Myoblast-differentiation and mRNA/miRNA Expression in Developmental Mouse Tongue

Objectives: Tongue myogenesis is initiated by the migration of myogenic precursor cells from occipital somites to the tongue primordium, followed by myoblasts/myotubes differentiation under the guidance of multiple myogenic regulatory factors. Previous studies using gene-targeting mice disclosed the unique as well as common aspects of tongue myogenesis in comparison with skeletal musculature development. The present study aimed to gain further insight into both coding and non-coding molecular cues governing myoblast-differentiation in developmental mouse tongue. Methods: ICR mouse embryos from E9.5 through E18.5 were used to collect tongue primordia and muscle tissues. Developmental stages and cell characterization were examined histomorphologically in conjunction with immunohistochemistry. For microarray analysis of mRNAs and miRNAs (Affymetrix Mouse Genome 430A 2.0 for mRNAs; Agilent Mouse miRBase v.12.0 for miRNAs), frozen frontal sections were prepared to collect restricted tissue areas corresponding to the localization of myogenic lineage cells by laser-capture microdissection. Expression profiles of candidate mRNAs/miRNAs selected from the array-data were validated by real-time quantitative PCR.  Results: The present array analysis showed that the initial stage of tongue myogenesis at E10.5 to E11.5 was characterized by the upregulation (>5-fold) of 19 transcription factor genes including Meox1/Meox2 and Msc/Nfix as well as the major myogenic regulatory genes (Pax3, Myod1, Myf5, Myog). Of particular interest was that Nfix is known to be a marker for transition from embryonic- to fetal-myoblast in limb myogenesis. In harmony with these mRNA expression, we also ascertained alterations in expression level of 124 miRNAs (77 of >2-fold upregulated; 47 of downregulated) including known muscle-specific miRNAs such as miR-1, miR-133a, and miR-206. Computer-assisted target prediction helped link those miRNAs to stage-specific mRNA expression involved in tongue myogenic pathways. Conclusions: The present results support the theory that embryonic to fetal myoblast differentiation in tongue is driven by a temporal-spatial coupling of miRNA and mRNA expression.