Biology subjects

Amacher, S. L.

Publications and source records attributed to Amacher, S. L..

6 recordsLinked to original sources

bioRxiv2025

Validation of Duchenne muscular dystrophy candidate modifiers using a CRISPR-Cas9-based approach in zebrafish

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease for which there is no cure. There is a critical need for additional therapeutics. Human genome-wide association studies (GWAS) have identified candidate DMD genetic modifiers that could serve as therapeutic targets. Because many GWAS-identified single nucleotide polymorphisms (SNPs) lie in noncoding, putative regulatory regions, it can be challenging to identify which gene(s) are regulated by these SNPs and how gene expression is altered to modify disease severity even with extensive in silico modeling. We analyzed expression of zebrafish orthologs of putative DMD modifiers and showed almost all are comparably expressed in wild-type and dmd mutant zebrafish at three different stages of disease. To model decreased expression of candidate modifiers, we pursued a zebrafish CRISPR-based screening approach, which we validated by testing zebrafish orthologs of two extensively studied DMD modifiers, LTBP4 and THBS1. We then tested candidates from the most recent GWAS and demonstrate that galntl6, man1a1, etaa1a;etaa1b, and adamts17 are bona fide DMD modifiers. Our findings demonstrate the utility of zebrafish for DMD genetic modifier screening and characterizing modifier function. Summary StatementZebrafish CRISPR-based screening approach validates new genetic modifiers of Duchenne muscular dystrophy.

genetics↗

bioRxiv2025

Pnrc2 promotes rapid mRNA decay and coordinately supports early development with P-body factors Ddx6 and Ddx61

Somitogenesis is controlled by the segmentation clock, a molecular oscillator that controls periodic gene expression in unsegmented mesoderm and is regulated by a negative feedback loop driven by Hes/Her transcriptional repressors. In zebrafish, Pnrc2 is required for decay of her1 transcript and additional oscillatory gene transcripts. Despite accumulation of numerous mRNAs including those encoding developmental regulators, overt embryonic phenotypes are absent in pnrc2 mutants. Our previous work suggested that accumulated mRNAs are not translated in pnrc2 mutants, though the underlying mechanism(s) was unknown. We show here that many overexpressed transcripts in pnrc2 mutants have shortened poly(A) tails and are disengaged from ribosomes, and that deadenylation inhibition leads to somite defects in pnrc2 mutants. Transcripts encoding the P-body-associated protein, Ddx61, are overexpressed and engaged with ribosomes in pnrc2 mutants, leading to an increase in Ddx61 protein that may compensate against the negative effects of transcript accumulation. Co-depletion of Ddx61 and Ddx6 in pnrc2 mutants increases her1 accumulation and leads to pleiotropic defects. Together, our results show that multiple post-transcriptional mechanisms ensure proper translation when mRNA decay is inhibited.

developmental biology↗

bioRxiv2024

Mylpf dosage is proportionate to fast-twitch myofibril size in the zebrafish embryo

Muscle becomes stronger by expanding sarcomeres from the cells edge to its center. Here we investigate how Mylpf (Myosin Light Chain Phosphorylatable Fast) abundance impacts sarcomere growth. The two zebrafish Mylpf genes (mylpfa and mylpfb) are exclusively expressed in fast-twitch muscle, with mylpfa expressed more abundantly than mylpfb. Mutations in both genes cause full mRNA and protein loss, and loss of sarcomere formation. The degree of sarcomere reduction in fast-twitch muscle is predicted by Mylpf protein dosage using a high-slope Hill equation. The sarcomere defect may be driven by thick filament localization, which is impaired in mylpfa-/-and obliterated in mylpfa-/-;mylpfb-/-. Slow-twitch muscle is spared, becoming larger and more active in the mutants. The mylpfa-/- defects can be rescued by Mylpfa-GFP, Mylpfb-GFP, and by human MYLPF-GFP with equivalent efficiency, but not by a MYLPF protein variant that causes Distal Arthrogryposis. These effects indicate that Mylpf dosage strengthens muscle by mediating myofilament localization, and thereby sarcomere and myofibril growth.

developmental biology↗

bioRxiv2021

SIX1 is a master regulator of the Rhabdomyosarcoma undifferentiated state

Rhabdomyosarcoma (RMS) is a pediatric skeletal muscle sarcoma characterized by the expression of the myogenic-lineage transcription factors (TF) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their function. Here, we demonstrate that the developmental TF, SIX1, is highly expressed in RMS and is critical to maintain a muscle progenitor-like state. SIX1 loss induces terminal differentiation of RMS cells into myotube-like cells and dramatically impedes tumor growth in vivo. We show that SIX1 maintains the RMS undifferentiated state by controlling enhancer activity and MYOD1 occupancy at loci more permissive to tumor growth over terminal muscle differentiation. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status in RMS and predicts RMS progression in human disease. Our findings demonstrate a master regulatory role for SIX1 in the repression of RMS differentiation via genome-wide alterations in MYOD1-mediated transcription. HighlightsO_LISIX1 prevents differentiation in RMS while it promotes differentiation during normal development C_LIO_LIFN-RMS are highly dependent on SIX1 for growth in both zebrafish and mouse xenograft models C_LIO_LILoss of SIX1 alters the transcriptional landscape of RMS cells, inducing a growth to differentiation switch C_LIO_LISIX1 knockdown in FN-RMS causes reduced super enhancer-based activity at stem-related genes and enhanced MYOD1 binding to differentiation loci, resulting in the activation of a myogenic differentiation program C_LIO_LIA gene signature derived from SIX1 loss strongly correlates with myogenic differentiation status and is predictive of advanced RMS. C_LI

cancer biology↗

bioRxiv2020

Mutations in MYLPF cause a novel segmental amyoplasia that manifests as distal arthrogryposis

We identified ten persons in six consanguineous families with Distal Arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T, p.(Cys157Phe) or c.469T>C, p.(Cys157Arg)) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A, p.(Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly-inherited DA, a c.98C>T, p.(Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb.

genetics↗

bioRxiv2019

Stop codon-proximal 3′UTR introns in vertebrates can elicit EJC-dependent Nonsense-Mediated mRNA Decay

The Exon Junction Complex (EJC) regulates many steps in post-transcriptional gene expression and is essential for cellular function and organismal development; however, EJC-regulated genes and genetic pathways during development remain largely unknown. To study EJC function during zebrafish development, we first established that zebrafish EJCs mainly bind [~]24 nucleotides upstream of exon-exon junctions, and are also detected at more distant non-canonical positions. We then generated mutations in two zebrafish EJC core genes, rbm8a and magoh, and observed that homozygous mutant embryos show paralysis, muscle disorganization, neural cell death, and motor neuron outgrowth defects. Coinciding with developmental defects, mRNAs subjected to Nonsense-Mediated mRNA Decay (NMD) due to translation termination [≥] 50 nts upstream of the last exon-exon junction are upregulated in EJC mutant embryos. Surprisingly, several transcripts containing 3'UTR introns (3'UI) < 50 nts downstream of a stop codon are also upregulated in EJC mutant embryos. These proximal 3'UI-containing transcripts are also upregulated in NMD-compromised zebrafish embryos, cultured human cells, and mouse embryonic stem cells. Loss of function of foxo3b, one of the upregulated proximal 3'UI-containing genes, partially rescues EJC mutant motor neuron outgrowth. In addition to foxo3b, 166 other genes contain a proximal 3'UI in zebrafish, mouse and humans, and these genes are enriched in nervous system development and RNA binding functions. A proximal 3'UI-containing 3'UTR from one of these genes, HNRNPD, is sufficient to reduce steady state transcript levels when fused to a {beta}-globin reporter in HeLa cells. Overall, our work shows that genes with stop codon-proximal 3'UIs encode a new class of EJC-regulated NMD targets with critical roles during vertebrate development.

molecular biology↗