Skip to main content

Advertisement

SpringerLink
Log in

Effects of Reduced Connexin43 Function on Mandibular Morphology and Osteogenesis in Mutant Mouse Models of Oculodentodigital Dysplasia

  • Original Research
  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Abstract

Mutations in the gene encoding the gap-junctional protein connexin43 (Cx43) are the cause of the human disease oculodentodigital dysplasia (ODDD). The mandible is often affected in this disease, with clinical reports describing both mandibular overgrowth and conversely, retrognathia. These seemingly opposing observations underscore our relative lack of understanding of how ODDD affects mandibular morphology. Using two mutant mouse models that mimic the ODDD phenotype (I130T/+ and G60S/+), we sought to uncover how altered Cx43 function may affect mandibular development. Specifically, mandibles of newborn mice were imaged using micro-CT, to enable statistical comparisons of shape. Tissue-level comparisons of key regions of the mandible were conducted using histomorphology, and we quantified the mRNA expression of several cartilage and bone cell differentiation markers. Both G60S/+ and I130T/+ mutant mice had altered mandibular morphology compared to their wildtype counterparts, and the morphological effects were similarly localized for both mutants. Specifically, the biggest phenotypic differences in mutant mice were focused in regions exposed to mechanical forces, such as alveolar bone, muscular attachment sites, and articular surfaces. Histological analyses revealed differences in ossification of the intramembranous bone of the mandibles of both mutant mice compared to their wildtype littermates. However, chondrocyte organization within the secondary cartilages of the mandible was unaffected in the mutant mice. Overall, our results suggest that the morphological differences seen in G60S/+ and I130T/+ mouse mandibles are due to delayed ossification and suggest that mechanical forces may exacerbate the effects of ODDD on the skeleton.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data Availability

Data are available from the authors upon request.

References

  1. Duan X, Bradbury SR, Olsen BR, Berendsen AD (2016) VEGF stimulates intramembranous bone formation during craniofacial skeletal development. Matrix Biol 52–54:127–140. https://doi.org/10.1016/j.matbio.2016.02.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Day TF, Guo X, Garrett-Beal L, Yang Y (2005) Wnt/β-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell 8(5):739–750. https://doi.org/10.1016/J.DEVCEL.2005.03.016

    Article  CAS  PubMed  Google Scholar 

  3. Gupta A, Anderson H, Buo AM, Moorer MC, Ren M, Stains JP (2016) Communication of cAMP by connexin43 gap junctions regulates osteoblast signaling and gene expression. Cell Signal 28(8):1048–1057. https://doi.org/10.1016/j.cellsig.2016.04.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zappitelli T, Aubin JE (2014) The ‘connexin’ between bone cells and skeletal functions. J Cell Biochem 115(10):1646–1658. https://doi.org/10.1002/jcb.24836

    Article  CAS  PubMed  Google Scholar 

  5. Lecanda F, Warlow PM, Sheikh S, Furlan F, Steinberg TH, Civitelli R (2000) Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J Cell Biol 151(4):931–943. https://doi.org/10.1083/jcb.151.4.931

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Donahue HJ et al (2009) Chondrocytes isolated from mature articular cartilage retain the capacity to form functional gap junctions. J Bone Miner Res 10(9):1359–1364. https://doi.org/10.1002/jbmr.5650100913

    Article  Google Scholar 

  7. Civitelli R, Beyer EC, Warlow PM, Robertson AJ, Geist ST, Steinberg TH (1993) Connexin43 mediates direct intercellular communication in human osteoblastic cell networks. J Clin Invest 91(5):1888–1896. https://doi.org/10.1172/JCI116406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chaible LM, Sanches DS, Cogliati B, Mennecier G, Lucia M, Dagli Z (2011) Delayed osteoblastic differentiation and bone development in Cx43 knockout mice. Toxicol Pathol 39(7):1046–1055. https://doi.org/10.1177/0192623311422075

    Article  CAS  PubMed  Google Scholar 

  9. Paznekas WA et al (2003) Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia. Am J Hum Genet 72(2):408–418. https://doi.org/10.1086/346090

    Article  CAS  PubMed  Google Scholar 

  10. Paznekas WA et al (2009) GJA1 mutations, variants, and connexin 43 dysfunction as it relates to the oculodentodigital dysplasia phenotype. Hum Mutat 30(5):724–733. https://doi.org/10.1002/humu.20958

