGRP staining (f), P0 jugular–nodose complex with Advillin staining (g), adult thoracic DRG (h), and P0 trigeminal ganglion with Advillin staining (i). TdTomato signal is present in nodose ganglia (g), but absent in lung cells and NEBs (f), jugular ganglia (g), DRG (h), and trigeminal ganglia (i). Arrows in f indicate tdTomato+ vagal nerve fibre innervating the lung epithelium. Dotted line in h demarcates the boundary of DRG. j–m, tdTomato epifluorescence in P0 Wnt1Cre;Ai9 lung with CGRP staining (j), P0 jugular–nodose complex (k), adult thoracic DRG (l), and adult jugular–nodose complex with Piezo2 staining (m). m′, m′′, Higher magnification images of m. Arrows show tdTomato expression in both jugular neuronal cell bodies and satellite cells. Arrowheads show tdTomato expression only in satellite cells. TdTomato signal is present in neuronal cell bodies of jugular ganglia (k, m) and DRG (l), and satellite cells in the jugular–nodose complex and DRG (k–m), but absent in lung cells and NEBs (j) and neuronal cell bodies of nodose ganglia (k, m). TdTomato+ nerve fibres innervate the lung (j). Dotted lines indicate boundaries of the ganglia. VII + VIII, facial-acoustic complex. Scale bars, 50 μm (b), 12.5 μm (m′, m′′), 100 μm (all other panels).

Extended Data Figure 5 Characterization of Piezo2 knockdown in Piezo2 conditional knockout mice.

a, Characterization of Piezo2 knockdown by qRT–PCR using FACS-sorted CD31+ (or PECAM1+) lung cells from adult wild-type and Tie2Cre;Piezo2 cKO mice. b, Piezo2 in situ hybridization in the jugular–nodose complex of adult wild-type and Phox2bCre;Piezo2 cKO mice. Dotted circles mark nodose ganglia (N). c, qRT–PCR using DRG isolated from P0 wild-type and Wnt1Cre;Piezo2 cKO pups. *P < 0.05, ***P < 0.001, ****P < 0.0001, unpaired Welch’s _t_-test for a, c. Data are presented as mean ± s.e.m. Scale bar, 100 μm.

Extended Data Figure 6 Optogenetic activation of Piezo2+ vagal sensory neurons in Piezo2 GFP ;lox-ChR2 mice.

a, A compound action potential response following brief optogenetic stimulation (blue lightning sign) of vagus nerve in Piezo2 GFP ;lox-ChR2 mice. b, A and C currents classified on the basis of corresponding peak area in the compound action potential. Dashed line: A–C ratio of 1. Data are presented as mean ± s.e.m. c, GFP, Nefh and IB4 co-staining in adult Piezo2 GFP jugular–nodose complex. Red arrows indicate GPF+ Nefh+ IB4− cells; blue arrows indicate GFP+ Nefh− IB4+ cells. d, Percentage of Nefh or IB4 positive cells among GFP+ cells in adult Piezo2 GFP jugular–nodose complex. e–g, State of respiratory trapping following optogenetic stimulation (50 Hz, 10 s) of vagus nerve in Piezo2 GFP ;lox-ChR2 mice. Representative trace showing changes in lung volume following optogenetic activation in Piezo2 GFP ;lox-ChR2 mice (e). Per cent change in total lung volume in Piezo2 GFP ;lox-ChR2 mice without and with light (f). The percentage of time in a high lung volume state (greater than mean volume during tidal breathing) in Piezo2 GFP ;lox-ChR2 mice without and with light (g). ***P < 0.001, ****P < 0.0001, paired _t_-test, mean ± s.e.m. h, Brainstem of Piezo2 _GFP_mice with AAV-flex-tdTomato injection to the jugular–nodose complex. Sol, solitary tract; CC, central canal; AP, area postrema; L-NTS; ventral, lateral, ventrolateral, interstitial, and intermediate NTS subnuclei; M-NTS; dorsolateral, dorsomedial, medial, and commissural NTS subnuclei. Scale bars, 100 μm.

Extended Data Figure 7 Characterization of AdvillinCreERT2 and PvalbCre activity via Ai9 reporter.

a, tdTomato epifluorescence and DAPI staining in the lung from adult AdvillinCreER T2 ;Ai9+Tam reporter mice. TdTomato is not expressed in lung cells. Instead, tdTomato+ nerve fibres innervate the lung. b, tdTomato epifluorescence, CGRP and DAPI staining in lungs from adult AdvillinCreER T2 ;Ai9+Tam reporter mice. TdTomato is not expressed in NEBs. Dotted line demarcates the lung epithelium. Bronch, bronchioles. c, d, tdTomato epifluorescence in adult jugular–nodose complex (c) and tdTomato epifluorescence and DAPI staining in adult thoracic DRG (d) from AdvillinCreER T2 ;Ai9+Tam reporter mice. TdTomato is expressed in both the jugular–nodose complex (c) and the DRG (d). e, f, tdTomato epifluorescence and DAPI staining in the lung (e) and jugular–nodose complex (f) of adult PvalbCre;Ai9 reporter mice. TdTomato is not expressed in lung cells and neuronal cell bodies in the jugular–nodose complex. Scale bars, 100 μm.

