4 diethylaminobenzaldehyde

MeCN (extra high purity, water content < 0.3%, Cryochrom) was used to prepare the solutions. The Bu₄NClO₄ (≥99%, for electrochemical analysis) was purchased from Sigma-Aldrich and used as the supporting electrolyte. The cyclobutanone, cyclopentanone, benzaldehyde, 4-methoxybenzaldehyde, 3,4-dimethoxybenzaldehyde, 4-(methylthio)benzaldehyde, 4-dimethylaminobenzaldehyde, and 4-diethylaminobenzaldehyde (Sigma–Aldrich) were used as received. The EtOH (chemically pure grade) was used without additional purification, (2E,5E)-2,5-bis(3,4-dimethoxybenzylidene)cyclopentanone (2) was prepared as described previously [34 (link)]. The DC-Alufolien Aluminiumoxid 60 F254 neutral was purchased from Merck.

Absolute ethanol (ETOH, Sigma-Aldrich, St. Louis, MO, USA) was administered in embryo media at 1% and 3%. Wildtype (WT) Casper strain embryos were dechorionated, and the treatments were administered from 24 to 48 hpf. Following treatment, the embryos were washed multiple times with embryo media and subsequently placed in fresh embryo media for the remainder of the time course as indicated. All-trans retinoic acid (RA; Sigma-Aldrich) and 4-diethylaminobenzaldehyde (DEAB, Sigma-Aldrich, pan-aldehyde dehydrogenase inhibitor of RA synthesis) were diluted in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to a 1000× final concentration. The pharmacological treatments were initiated between 24 and 27 hpf (as described in the Results). Dose curves were conducted for each pharmacologic treatment (1, 10, 25, 100 nM RA; 5, 10, 20 μM DEAB), and final concentrations were chosen based on LD50 and consistency of phenotype (data not shown). Final concentrations were as follows: 0.1% DMSO (vehicle control), 100 nM RA, and 10 μM DEAB. Experiments used 50 to 100 embryos per treatment group and were replicated 4 to 6 times. Phenotypes were assessed at 24, 36, 48, 60, 72 and 96 hpf.