    Article  CAS  PubMed  Google Scholar 

  11. van Es RJJ, Wittebol-Post D, Beemer FA (2007) Oculodentodigital dysplasia with mandibular retrognathism and absence of syndactyly: a case report with a novel mutation in the connexin 43 gene. Int J Oral Maxillofac Surg 36(9):858–860. https://doi.org/10.1016/j.ijom.2007.03.004

    Article  PubMed  Google Scholar 

  12. Laird DW (2014) Syndromic and non-syndromic disease-linked Cx43 mutations. FEBS Lett 588(8):1339–1348. https://doi.org/10.1016/j.febslet.2013.12.022

    Article  CAS  PubMed  Google Scholar 

  13. Flenniken AM et al (2005) A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia. Development 132(19):4375–4386. https://doi.org/10.1242/dev.02011

    Article  CAS  PubMed  Google Scholar 

  14. Kalcheva N et al (2007) Gap junction remodeling and cardiac arrhythmogenesis in a murine model of oculodentodigital dysplasia. Proc Natl Acad Sci USA 104(51):20512–20516. https://doi.org/10.1073/pnas.90.18.8424

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dobrowolski R et al (2008) The conditional connexin43G138R mouse mutant represents a new model of hereditary oculodentodigital dysplasia in humans. Hum Mol Genet 17(4):539–554. https://doi.org/10.1093/hmg/ddm329

    Article  CAS  PubMed  Google Scholar 

  16. Stewart MKG, Gong X-Q, Barr KJ, Bai D, Fishman GI, Laird DW (2013) The severity of mammary gland developmental defects is linked to the overall functional status of Cx43 as revealed by genetically modified mice. Biochem J 449(2):401–413. https://doi.org/10.1042/BJ20121070

    Article  CAS  PubMed  Google Scholar 

  17. McLachlan E et al (2008) ODDD-linked Cx43 mutants reduce endogenous Cx43 expression and function in osteoblasts and inhibit late stage differentiation. J Bone Miner Res 23(6):928–938. https://doi.org/10.1359/jbmr.080217

    Article  CAS  PubMed  Google Scholar 

  18. Jarvis SE et al (2020) Effects of reduced connexin43 function on skull development in the Cx43I130T/+ mutant mouse that models oculodentodigital dysplasia. Bone. https://doi.org/10.1016/j.bone.2020.115365

    Article  PubMed  PubMed Central  Google Scholar 

  19. Watkins M, Grimston SK, Yi J, Guillotin B, Shaw A (2011) Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling. Mol Biol Cell. https://doi.org/10.1091/mbc.E10-07-0571

    Article  PubMed  PubMed Central  Google Scholar 

  20. Atchley WR, Hall BK (1991) A model for development and evolution of complex morphological structures. Biol Rev 66(2):101–157. https://doi.org/10.1111/j.1469-185X.1991.tb01138.x

    Article  CAS  PubMed  Google Scholar 

  21. Shibata S et al (2004) Runx2-deficient mice lack mandibular condylar cartilage and have deformed Meckel’s cartilage. Anat Embryol (Berlin) 208(4):273–280. https://doi.org/10.1007/s00429-004-0393-2

    Article  CAS  Google Scholar 

  22. Shibata S, Suda N, Suzuki S, Fukuoka H, Yamashita Y (2006) An in situ hybridization study of Runx2, Osterix, and Sox9 at the onset of condylar cartilage formation in fetal mouse mandible. J Anat 208(2):169–177. https://doi.org/10.1111/j.1469-7580.2006.00525.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Willmore KE, Leamy L, Hallgrímsson B (2006) Effects of developmental and functional interactions on mouse cranial variability through late ontogeny. Evol Dev 8(6):550–567. https://doi.org/10.1111/j.1525-142X.2006.00127.x

    Article  PubMed  Google Scholar 

  24. Klingenberg CP (2010) Evolution and development of shape: integrating quantitative approaches. Nat Rev Genet 11(9):623–635. https://doi.org/10.1038/nrg2829

    Article  CAS  PubMed  Google Scholar 

  25. Cardini A, Elton S (2007) Sample size and sampling error in geometric morphometric studies of size and shape. Zoomorphology 126(2):121–134. https://doi.org/10.1007/s00435-007-0036-2

