Double deletion of Panx1 and Panx3 affects skin and bone but not hearing

  • J. M. Abitbol
  • B. L. O’Donnell
  • C. B. Wakefield
  • E. Jewlal
  • J. J. Kelly
  • K. Barr
  • K. E. Willmore
  • B. L. Allman
  • S. PenuelaEmail author
Original Article


Pannexins (Panxs), large-pore channel forming glycoproteins, are expressed in a wide variety of tissues including the skin, bone, and cochlea. To date, the use of single knock-out mouse models of both Panx1 and Panx3 have demonstrated their roles in skin development, bone formation, and auditory phenotypes. Due to sequence homology between Panx1 and Panx3, when one Panx is ablated from germline, the other may be upregulated in a compensatory mechanism to maintain tissue homeostasis and function. To evaluate the roles of Panx1 and Panx3 in the skin, bone, and cochlea, we created the first Panx1/Panx3 double knock-out mouse model (dKO). These mice had smaller litters and reduced body weight compared to wildtype controls. The dKO dorsal skin had decreased epidermal and dermal area as well as decreased hypodermal area in neonatal but not in older mice. In addition, mouse skull shape and size were altered, and long bone length was decreased in neonatal dKO mice. Finally, auditory tests revealed that dKO mice did not exhibit hearing loss and were even slightly protected against noise-induced hearing damage at mid-frequency regions. Taken together, our findings suggest that Panx1 and Panx3 are important at early stages of development in the skin and bone but may be redundant in the auditory system.

Key messages

  • Panx double KO mice had smaller litters and reduced body weight.

  • dKO skin had decreased epidermal and dermal area in neonatal mice.

  • Skull shape and size changed plus long bone length decreased in neonatal dKO mice.

  • dKO had no hearing loss and were slightly protected against noise-induced damage.


Pannexin Hearing Skin Bone Panx1 Panx3 



We thank Genentech Inc. (San Francisco, CA) for the gift of the Panx1 knockout mouse, Rafael Sanchez Pupo for technical assistance, and Quintyn Farrar for his help collecting long bone data. JMA was funded by a Natural Sciences and Engineering Research Council (NSERC) Scholarship. BO and CBW were funded by an Ontario Graduate Scholarship (OGS). EJ and CBW received the Collaborative Specialization in Musculoskeletal Health Research (CMHR) award. NSERC Discovery Grant to KW. CIHR Project grant to BLA. NSERC Discovery Grant to SP.

Author contributions

JMA: Mouse genotyping, conducted all hearing experiments, data collection, and analysis, compilation of all experimental data, and wrote the manuscript draft.

BO’D: Mouse genotyping, expression analyses of skin and hindlimb, histological analyses of skin and paw.

CBW: Mouse genotyping, qPCR and protein experiments, data collection, and analysis.

EJ: Skull data collection and analysis, histological analysis of tibial growth plate and long bone cross-sectional analyses.

JJK: Conceptualization of the dKO hearing study, student training, data analysis.

KB: Generated the dKO mice, performed initial characterization and limb bone length comparisons, and contributed to skull landmarking.

KW: Skull and limb phenotypic analyses, provided funding.

BLA: Analyses and interpretation of hearing testing, provided funding.

SP: Generation of the dKO mice, initial characterization experiments, study design, provided funding, supervised and coordinated all participants, data analysis, manuscript editing.

All authors participated in manuscript editing.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

109_2019_1779_MOESM1_ESM.pdf (3.1 mb)
ESM 1 (PDF 3.10 MB)


  1. 1.
    Panchin Y, Kelmanson I, Matz M, Lukyanov K, Usman N, Lukyanov S (2000) A ubiquitous family of putative gap junction molecules. Curr Biol 10:R473–R474CrossRefGoogle Scholar
  2. 2.
    Baranova A, Ivanov D, Petrash N, Pestova A, Skoblov M, Kelmanson I, Shagin D, Nazarenko S, Geraymovych E, Litvin O, Tiunova A, Born TL, Usman N, Staroverov D, Lukyanov S, Panchin Y (2004) The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins. Genomics 83:706–716CrossRefGoogle Scholar
  3. 3.
    Penuela S, Gehi R, Laird DW (2013) The biochemistry and function of pannexin channels. Biochim Biophys Acta 1828:15–22CrossRefGoogle Scholar
  4. 4.
    Sosinsky GE, Boassa D, Dermietzel R, Duffy HS, Laird DW, MacVicar B, Naus CC, Penuela S, Scemes E, Spray DC, Thompson RJ, Zhao HB, Dahl G (2011) Pannexin channels are not gap junction hemichannels. Channels (Austin) 5:193–197CrossRefGoogle Scholar
  5. 5.
    Scemes E, Spray DC, Meda P (2009) Connexins, pannexins, innexins: novel roles of “hemi-channels”. Pflugers Arch 457:1207–1226CrossRefGoogle Scholar
  6. 6.
    Bruzzone R, Hormuzdi SG, Barbe MT, Herb A, Monyer H (2003) Pannexins, a family of gap junction proteins expressed in brain. Proc Natl Acad Sci U S A 100:13644–13649CrossRefPubMedCentralGoogle Scholar
  7. 7.
    Penuela S, Bhalla R, Nag K, Laird DW (2009) Glycosylation regulates pannexin intermixing and cellular localization. Mol Biol Cell 20:4313–4323CrossRefPubMedCentralGoogle Scholar
  8. 8.
    Ray A, Zoidl G, Weickert S, Wahle P, Dermietzel R (2005) Site-specific and developmental expression of pannexin1 in the mouse nervous system. Eur J Neurosci 21:3277–3290CrossRefGoogle Scholar
  9. 9.
    Wang XH, Streeter M, Liu YP, Zhao HB (2009) Identification and characterization of pannexin expression in the mammalian cochlea. J Comp Neurol 512:336–346CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Tang W, Ahmad S, Shestopalov VI, Lin X (2008) Pannexins are new molecular candidates for assembling gap junctions in the cochlea. Neuroreport 19:1253–1257CrossRefPubMedCentralGoogle Scholar
  11. 11.
    Le Vasseur M, Lelowski J, Bechberger JF, Sin WC, Naus CC (2014) Pannexin 2 protein expression is not restricted to the CNS. Front Cell Neurosci 8:392CrossRefPubMedCentralGoogle Scholar
  12. 12.
    Turmel P, Dufresne J, Hermo L, Smith CE, Penuela S, Laird DW, Cyr DG (2011) Characterization of pannexin1 and pannexin3 and their regulation by androgens in the male reproductive tract of the adult rat. Mol Reprod Dev 78:124–138CrossRefGoogle Scholar
  13. 13.
    Bao L, Locovei S, Dahl G (2004) Pannexin membrane channels are mechanosensitive conduits for ATP. FEBS Lett 572:65–68CrossRefGoogle Scholar
  14. 14.
    Locovei S, Wang J, Dahl G (2006) Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium. FEBS Lett 580:239–244CrossRefGoogle Scholar
  15. 15.
    Chekeni FB, Elliott MR, Sandilos JK, Walk SF, Kinchen JM, Lazarowski ER, Armstrong AJ, Penuela S, Laird DW, Salvesen GS, Isakson BE, Bayliss DA, Ravichandran KS (2010) Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis. Nature 467:863–867CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Penuela S, Bhalla R, Gong XQ, Cowan KN, Celetti SJ, Cowan BJ, Bai D, Shao Q, Laird DW (2007) Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins. J Cell Sci 120:3772–3783CrossRefGoogle Scholar
  17. 17.
    Penuela S, Kelly JJ, Churko JM, Barr KJ, Berger AC, Laird DW (2014) Panx1 regulates cellular properties of keratinocytes and dermal fibroblasts in skin development and wound healing. J Invest Dermatol 134:2026–2035CrossRefGoogle Scholar
  18. 18.
