Mechanisms Of Microglossia Caused By Inactivation Of Fam20B In Neural Crest-Derived Mesenchyme

Background:Microglossia is a rare congenital malformation of the tongue that was often associated with maxillofacial and systemic genetic syndromes such as Piriformis,Mobius syndrome,and recessive pedal keratosis of the hand.However,the scarcity of clinical cases and the complex tissue composition of the tongue had slowed progress in the study of the pathogenesis of malformations.Two new technologies that have emerged in recent years had brought about a turnaround in this dilemma:1.Cre-Loxp conditional knockout systems:conditional knockout of genes of interest could explain their spatial and temporal roles in organ development,and it could also explained inter-tissue interactions in organ development based on conditional knockout of tissue-specific marker genes;2.development of high-throughput sequencing technologies:high-throughput sequencing could capture the extent of genetic alterations across the whole genome and thus provide massive amounts of genomic data that could be combined with mathematical and computer science modeling to identify the molecular features behind disease phenotypes.In our group,we found that specific knockdown of Fam20B in Cranial Neural Crest-Derived Cell（CNCC）leads to Microglossia,however,there is a lack of systematic and integrated mechanistic studies on the cause.Objective:A conditional knockout mouse Wnt1-Cre;Fam20B^f/fwas constructed using the Cre-loxp system to systematically and integrally analyze the pathological mechanism of microglossia caused by Fam20B deletion in CNCC based on histological characterization and transcriptome sequencing,combined with bioinformatics and cybernetics analysis.Methods:Adult mice of two genotypes(Wnt1-Cre;Fam20B^f/+vs.Fam20B^f/f)were crossed to produce Wnt1-Cre;Fam20B^f/fmice,pregnant mice were killed to obtain Wnt1-Cre;Fam20B^f/fembryos,and tongue tissue was extracted for fixation,dehydration,embedding,and sectioning.Immunohistochemical assay of Myosin for observation of morphological changes in lingual muscle fibers and determination of Microglossia.Myosin/KI67 immunodouble fluorescence staining assay was used to observe changes in the number of myofibroblasts in tongue,and in situ hybridization assay for Myomaker,a myofiber fusion marker,was used to detect tongue myofiber fusion in embryonic mice.In situ hybridization assays for the tendon markers:Scx,Tenascin-C and Colla1 were used to detect the development of tongue tendons in embryonic mice.m RNA from tongue tissue of E11.5 and E12.5 was extracted for RNA sequencing and thus whole transcriptome wide detection of altered gene expression levels.T-test was used to identify the differentially expressed genes between Wnt1-Cre;Fam20B^f/fand control.Gene set enrichment analysis（GSEA）was used to identify pathways or gene set enrichment trends for differential gene expression rank,and GO and KEGG were used to identify the pathway enrichment of specific gene sets.The Random Walk Restart algorithm was used to confirm the construction of co-regulatory networks for lingual tendon cell development and myofibroblast fusion and to identify the perturbation of Fam20B on them,and three network centrality parameters（Degree,Closeness Centrality and Betweeness Centrality）were used to confirm the importance of nodes in the network.Immunohistochemical experiments with phosphorylated Erk1/2and Akt1 were used to verify the expression of network key genes（Hub genes）in the tongue.Results:（1）Immunohistochemical staining for the mature myofibrillar marker Myosin revealed a reduced number,shorter length,and disorganized arrangement of myofibrils in the tongue of Wnt1-Cre;Fam20B^f/fmice,suggesting abnormal pattern formation and morphogenesis of the tongue muscle;（2）Significantly reduced myofibrillar fusion marker Myomaker in the tongue of Wnt1-Cre;Fam20B^f/fmice expression and narrowed expression range suggested abnormal myofiber fusion in the tongue;（3）Dysregulation of the Tgfβ-based pathway network（also including Wnt,Fgf and Shh）controlling myofiber fusion in the tongue of Wnt1-Cre;Fam20B^f/fmice was demonstrated by downregulated gene expression in Tgfβand Fam20B network perturbation experiments,in which reduced activity of the Shh and Wnt pathways were further verified by overall in situ hybridization and X-GAL staining,respectively;（4）The abnormal expression pattern of Scx in the tongue of Wnt1-Cre;Fam20B^f/fmice suggested that the tongue tendon and dorsal submucosal lingual aponeurosis were dysplastic;（5）Scx,Myomaker,and Myomixer were selected as seed nodes to obtain the top 500 genes of their respective scores after iterating to a steady state using the random walk restart algorithm,and 145 genes were obtained by taking the intersection set and mapping them to the global（protein interaction network PPI and gene regulatory network GRN）biological network to form a co-regulatory network for tongue tendon cell development and myofibroblast fusion.（6）Fam20B were selected as seed node to obtain the top 500genes and intersect them with the regulatory network constructed in step（5）to obtain33 Fam20B perturbed genes,among which the Hub genes Mapk1 and Akt1 were verified by immunohistochemistry.Conclusion:（1）Fam20B deficiency in CNCC leads to shortening of muscle fibers by reducing lingual myocyte fusion;（2）The pattern and morphology of the internal transverse lingual muscle is essential for maintaining the development of the lingual septal tendon and submucosal lingual aponeurosis;（3）Fam20B deficiency in CNCC disrupts the co-regulatory network of lingual tendon cell development and myofibroblast fusion leading to Microglossia.