Figure 2: Minion is required for skeletal muscle development.
From: The microprotein Minion controls cell fusion and muscle formation
(a) Strategy for CRISPR/Cas9 mutagenesis of the gm7325/Minion locus using a dual sgRNA approach. Grey box, Minion ORF; white box, non-coding exons; sgRNA, single gRNA; Fwd and Rev, forward and reverse genotyping primers. (b) Left: representative genotyping PCR of Minion wild type (+/+) and heterozygous (Δ/+) mice carrying the 135-bp deletion depicted in a. n=20 (more than 300 total adult mice of both sex). Right: representative sequence traces. Black line indicates 5′ boundary of the deletion. WT, wild-type allele; KO, knockout allele (135-bp deletion). (c) Photographs of skinned Minion+/+ and MinionΔ/Δ P0 mice. Cyan arrows and Blue asterisks indicate forelimb and intercostal musculature, respectively. Three litters of embryos. (d) Histological and immunofluorescence (IF) analyses of embryonic tongue skeletal muscle from E18.5 embryos. Yellow arrowheads and yellow arrows indicate multinuclear myofibres and unfused differentiating elongating myoblasts, respectively. Top row: hematoxylin and eosin (H&E) staining of sagittal tongue sections. Inset demonstrates the originating region and orientation of the provided tongue sections. Bottom row: Immunofluorescence staining for the muscle marker MHC (red), with DNA counterstain DAPI (4′,6-diamidino-2-phenylindole; blue). Insets demonstrate magnification of the boxed areas. Three litters of embryos. (e) Histological images of H&E-stained E19.5 forelimb longitudinal sections of indicated genotypes. Two litters of embryos. (f) Immunofluorescence images of forelimb longitudinal sections for E19.5 embryos with indicated genotypes. MHC (red) and DAPI (blue) staining are shown. Two litters of embryos. (d–f) Paraffin-embedded embryos of different stages were examined. (c–f) Embryos from the same litter were compared with 1–2 embryos for each genotype in each experimental repeat. Scale bars, 1 mm (c), 100 μm (d–f).