    Celetti SJ, Cowan KN, Penuela S, Shao Q, Churko J, Laird DW (2010) Implications of pannexin 1 and pannexin 3 for keratinocyte differentiation. J Cell Sci 123:1363–1372CrossRefGoogle Scholar
  19. 19.
    Cowan KN, Langlois S, Penuela S, Cowan BJ, Laird DW (2012) Pannexin1 and Pannexin3 exhibit distinct localization patterns in human skin appendages and are regulated during keratinocyte differentiation and carcinogenesis. Cell Commun Adhes 19:45–53CrossRefGoogle Scholar
  20. 20.
    Zhang P, Ishikawa M, Rhodes C, Doyle A, Ikeuchi T, Nakamura K, Chiba Y, He B, Yamada Y (2018) Pannexin-3 deficiency delays skin wound healing in mice due to defects in channel functionality. J Invest Dermatol 139(4):909–918Google Scholar
  21. 21.
    Bond SR, Lau A, Penuela S, Sampaio AV, Underhill TM, Laird DW, Naus CC (2011) Pannexin 3 is a novel target for Runx2, expressed by osteoblasts and mature growth plate chondrocytes. J Bone Miner Res 26:2911–2922CrossRefGoogle Scholar
  22. 22.
    Ishikawa M, Yamada Y (2017) The role of Pannexin 3 in bone biology. J Dent Res 96:372–379CrossRefGoogle Scholar
  23. 23.
    Ishikawa M, Iwamoto T, Nakamura T, Doyle A, Fukumoto S, Yamada Y (2011) Pannexin 3 functions as an ER Ca(2+) channel, hemichannel, and gap junction to promote osteoblast differentiation. J Cell Biol 193:1257–1274CrossRefPubMedCentralGoogle Scholar
  24. 24.
    Moon PM, Penuela S, Barr K, Khan S, Pin CL, Welch I, Attur M, Abramson SB, Laird DW, Beier F (2015) Deletion of Panx3 prevents the development of surgically induced osteoarthritis. J Mol Med (Berl) 93:845–856CrossRefGoogle Scholar
  25. 25.
    Caskenette D, Penuela S, Lee V, Barr K, Beier F, Laird DW, Willmore KE (2016) Global deletion of Panx3 produces multiple phenotypic effects in mouse humeri and femora. J Anat 228:746–756CrossRefPubMedCentralGoogle Scholar
  26. 26.
    Abitbol JM, Kelly JJ, Barr K, Schormans AL, Laird DW, Allman BL (2016) Differential effects of pannexins on noise-induced hearing loss. Biochem J 473:4665–4680CrossRefGoogle Scholar
  27. 27.
    Chen J, Zhu Y, Liang C, Zhao HB (2015) Pannexin1 channels dominate ATP release in the cochlea ensuring endocochlear potential and auditory receptor potential generation and hearing. Sci Rep 5:10762CrossRefPubMedCentralGoogle Scholar
  28. 28.
    Zhao HB, Zhu Y, Liang C, Chen J (2015) Pannexin 1 deficiency can induce hearing loss. Biochem Biophys Res Commun 463:143–147CrossRefPubMedCentralGoogle Scholar
  29. 29.
    Bargiotas P, Krenz A, Hormuzdi SG, Ridder DA, Herb A, Barakat W, Penuela S, von Engelhardt J, Monyer H, Schwaninger M (2011) Pannexins in ischemia-induced neurodegeneration. Proc Natl Acad Sci U S A 108:20772–20777CrossRefPubMedCentralGoogle Scholar
  30. 30.
    Lohman AW, Billaud M, Straub AC, Johnstone SR, Best AK, Lee M, Barr K, Penuela S, Laird DW, Isakson BE (2012) Expression of pannexin isoforms in the systemic murine arterial network. J Vasc Res 49:405–416CrossRefPubMedCentralGoogle Scholar
  31. 31.
    Whyte-Fagundes P, Kurtenbach S, Zoidl C, Shestopalov VI, Carlen PL, Zoidl G (2018) A potential compensatory role of Panx3 in the VNO of a Panx1 Knock out mouse model. Front Mol Neurosci 11:135CrossRefPubMedCentralGoogle Scholar
  32. 32.
    Qu Y, Misaghi S, Newton K, Gilmour LL, Louie S, Cupp JE, Dubyak GR, Hackos D, Dixit VM (2011) Pannexin-1 is required for ATP release during apoptosis but not for inflammasome activation. J Immunol 186:6553–6561CrossRefGoogle Scholar
  33. 33.
    Hill CA, Sussan TE, Reeves RH, Richtsmeier JT (2009) Complex contributions of Ets2 to craniofacial and thymus phenotypes of trisomic “Down syndrome” mice. Am J Med Genet A 149A:2158–2165CrossRefPubMedCentralGoogle Scholar
  34. 34.
