Expression of DUX4 in zebrafish development recapitulates facioscapulohumeral muscular dystrophy

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy characterized by an asymmetric progressive weakness and wasting of the facial, shoulder and upper arm muscles, frequently accompanied by hearing loss and retinal vasculopathy. FSHD is an autosomal dominant disease linked to chromosome 4q35, but the causative gene remains controversial. DUX4 is a leading candidate gene as causative of FSHD. However, DUX4 expression is extremely low in FSHD muscle, and there is no DUX4 animal model that mirrors the pathology in human FSHD. Here, we show that the misexpression of very low levels of human DUX4 in zebrafish development recapitulates the phenotypes seen in human FSHD patients. Microinjection of small amounts of human full-length DUX4 (DUX4-fl) mRNA into fertilized zebrafish eggs caused asymmetric abnormalities such as less pigmentation of the eyes, altered morphology of ears, developmental abnormality of fin muscle, disorganization of facial musculature and/or degeneration of trunk muscle later in development. Moreover, DUX4-fl expression caused aberrant localization of myogenic cells marked with α-actin promoter-driven enhanced green fluorescent protein outside somite boundary, especially in head region. These abnormalities were rescued by coinjection of the short form of DUX4 (DUX4-s). Our results suggest that the misexpression of DUX4-fl, even at extremely low level, can recapitulate the phenotype observed in FSHD patients in a vertebrate model. These results strongly support the current hypothesis for a role of DUX4 in FSHD pathogenesis. We also propose that DUX4 expression during development is important for the pathogenesis of FSHD.

DUX4 induces a transcriptome more characteristic of a less-differentiated cell state and inhibits myogenesis

“…DUX4-fl and splice variants inhibit myoblast differentiation (Bosnakovski et al, 2008b;Snider et al, 2009), and DUX4-fl is also apoptotic (Bosnakovski et al, 2008b;Mitsuhashi et al, 2013;Wallace et al, 2010). DUX4-fl contains double homeobox DNA-binding domains and an evolutionarily conserved peptide sequence at the C-terminus (Clapp et al, 2007) that acts as a strong transcriptional activation domain (Kawamura-Saito et al, 2006).…”

“…Mutation of a DUX4 homeodomain or competitive inhibition by shortened DUX4 splice variants inhibits DUX4 target gene activation and abrogates DUX4-induced cell death (Ferri et al, 2015;Geng et al, 2012;Mitsuhashi et al, 2013;Wallace et al, 2010). Although DUX4 binding motifs have been identified (Dixit et al, 2007;Ferri et al, 2015;Geng et al, 2012;Young et al, 2013;Choi et al, 2016), and ChIP-Seq performed (Geng et al, 2012;Choi et al, 2016), a set of target genes that explains both anti-myogenic and apoptotic phenotypes induced by DUX4 has not been comprehensibly defined.…”

A Pentanucleotide ATTTC Repeat Insertion in the Non-coding Region of DAB1, Mapping to SCA37, Causes Spinocerebellar Ataxia

“…experimentation. Zebrafish were crossed for the generation of embryos for RNA injections; 5 nL in-vitro-synthesized RNA for each of the normal N(ATTTT) 7 and N(ATTTT) 139 alleles and the pathological ins(ATTTC) 58 allele and control RNA was microinjected in the yolk of 1-to 2-cell stage zebrafish embryos 27 at a concentration of 100 ng/mL. Each RNA sequence was microinjected three times in at least 200 embryos per injection.…”

Altered Tnnt3 characterizes selective weakness of fast fibers in mice overexpressing FSHD region gene 1 (FRG1)

“…To better understand the function of these genes and their potential role in FSHD pathogenesis, animal models have been generated (13,20). Although the misregulation of DUX4 expression in zebrafish, whose genome does not contain a DUX4 homologue, seems to recapitulate some of the phenotypes seen in human FSHD patients (29), overexpression of DUX4 in mice, at extents similar to that of FSHD patients, does not show any obvious muscle phenotype (20). On the other hand, FRG1 is a highly conserved gene whose proper expression has been shown to be crucial for muscle development in vertebrates and invertebrates (17,24).…”