    Article  Google Scholar 

  26. Ho T-V et al (2015) Integration of comprehensive 3D microCT and signaling analysis reveals differential regulatory mechanisms of craniofacial bone development. Dev Biol 400(2):180–190. https://doi.org/10.1016/J.YDBIO.2015.02.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. von Cramon-Taubadel N, Frazier BC, Lahr MM (2007) The problem of assessing landmark error in geometric morphometrics: Theory, methods, and modifications. Am J Phys Anthropol 134(1):24–35. https://doi.org/10.1002/ajpa.20616

    Article  Google Scholar 

  28. Adams D, Collyer M, Kaliontzopoulou A (2018) Geometric morphometric analyses of 2D/3D landmark data. https://cran.r-project.org/web/packages/geomorph/geomorph.pdf. Accessed 22 Oct 2018

  29. Gower JC (1975) Generalized procrustes analysis. Psychometrika 40(1):33–51. https://doi.org/10.1007/BF02291478

    Article  Google Scholar 

  30. Green RM et al (2017) Developmental nonlinearity drives phenotypic robustness. Nat Commun. https://doi.org/10.1038/s41467-017-02037-7

    Article  PubMed  PubMed Central  Google Scholar 

  31. Collyer ML, Sekora DJ, Adams DC (2015) A method for analysis of phenotypic change for phenotypes described by high-dimensional data. Heredity (Edinb) 115(4):357–365. https://doi.org/10.1038/hdy.2014.75

    Article  CAS  Google Scholar 

  32. Lele S, Richtsmeier JT (1991) Euclidean distance matrix analysis: a coordinate-free approach for comparing biological shapes using landmark data. Am J Phys Anthropol 86:415–427

    Article  CAS  PubMed  Google Scholar 

  33. Lele SR, Richtsmeier JT (2001) An invariant approach to statistical analysis of shapes. Chapman & Hall, Philadelphia

    Book  Google Scholar 

  34. Klingenberg CP (2016) Size, shape, and form: concepts of allometry in geometric morphometrics. Dev Genes Evol 226(3):113–137. https://doi.org/10.1007/s00427-016-0539-2

    Article  PubMed  PubMed Central  Google Scholar 

  35. Schmitz N, Laverty S, Kraus VB, Aigner T (2010) Basic methods in histopathology of joint tissues. Osteoarthr Cartil 18:S113–S116. https://doi.org/10.1016/J.JOCA.2010.05.026

    Article  Google Scholar 

  36. Zhao Q, Eberspaecher H, Lefebvre V, de Crombrugghe B (1997) Parallel expression of Sox9 andCol2a1 in cells undergoing chondrogenesis. Dev Dyn 209(4):377–386. https://doi.org/10.1002/(SICI)1097-0177(199708)209:4<377:AID-AJA5>3.0.CO;2-F

    Article  CAS  PubMed  Google Scholar 

  37. Akiyama H, Chaboissier M-C, Martin JF, Schedl A, de Crombrugghe B (2002) The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev 16(21):2813–2828. https://doi.org/10.1101/gad.1017802

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Shibata S, Fukada K, Suzuki S, Yamashita Y (1997) Immunohistochemistry of collagen types II and X, and enzyme-histochemistry of alkaline phosphatase in the developing condylar cartilage of the fetal mouse mandible. J Anat 191(4):561–570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Schmid TM, Linsenmayer TF (1985) Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues. J Cell Biol 100(2):598–605. https://doi.org/10.1083/JCB.100.2.598

    Article  CAS  PubMed  Google Scholar 

  40. Otto F et al (1997) Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89(5):765–771. https://doi.org/10.1016/S0092-8674(00)80259-7

    Article  CAS  PubMed  Google Scholar 

  41. Takeda S, Bonnamy JP, Owen MJ, Ducy P, Karsenty G (2001) Continuous expression of Cbfa1 in nonhypertrophic chondrocytes uncovers its ability to induce hypertrophic chondrocyte differentiation and partially rescues Cbfa1-deficient mice. Genes Dev 15(4):467–481. https://doi.org/10.1101/gad.845101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Nakashima K et al (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108(1):17–29. https://doi.org/10.1016/S0092-8674(01)00622-5

