Mandibular Explant Assay for Investigating Extrinsic Stimuli on Bone and Cartilage Development   

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A brief version of this protocol appeared in:
Scientific Reports
Aug 2017


A major issue in developmental biology is to determine how embryonic tissues respond to molecular signals in a timely manner and given the position-restricted instructions during morphogenesis, of which Meckel’s cartilage is a classical example. The ex-vivo explant model is a practical and convenient system that allows investigation of bone and cartilage responses to specific stimuli under a controlled manner that closely mimics the in vivo conditions. In this protocol, the explant model was applied to test whether Meckel’s cartilage and surrounding tissues are responsive to the Endothelin1 (Edn1) signaling molecule and whether it would rescue the phenotype of genetic mutations. The system allows a high degree of manipulation in terms of the concentrations of exogenous compounds added to the explant, time points with regards to measuring mandibular development, and the method of application of exogenous molecules and teratogens.

Keywords: Ex-vivo mandibular explant, Craniofacial development, Mandibular dysplasia, Mandibular hypoplasia, Agnathia, Mandibular patterning, Meckel’s cartilage, Embryonic development


Craniofacial malformations are among the most frequent congenital birth defects in humans (Miettinen et al., 1999). Many of these malformations occur during facial morphogenesis, a complex multi-step process in which cranial neural crest cells migrate to pharyngeal arches to give rise to many facial structures (Jin et al., 2011).

Both variations within specific genes as well as gene-gene interactions can lead to craniofacial deformations. Mutations in IRF6, a gene that contributes to the formation of ectoderm and epithelium in the head and face, can result in cleft lip, cleft palate, and mandibular abnormalities. In comparison to IRF6, the TWIST1 gene regulates neural tube closure during embryonic development and cranial suture fusion during skull development. Mutations in TWIST1 can cause craniosynostosis, mandibular hypoplasia, and cleft palate (Fakhouri et al., 2017). Inhibition or alteration of IRF6 and TWIST1 expression can be done similar to the methods performed by Miettinen et al. (1999) with EGF receptors in order to examine their roles in craniofacial development. However, difficulties arise in in vivo experiments when the study begins to incorporate genetic interactions and rescue experiments of two or more allelic mutations. In our recent study, the genetic interaction between Irf6 and Twist1 causes severe mandible abnormality and cleft of the secondary palate in the mouse model (Fakhouri et al., 2017).

Various studies have used in vivo experiments to test for the expression of various genes involved in craniofacial development. The presence of TGF-β subtypes was studied by using an ex vivo culture model in a serumless, chemically defined medium during mandibular morphogenesis (Chai et al., 1994). Similarly, a study used Alcian blue staining of cultured mandible explants to examine Meckel’s cartilage during morphogenesis in Egfr-/- embryos (Miettinen et al., 1999). A combination of the methodologies from these studies, including an ex vivo mandibular explant described in this report, is useful to characterize the phenotype and signaling pathway in mammalian systems.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Jiang, J., Bertol, J. W. and Fakhouri, W. D. (2017). Mandibular Explant Assay for Investigating Extrinsic Stimuli on Bone and Cartilage Development. Bio-protocol 7(23): e2641. DOI: 10.21769/BioProtoc.2641.

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