Inactivation Of Fam20B In Neural Crest Mesenchyme Leads To Microglossia

Background:The tendons and connective tissues of mammalian tongue are CNCC-derived mesenchyme cells,while tongue myogenic cells migrated from occipital somites and differentiated into muscles.The development of connective tissue and tongue muscle are not completely independent of each other,but require the reciprocal interaction between them.PGs are among the most important structural and functional biomacromolecules in connective tissue extracellular matrix?ECM?.They interact with numerous growth factors,chemokines and cell surface receptors through their GAG chains to participate in several cell functional properties,such as cell adhesion,migration,proliferation and differentiation.Fam20B is a newly identified kinase phosphorylating the xylose in the common linkage region connecting the GAGs to the protein core of PGs,which is essential for the subsequent elongation and stability of GAGs.Studies have shown that inactivation of Fam20B led to the loss of GAG chains in multiple PGs?including heparan sulfate,chondroitin sulfateand dermatan sulfate?and defects in skeleton and tooth.Objective:In this study,we used Wnt1-Cre;Fam20B^f/fmice,in which Fam20B was specifically knocked out in the mesenchymal cells derived from CNCC,to explore the role of GAG chains in the development of tongue connective tissue and the mechanism regulating the interaction between tongue connective tissue and muscles.Methods:Wnt1-Cre;Fam20B^f/+mice were crossbred with Fam20B^f/fmice to obtain timed Wnt1-Cre;Fam20B^f/fembryos and their wild type littermates?WT?,proceeding tissue fixation,dehydration,embedding and section.The accumulation of GAG chains in tongue was observed by Alcian blue staining and tongue morphology of embryos was observed by Masson staining.Immunohistochemistry assay was performed to evaluate the tongue muscle fiber development by using primary antibodies against Myo D,Myogenin and Myosin,and to further observe the development of tongue muscle through Myf5-Cre;Fam20B^f/fmice.Real-Time PCR was performed for the transcription of specific markers during the development of connective tissue in tongue.Cell proliferation and apoptosis of tongue was detected by Brd U labeling assay and TUNEL assay.The Lac Z staining of Wnt1-Cre;Fam20B^f/f;BATGAL mice was applied to test the activity of Wnt/?-catenin signaling.Masson staining of Wnt1-Cre;Fam20B^f/f;Ctnnb1^ex3fmice,in which Wnt/?-catenin signaling was constitutively activated in CNCC-derived cells,was generated to rescue the microglossia of Wnt1-Cre;Fam20B^f/fmice.Results:?1?The intensity of Alcian blue staining was apparently reduced in E13.5 Wnt1-Cre;Fam20B^f/ftongue mesenchyme comparing with WT littermates,which suggested that the GAG chains accumulation was decreased in mutant embryos.?2?The size of the Wnt1-Cre;Fam20B^f/ftongue was decreased.Masson staining showed that mutant tongue was reduced both in length and width.In the sagittal plane,the free end of the tongue became shorter,suggesting the shortened sublingual ligament.And the septum of Wnt1-Cre;Fam20B^f/ftongue became narrower,suggesting the degeneration of tongue connective tissue,especially the tendon.?3?Immunohistochemistry assay showed there was no obvious abnormalities of Myo D,Myogenin and Myosin staining in Wnt1-Cre;Fam20B^f/ftongue muscle fibers comparing with WT.However,in sagittal plane,in anterior part of the Wnt1-Cre;Fam20B^f/ftongue,the connective tissue separating transverse fibers into lobes was diminished.In posterior part,the fibers of vertical muscles and the superior longitudinal muscles crossover without clear boundary.Masson staining showed that Myf5-Cre;Fam20B^f/ftongue showed no difference comparing with the WT group.These results suggested that the loss of Fam20B in CNCC-derived cells did not affect the development of the tongue muscles,but only impaired the development of tongue connective tissue.?4?Real-Time PCR results showed that the m RNA expression of tendon development markers Scx and Tnmd in Wnt1-Cre;Fam20B^f/fmice were significantly decreased.The expression of Col4 in the tongue and Tcf4,an early specialized marker of irregular connective tissue,were also significantly reduced in the tongue of Wnt1-Cre;Fam20B^f/fmice,all of which suggesting that the decreased GAG chains in tongue mesenchyme affected the normal development of connective tissue and further caused tendon degeneration in the late stage of tongue development.?5?Brd U labeling showed that at E13.5 Wnt1-Cre;Fam20B^f/ftongue,the cell proliferation in tongue muscles showed no difference,while the mesenchymal cell proliferation in septum was significantly decreased,suggesting that CNCC-derived connective tissue cell proliferation was affected.Meanwhile,loss of Fam20B in CNCCs did not affect tongue cell apoptosis.?6?Wnt/?-catenin signaling activity was down-regulated in Wnt1-Cre;Fam20B^f/f;BATGAL tongue.At the coronal plane,the Lac Z activity was significantly reduced in tongue connective tissues,especially in septum of posterior part of tongue,implying that inactivation of Fam20B in neural crest mesenchyme could inhibit Wnt/?-catenin signaling activity.?7?Masson staining of Wnt1-Cre;Fam20B^f/f;Ctnnb1^ex3fmice showed neither microglossia nor cleft palates,implying that tongue dysplasia in Wnt1-Cre;Fam20B^f/fwas related to reduced Wnt/?-catenin signaling,but it still needed more repeated investigations to test our hypothesis.Conclusion:?1?The loss of Fam20B in CNCC-derived cells does not affect the development of the tongue muscles,but only impairs development of tongue connective tissue by inhibiting its cell proliferation.?2?The decrease of GAG chains in tongue mesenchyme leads to the abnormal development of connective tissue by down regulating Wnt/?-catenin signaling activity,which eventually results in microglossia.