Digoxigenin‐labeled RNA probes for untranslated regions enable the isoform‐specific gene expression analysis of myosin heavy chains in whole‐mount in situ hybridization. (22nd December 2022)

• Record Type: Journal Article
• Authors: Tanji, Masafumi; Wada, Keitaro; Sakamoto, Keita; Ono, Yudai; Inui, Masafumi
• Other Names: Ochi H. guestEditor.; Michiue T. guestEditor.; Kato T. guestEditor.; Zorn A. guestEditor.; Hayashi T. guestEditor.; Inoue T. guestEditor.; Kondo M. guestEditor.; Taira M. guestEditor.
• Is Part Of: Development growth and differentiation. Volume 65:Number 1(2023)
• Page Start: 48
• Page End: 55
• Publication Date: 2022-12-22
• Subjects: fiber type -- myosin heavy chain -- skeletal muscle; Embryology -- Periodicals; Developmental biology -- Periodicals; Growth -- Periodicals; 574.3
• DOI: 10.1111/dgd.12832
• Languages: English
• ISSNs: 0012-1592

Abstract

Abstract: Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain ( Myh ) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio‐temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole‐mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost‐effective and specific method for analyzing the Myh gene expression in whole‐mount samples. Digoxigenin (DIG)‐labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene‐specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG‐labeled UTR probe is a cost‐effective and versatile method to specifically detect skeletal muscle Myh genes in a whole‐mount analysis. Abstract : The expression profile of myosin heavy chain ( Myh ) isoform genes is considered as molecular signatures of skeletal muscle cell/fiber types. Immunohistochemistory has been used to detect myh gene expression pattern. In this study, we showed that DIG‐labeled probe with UTR sequence, but not protein coding sequence, could be useful for detecting isoform gene specifc myh expression pattern in both embryo and adult whole‐mount samples.