    Motch Perrine SM, Cole TM 3rd, Martinez-Abadias N, Aldridge K, Jabs EW, Richtsmeier JT (2014) Craniofacial divergence by distinct prenatal growth patterns in Fgfr2 mutant mice. BMC Dev Biol 14:8CrossRefPubMedCentralGoogle Scholar
  35. 35.
    Martinez-Abadias N, Holmes G, Pankratz T, Wang Y, Zhou X, Jabs EW, Richtsmeier JT (2013) From shape to cells: mouse models reveal mechanisms altering palate development in Apert syndrome. Dis Model Mech 6:768–779CrossRefPubMedCentralGoogle Scholar
  36. 36.
    Adams DC, Otarola-Castillo E (2013) Geomorph: an r package for the collection and analysis of geometric morphometric shape data. Methods Ecol Evol 4:393–399CrossRefGoogle Scholar
  37. 37.
    Adams DC, Collyer ML, Kaliontzopoulou A (2018) Geometric morphometric analyses of 2D/3D landmark data (Version 3.07)Google Scholar
  38. 38.
    Collyer ML, Sekora DJ, Adams DC (2015) A method for analysis of phenotypic change for phenotypes described by high-dimensional data. Heredity (Edinb) 115:357–365CrossRefGoogle Scholar
  39. 39.
    McLeod MJ (1980) Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology 22:299–301CrossRefGoogle Scholar
  40. 40.
    Lee V, Barr KJ, Kelly J, Johnston D, Brown C, Robb K, Sayedyahossein S, Huang K, Gros R, Flynn L et al (2018) Pannexin 1 regulates adipose stromal cell differentiation and fat accumulation. Sci Rep 8:16166CrossRefPubMedCentralGoogle Scholar
  41. 41.
    Oh SK, Shin JO, Baek JI, Lee J, Bae JW, Ankamerddy H, Kim MJ, Huh TL, Ryoo ZY, Kim UK, Bok J, Lee KY (2015) Pannexin 3 is required for normal progression of skeletal development in vertebrates. FASEB J 29:4473–4484CrossRefGoogle Scholar
  42. 42.
    Ishikawa M, Williams GL, Ikeuchi T, Sakai K, Fukumoto S, Yamada Y (2016) Pannexin 3 and connexin 43 modulate skeletal development through their distinct functions and expression patterns. J Cell Sci 129:1018–1030CrossRefPubMedCentralGoogle Scholar
  43. 43.
    Vora SR, Camci ED, Cox TC (2015) Postnatal ontogeny of the cranial base and craniofacial skeleton in male C57BL/6J mice: a reference standard for quantitative analysis. Front Physiol 6:417Google Scholar
  44. 44.
    Shao Q, Lindstrom K, Shi R, Kelly J, Schroeder A, Juusola J, Levine KL, Esseltine JL, Penuela S, Jackson MF, Laird DW (2016) A germline variant in the PANX1 gene has reduced channel function and is associated with multisystem dysfunction. J Biol Chem 291:12432–12443CrossRefPubMedCentralGoogle Scholar
  45. 45.
    Chen J, Liang C, Zong L, Zhu Y, Zhao HB (2018) Knockout of Pannexin-1 induces hearing loss. Int J Mol Sci 19Google Scholar
  46. 46.
    Zorzi V, Paciello F, Ziraldo G, Peres C, Mazzarda F, Nardin C, Pasquini M, Chiani F, Raspa M, Scavizzi F, Carrer A, Crispino G, Ciubotaru CD, Monyer H, Fetoni AR, M. Salvatore A, Mammano F (2017) Mouse Panx1 is dispensable for hearing acquisition and auditory function. Front Mol Neurosci 10:379CrossRefPubMedCentralGoogle Scholar
  47. 47.
    Forge A, Becker D, Casalotti S, Edwards J, Marziano N, Nevill G (2003) Gap junctions in the inner ear: comparison of distribution patterns in different vertebrates and assessement of connexin composition in mammals. J Comp Neurol 467:207–231CrossRefGoogle Scholar
  48. 48.
    Plotkin LI, Bellido T (2013) Beyond gap junctions: Connexin43 and bone cell signaling. Bone 52:157–166CrossRefGoogle Scholar
  49. 49.
    Xu J, Nicholson BJ (2013) The role of connexins in ear and skin physiology - functional insights from disease-associated mutations. Biochim Biophys Acta 1828:167–178CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Anatomy and Cell Biology, Schulich School of Medicine and DentistryUniversity of Western OntarioLondonCanada

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