    Article  CAS  PubMed  Google Scholar 

  43. Poole KES et al (2005) Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation. FASEB J 19(13):1842–1844. https://doi.org/10.1096/fj.05-4221fje

    Article  CAS  PubMed  Google Scholar 

  44. Untergasser A et al (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40(15):e115. https://doi.org/10.1093/nar/gks596

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Koressaar T, Remm M (2007) Enhancements and modifications of primer design program Primer3. Bioinformatics 23(10):1289–1291. https://doi.org/10.1093/bioinformatics/btm091

    Article  CAS  PubMed  Google Scholar 

  46. Buo AM, Tomlinson RE, Eidelman ER, Chason M, Stains JP (2017) Connexin43 and Runx2 interact to affect cortical bone geometry, skeletal development, and osteoblast and osteoclast function. J Bone Miner Res 32(8):1727–1738. https://doi.org/10.1002/jbmr.3152

    Article  CAS  PubMed  Google Scholar 

  47. Willems E, Leyns L, Vandesompele J (2008) Standardization of real-time PCR gene expression data from independent biological replicates. Anal Biochem 379(1):127–129. https://doi.org/10.1016/j.ab.2008.04.036

    Article  CAS  PubMed  Google Scholar 

  48. Plotkin LI, Speacht TL, Donahue HJ (2015) Cx43 and mechanotransduction in bone. Curr Osteoporos Rep 13(2):67–72. https://doi.org/10.1007/s11914-015-0255-2

    Article  PubMed  PubMed Central  Google Scholar 

  49. Batra N et al (2012) Mechanical stress-activated integrin α5β1 induces opening of connexin 43 hemichannels. Proc Natl Acad Sci USA 109(9):3359–3364. https://doi.org/10.1073/pnas.1115967109

    Article  PubMed  PubMed Central  Google Scholar 

  50. Shen H, Grimston S, Civitelli R, Thomopoulos S (2015) Deletion of connexin43 in osteoblasts/osteocytes leads to impaired muscle formation. J Bone Miner Res 30(4):596–605. https://doi.org/10.1002/jbmr.2389

    Article  CAS  PubMed  Google Scholar 

  51. Shen H, Schwartz AG, Civitelli R, Thomopoulos S (2020) Connexin 43 is necessary for murine tendon enthesis formation and response to loading. J Bone Miner Res 35(8):1494–1503. https://doi.org/10.1002/jbmr.4018

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Mariyan Jeyarajah for sharing his qPCR expertise, Chris Norley for help with acquiring micro-computed tomography images, and Annie Lin and Ryan Masney for their assistance in sample preparation. This work was supported by a Natural Sciences and Engineering Research Council Discovery Grant (R5211A02) to KEW and a Canadian Institute of Health Research operating Grant (148630) to DWL.

Funding

None.

Author information

Authors and Affiliations

  1. Department of Anatomy & Cell Biology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada

    Alyssa C. Moore, Elizabeth Jewlal, Kevin Barr, Dale W. Laird & Katherine E. Willmore

  2. Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Canada

    Jessica Wu & Dale W. Laird

Authors
  1. Alyssa C. Moore
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jessica Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Elizabeth Jewlal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kevin Barr
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dale W. Laird
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Katherine E. Willmore
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

AM: conceptualization, data collection, data analysis, writing—Original draft, review, and editing. JW, EJ, and KB: data collection, review, and editing. . DL: resources, review and editing, funding. KW: conceptualization, resources, writing—original draft, writing—editing and review, and validation.

Corresponding author

Correspondence to Katherine E. Willmore.

Ethics declarations

Conflict of interest

Alyssa Moore, Jessica Wu, Elizabeth Jewlal, Kevin Barr, Dale Laird, and Katherine Willmore declare that they have no conflict of interest.

Human and Animal Rights

All experiments were approved by the Animal Care Committee at the University of Western Ontario (2018-061) and abide by the guidelines of the Canadian Council on Animal Care.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Moore, A.C., Wu, J., Jewlal, E. et al. Effects of Reduced Connexin43 Function on Mandibular Morphology and Osteogenesis in Mutant Mouse Models of Oculodentodigital Dysplasia. Calcif Tissue Int 107, 611–624 (2020). https://doi.org/10.1007/s00223-020-00753-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00223-020-00753-9

Keywords

Search

Navigation