Extended Data Figure 8 Respiratory properties of various _Piezo2_-deficient mouse lines.

a, b, Average frequency (a) and average tidal volume (b) of adult wild-type and Phox2bCre;Piezo2 cKO mice under anaesthesia. *P < 0.05, unpaired Student’s _t_-test, mean ± s.e.m. c, Respiration activity during lung inflation (0.3 ml air) normalized to baseline in adult wild-type and AdvillinCreER T2 ;Piezo2 cKO mice. *P < 0.05, unpaired Welch’s _t_-test, mean ± s.e.m. d, e, Phenylbiguanide (PBG)-induced chemoreflex in adult AdvillinCreER T2 ;Piezo2 cKO mice. Representative traces of respiratory air flow from wild-type and AdvillinCreER T2 ;Piezo2 cKO mice with 2.0 μg PBG intravenous injection (d). Baseline and longest breath interval after PBG injection. Bars represent mean ± s.e.m. (e). f, g, Average frequency (f) and average tidal volume (g) of adult wild-type and PvalbCre;Piezo2 cKO mice under anaesthesia. Unpaired Student’s _t_-test, mean ± s.e.m. h, Respiration activity during lung inflation (0.3 ml air) normalized to baseline in adult wild-type and PvalbCre;Piezo2 cKO mice. Unpaired Welch’s _t_-test, mean ± s.e.m. NS, statistically not significant.

Extended Data Figure 9 Roles of Piezo2 in respiratory system.

a, Piezo2 in nodose sensory neurons. It has been widely reported that nodose sensory neurons contain low-threshold mechanosensors innervating the lower airway tract including the lungs, while jugular sensory neurons contain high-threshold mechanosensors that innervate the upper airway tract, such as the larynx and the trachea. In addition, nodose sensory neurons project to the NTS6,53, a synaptic station required for the Hering–Breuer reflex in the brainstem54. Consistent with these findings, Piezo2 expression is detected in the jugular–nodose ganglia complex, and Piezo2+ nerve fibres project to the NTS. Moreover, adult mice lacking Piezo2 in the nodose ganglion show abolished vagal nerve responses to lung inflation, increased tidal volume, and an impaired Hering–Breuer inspiratory reflex. In addition to the nodose ganglia, Piezo2 is also expressed in the jugular, trigeminal, and dorsal root ganglia. We observed similar phenotypes in mice with Piezo2 depletion induced in virtually all sensory neurons in the adult. These data suggest that Piezo2 in nodose sensory neurons is the major stretch sensor required for lung volume regulation and the Hering–Breuer reflex response in adult mice. b, Piezo2 in jugular, trigeminal and/or spinal sensory neurons. In newborn mice, Piezo2 in sensory neurons of the neural crest origin is required for proper lung expansion and establishing efficient respiration as both global Piezo2 knockout and neural crest-derived sensory neuron-specific Piezo2 conditional knockout newborn mice showed hypoventilation, decreased inspiratory activity, altered expiratory pattern and unexpanded lungs. Our genetic studies also suggest that Piezo2 is not required in nodose ganglia for newborn lung expansion and respiration; however, this lack of requirement does not imply lack of involvement, and could be due to functional redundancy (that is, Piezo2 in jugular, trigeminal and/or dorsal root ganglia can compensate for Piezo2 deficiency in nodose ganglia). Although sensory neuronal control of respiration in newborn animals remains largely unknown, the newborn airway experiences a large pressure change in the course of lung expansion55. Piezo2-mediated mechanosensory feedback of the airway might be crucial for subsequent motor output (for example, control of diaphragm discharge or prevention of upper airway narrowing) to establish proper breathing patterns in newborn animals. The data presented here do not identify the exact cause of lethality in Piezo2-deficient newborn animals; however, we speculate that lethality in pups might be due to a combined effect of hypoventilation and lack of nutrients owing to inability to suckle. c, Piezo2 in NEBs. In addition to airway-innervating sensory neurons, Piezo2 is also expressed in pulmonary NEB cells, which are likely to be innervated by Piezo2+ afferents (Extended Data Fig. 3b, arrow). NEBs are enigmatic pulmonary cells whose physiological function is unclear3,22,23. Previous studies have suggested that the inflation-induced vagal nerve responses that are responsible for the Hering–Breuer reflex are slowly adapting2,4,5,7. While Piezo2 channels are generally rapidly adapting when assayed in cultured cells16, Piezo2 channels in Merkel cell–neurite complexes in the skin give rise to slowly adapting firing responses21,56 that are proposed to be caused by dual Piezo2 expression in both epidermal Merkel cells and associated afferents21,56. Therefore, it is possible that NEBs also contribute to sensing lung inflation in concert with Piezo2+ mechanosensory afferents. Future efforts will explore whether pulmonary NEBs function as mechanosensory cells, similar to Merkel cells.

Extended Data Table 1 Respiratory properties of anaesthetized wild-type and AdvillinCreER T2 ;Piezo2 cKO mice

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Nonomura, K., Woo, SH., Chang, R. et al. Piezo2 senses airway stretch and mediates lung inflation-induced apnoea.Nature 541, 176–181 (2017). https://doi.org/10.1038/nature20793

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