Ro 48 8071

Wnt agonist‐1 was dissolved in 100% DMSO and embryos were treated with a final concentration ranging from 0.1–1.0 μM. Concentrations were developed from recommendations previously described (Liu et al., 2005). Final concentration of DMSO was less than or equal to 0.01% in all treated samples and the vehicle control treatment. Wnt agonist‐1 treatment was initiated at 30 HPF, consistent with the onset of facial defects (Quintana et al., 2017) and removed at 54 HPF, for a total treatment time of no more than 24 hrs at 28 °C. Following treatment, embryos were maintained at 28 °C until 4 days post fertilization (DPF) in system water. For all other drug treatments, each drug was added at the indicated concentration at the sphere stage as previously described (Hernandez et al., 2019; Quintana et al., 2017; Quintana, Picchione, Klein Geltink, Taylor, & Grosveld, 2014). Embryos were treated with 1, 1.5, 2, or 3 μM Ro 488071 (Santa Cruz Biotechnology), 8 μM lonafarnib (Millipore‐Sigma), or 2 μM atorvastatin (Millipore‐Sigma); all concentrations were empirically derived and previously published and validated (Hernandez et al., 2019; Quintana, Picchione, et al., 2014, p. 7; Quintana et al., 2017). All larvae were harvested at 30 HPF or 54 HPF in each treatment group for qPCR analysis.