Zebrafish mbnl mutants model physical and molecular phenotypes of myotonic dystrophy

ABSTRACT The muscleblind RNA-binding proteins (MBNL1, MBNL2 and MBNL3) are highly conserved across vertebrates and are important regulators of RNA alternative splicing. Loss of MBNL protein function through sequestration by CUG or CCUG RNA repeats is largely responsible for the phenotypes of the human genetic disorder myotonic dystrophy (DM). We generated the first stable zebrafish (Danio rerio) models of DM-associated MBNL loss of function through mutation of the three zebrafish mbnl genes. In contrast to mouse models, zebrafish double and triple homozygous mbnl mutants were viable to adulthood. Zebrafish mbnl mutants displayed disease-relevant physical phenotypes including decreased body size and impaired movement. They also exhibited widespread alternative splicing changes, including the misregulation of many DM-relevant exons. Physical and molecular phenotypes were more severe in compound mbnl mutants than in single mbnl mutants, suggesting partially redundant functions of Mbnl proteins. The high fecundity and larval optical transparency of this complete series of zebrafish mbnl mutants will make them useful for studying DM-related phenotypes and how individual Mbnl proteins contribute to them, and for testing potential therapeutics. This article has an associated First Person interview with the first author of the paper.

. Effects of mbnl mutations on mbnl mRNA levels.
A-C. qRT-PCR showing the levels of (A) mbnl1, (B) mbnl2, and (C) mbnl3 mRNAs in WT and mbnl mutant 5 days post fertilization whole larval zebrafish. D-F. qRT-PCR showing relative levels of (D) mbnl1 mRNA, (E) mbnl2 mRNA, and (F) mbnl3 mRNA in WT 5 dpf whole larvae, adult skeletal muscle, heart, brain, cornea, and intestine. G-J. RNA-Seq data showing the normalized counts of WT mbnl1, mbnl2, and mbnl3 RNAs in unfertilized eggs (from Mehjabin et al., 2019), and (H) mbnl1, (I) mbnl2, and (J) mbnl3 mRNAs in WT and mbnl mutant adult zebrafish skeletal muscle. Data information: In (A-J) data are presented as mean ± SEM. Each dot represents RNA from a pool of 5 larvae or one adult fish. Data were analyzed by ordinary one-way ANOVA with Tukey's multiple comparisons test. Data bars that do not share a letter above them are significantly different from one another. Raw data and statistical analysis details are in Table S5.   Figure S2. Single mbnl mutant zebrafish were not decreased in size. A-C. Standard length of (A) 83 dpf clutchmates from an incross of 1 +/B fish, (B) 83 dpf clutchmates from an incross of 2 +/A fish, and (C) 78 dpf clutchmates from an incross of 3 +/C fish. Example images of fish of each genotype are next to each graph. Scale bar = 10 mm. D. Standard length of 7 dpf larval fish. Data information: In (A-D) mbnl1 mutant alleles are denoted as 1 A and 1 B , mbnl2 alleles as 2 A , and mbnl3 alleles as 3 A , 3 B , and 3 C . Data are presented as mean ± standard deviation. Each dot represents one fish. Data were analyzed by ordinary one-way ANOVA with Tukey's multiple comparisons test. Data bars that do not share a letter above them are significantly different from one another. Raw data and statistical analysis details are in Table S5.  Figure S4. The introns surrounding zebrafish mbnl1 exon 5 contain potential Mbnl protein binding sites.
