June 1, 2009;
Muscular dystrophy candidate gene FRG1 is critical for muscle development.
The leading candidate gene responsible for facioscapulohumeral muscular dystrophy (FSHD) is FRG1
(FSHD region gene 1). However, the correlation of altered FRG1
expression levels with disease pathology has remained controversial and the precise function of FRG1
is unknown. Here, we carried out a detailed analysis of the normal expression patterns and effects of FRG1
misexpression during vertebrate embryonic development using Xenopus laevis. We show that frg1
is expressed in and essential for the development of the tadpole
morpholino injection disrupted myotome
organization and led to inhibited myotome
growth, while elevated FRG1
led to abnormal epaxial and hypaxial muscle
formation. Thus, maintenance of normal FRG1
levels is critical for proper muscle
development, supportive of FSHD disease models whereby misregulation of FRG1
plays a causal role underlying the pathology exhibited in FSHD patients. Developmental Dynamics 238:1502-1512, 2009. (c) 2008 Wiley-Liss, Inc.
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Figure 1. Expression of FRG1 transcript and protein during development. A: Analysis of Xenopus laevis frg1 transcript levels across developmental stages using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). B-G: In situ hybridization using the Xenopus laevis frg1 transcript at stage (St.) 26 (B), St. 32 (C), and St. 38 (E,F). Sense frg1 controls are shown for St. 32 (D) and St. 38 (G). Arrowheads point to migrating hypaxial muscle. H-J: FRG1 immunostaining on St. 18 (H), St. 24 (I), and St. 38 (J) embryos showing FRG1 antibody (upper) and secondary antibody alone control (lower). K,L: Stage 30 FRG1 immunostained embryos were sectioned sagitally (K) or transversely (L). Arrows point to aligned nuclei in the somite stained with FRG1 antibody. np, neural plate; ps, pronephric sinus; nt, neural tube; nc, notochord; is, intersomitic region.
Figure 4. Decreased expression of myotome markers, decreased segmentation, and loss of hypaxial muscle in FRG1-depleted embryos. A,B: Stage 18 embryos injected (i, injected side) with either 40 ng of FMO1 or 40 ng of CMO respectively, stained for pax3 (dm dermomytome). C: Transverse section of a pax3-stained embryo injected with 40 ng of FMO1 showing less organized pax3 expression on the injected side (i) in the dermomyotome (arrowheads). D,E: myoD staining of stage 18 embryos injected with 20 ng of FMO1 and 40 ng of CMO, respectively. F: Graph showing percent stage 20 embryos with reduced pax3 in the dermomyotome and diffuse myoD staining. G,H: pax3 staining of injected (i) and uninjected sides of stage 34 embryos injected with 20 ng of FMO1, with close-up view of hypaxial muscle region. J,K: myoD staining of injected and uninjected sides of stage 34 embryos injected with 20 ng of FMO1. I,L: pax3 and myoD staining, respectively, of embryos injected with 40 ng of CMO. M,N: Vimentin antibody staining of injected (20 ng of FMO1) and uninjected side embryo (pm, pronephric mesenchyme). O: Percentage of embryos with defects in segmentation, hypaxial muscle, and mesenchyme with total numbers of embryos analyzed above each bar. P: Embryo injected with 40 ng of FMO1 and probed for myf5 shows no change in myf5 expression levels, despite a clear reduction in myotome width (double head arrow).
Figure 6. Elevated FRG1 leads to somite and hypaxial muscle disruptions. A,B: Stage 20 embryos injected with 1 ng of frg1 were analyzed by in situ hybridization with pax3 (A) or myoD (B). C,D: Transverse sections of pax3 and myoD stained embryos are shown in C and D, respectively. The injected sides are denoted by ldquo i rdquo . E-J: Somite disruptions and hypaxial muscle were analyzed in stage 36 embryos injected with 500 pg of frg1 (E,I) and 1 ng of frg1 (G) compared to the uninjected sides F, J, and H, respectively, by myoD (E-H) or pax3 (I,J) in situ hybridization. A graphic summary of somite and hypaxial abnormalities for 500 pg of frg1, 1 ng of frg1, and tracer is shown in K.
vim (vimentin) gene expression in Xenopus laevis embryos, NF stage 34, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up. Image extracted from XB-IMG-25099
A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus.