The mesodermal germ layer is patterned into mediolateral subtypes by signaling factors including BMP and FGF. How these pathways are integrated to induce specific mediolateral cell fates is not well understood. We used mesoderm derived from post-gastrulation neuromesodermal progenitors (NMPs), which undergo a binary mediolateral patterning decision, as a simplified model to understand how FGF acts together with BMP to impart mediolateral fate. Using zebrafish and mouse NMPs, we identify an evolutionarily conserved mechanism of BMP and FGF-mediated mediolateral mesodermal patterning that occurs through modulation of basic helix-loop-helix (bHLH) transcription factor activity. BMP imparts lateral fate through induction of Id helix loop helix (HLH) proteins, which antagonize bHLH transcription factors, induced by FGF signaling, that specify medial fate. We extend our analysis of zebrafish development to show that bHLH activity is responsible for the mediolateral patterning of the entire mesodermal germ layer.

Introduction: Animal models have enabled great progress in the discovery and understanding of pharmacological approaches for treating muscle diseases like Duchenne muscular dystrophy.Areas covered:...

Background: Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder and is one of the most common muscular dystrophies. There are currently few effective therapies to treat the disease, although many small-molecule approaches are being pursued. Certain histone deacetylase inhibitors (HDACi) have been shown to ameliorate DMD phenotypes in mouse and zebrafish animal models. The HDACi givinostat has shown promise for DMD in clinical trials. However, beyond a small group of HDACi, other classes of epigenetic small molecules have not been broadly and systematically studied for their benefits for DMD. Methods: We used an established animal model for DMD, the zebrafish dmd mutant strain sapje. A commercially available library of epigenetic small molecules was used to treat embryonic-larval stages of dmd mutant zebrafish. We used a quantitative muscle birefringence assay in order to assess and compare the effects of small-molecule treatments on dmd mutant zebrafish skeletal muscle structure. Results: We performed a novel chemical-combination screen of a library of epigenetic compounds using the zebrafish dmd model. We identified candidate pools of epigenetic compounds that improve skeletal muscle structure in dmd mutant zebrafish. We then identified a specific combination of two HDACi compounds, oxamflatin and salermide, that ameliorated dmd mutant zebrafish skeletal muscle degeneration. We validated the effects of oxamflatin and salermide on dmd mutant zebrafish in an independent laboratory. Furthermore, we showed that the combination of oxamflatin and salermide caused increased levels of histone H4 acetylation in zebrafish larvae. Conclusions: Our results provide novel, effective methods for performing a combination of small-molecule screen in zebrafish. Our results also add to the growing evidence that epigenetic small molecules may be promising candidates for treating DMD.

Zebrafish are an excellent model for Duchenne muscular dystrophy. In particular, zebrafish provide a system for rapid, easy, and low-cost screening of small molecules that can ameliorate muscle damage in dystrophic larvae. Here we identify an optimal anti-sense morpholino cocktail that robustly knocks down zebrafish Dystrophin (dmd-MO). We use two approaches, muscle birefringence and muscle actin expression, to quantify muscle damage and show that the dmd-MO dystrophic phenotype closely resembles the zebrafish dmd mutant phenotype. We then show that the histone deacetylase (HDAC) inhibitor TSA, which has been shown to ameliorate the mdx mouse Duchenne model, can rescue muscle fiber damage in both dmd-MO and dmd mutant larvae. Our study identifies optimal morpholino and phenotypic scoring approaches for dystrophic zebrafish, further enhancing the zebrafish dmd model for rapid and cost-effective small molecule screening.