Zebrafish mbnl1 exon5 (bold upper case letters) and its surrounding intronic sequences (lower case letters) were aligned with orthologous human sequences using the EMBOSS Water program. Potential YGCY (Y=pyrimidine) Mbnl protein binding sites are indicated in red.   (Tabs 5A and S7A of Table S5). Data information: In (A-F) mbnl1 mutant alleles are denoted as 1 A and 1 B , mbnl2 alleles as 2 A , and mbnl3 alleles as 3 A , 3 B , and 3 C . Representative RT-PCR gels are shown above each graph with band sizes in bp shown on the left. White boxes represent constitutive exons and gray boxes represent alternative exons. Dividing lines indicate samples run on separate gels. Each dot represents RNA from one adult fish or a pool of five larvae. Data are presented as mean ± SEM. Data were analyzed by ordinary oneway ANOVA with Tukey's multiple comparisons test. Data bars that do not share a letter above them are significantly different from one another. Raw data and statistical analysis details are in Table S5.  Table S5). Data information: In (A) ρ is the Spearman's rank correlation coefficient. In (A-F) homozygous mbnl1 mutant alleles are denoted as 1 A and 1 B , mbnl2 alleles as 2 A , and mbnl3 alleles as 3 A , 3 B , and 3 C . Representative RT-PCR gels are shown above each graph with band sizes in bp shown on the left. White boxes represent constitutive exons and gray boxes represent alternative exons. Each dot represents RNA from one fish. Data are presented as mean ± s.e.m. Raw data and statistical analysis details are in Table S5.  Figure S8. Zebrafish aplp2 exon 7 and its surrounding introns contain potential Mbnl protein binding sites. Zebrafish aplp2 exon 7 (bold upper case letters) and its surrounding intronic sequences (lower case letters) were aligned with orthologous human sequences using the EMBOSS Water program. Potential YGCY (Y=pyrimidine) Mbnl protein binding sites are indicated in red.  Figure S9. Zebrafish atp6v1h exon 6 and its surrounding introns contain potential Mbnl protein binding sites. Zebrafish atp6v1h exon 6 (bold upper case letters) and its surrounding intronic sequences (lower case letters) were aligned with orthologous human sequences using the EMBOSS Water program. Potential YGCY (Y=pyrimidine) Mbnl protein binding sites are indicated in red. igure S10. The introns surrounding zebrafish atp2a1l exon 23 contain potential Mbnl protein binding sites. Zebrafish atp2a1l exon 23 (bold upper case letters) and its surrounding intronic sequences (lower case letters) were aligned with orthologous human sequences using the EMBOSS Needle program. Potential YGCY (Y=pyrimidine) Mbnl protein binding sites are indicated in red.  Figure S13. Differentially expressed genes in zebrafish mbnl mutants and human DM1 myotubes belong to overlapping gene ontology pathways.
Significantly enriched gene ontology (GO) pathways for genes that were differentially expressed in 1 B/B ; 2 A/A ;3 C/C vs. WT adult zebrafish skeletal muscle. Red asterisks indicate those GO pathways that were also enriched in differentially expressed genes between human DM1 vs. WT myotubes. See Table S6 for complete lists of differentially expressed genes and enriched GO pathways in zebrafish and human. Data information: p. adjust indicates adjusted p-value. Count indicates number of differentially expressed genes belonging to each GO pathway. Raw data are in Table S6. Data information: mbnl1 mutant alleles are denoted as 1 A and 1 B , mbnl2 alleles as 2 A , and mbnl3 alleles as 3 A , 3 B , and 3 C . In (A-I) each dot represents one fish and data are presented as mean ± s.e.m. In (A,B,D,E,G,H) data were analyzed by ordinary one-way ANOVA with Tukey's multiple comparisons test and in (C,F,I) data were analyzed by an unpaired Student's t-test. Data bars that do not share the same letter above them are significantly different from one another. Raw data and statistical analysis details are in Table S5. For each zebrafish mbnl mutant used in this study, this table lists the University of Oregon allele number, exon that was mutated within the target gene, guide RNA (gRNA) used to generate the mutant, forward primers, reverse primers, and restriction sites used to genotype the mutants by restriction fragment length polymorphism, ZFIN Gene ID for the targeted gene, GRCz11 genome coordinates and coding sequence of the targeted exon, and predicted protein coding sequence. Sequences that are changed compared to WT are indicated in bold. Sequences of primers from this table are listed in Table S1.
Click here to download Table S2   Table S3. Zebrafish single, double, and triple homozygous mbnl mutants are viable to adulthood.
For each zebrafish single, double, and triple homozygous mutant mbnl genotype, this table lists the cross that was used to generate the genotype, the percent of adult offspring expected of the genotype according to Mendelian genetics, and the actual percent and number of adult offspring observed with the genotype.
Click here to download Table S3   Table S4. Significantly misregulated alternative splicing events identified by RNA-Seq in zebrafish mbnl mutants.
Each tab contains a list of alternative splicing events (cassette exon, mutually exclusive exon, retained intron, alternative 5' splice site, or alternative 3' splice site) that were identified by RNA -Seq analysis as being significantly misregulated in adult zebrafish mbnl mutant skeletal muscle compared to WT. All genome coordinates are from the GRCz11 genome assembly, except for WT vs. 1B/B;2A/A;3C/C, for which a list of significantly misregulated cassette exons identified from a preliminary analysis of the GRCz10 genome assembly is also shown. Complete RNA-Seq data are available from NCBI GEO #GSE145270. Each tab contains the source data and details of statistical analysis for the indicated figure.
Click here to download Table S5   Table S6. Differential gene expression and gene ontology (GO) analysis of zebrafish adult skeletal muscle WT vs. 1B/B;2A/A;3C/C and unaffected vs. DM1 human myotubes The first two tables contain lists of genes that were significantly differentially expressed in WT vs. 1B/B;2A/A;3F/F adult zebrafish skeletal muscle or in unaffected vs. DM1 human myotubes. The second two tables contain lists of significantly enriched GO terms for the differentially expressed zebrafish or human genes.