Trehalose

Sorbitol, sucrose, and trehalose were acquired from Wako Pure Chemicals (Tokyo, Japan). Guanidine hydrochloride (GdnHCl), 1-anilino-8-naphthalenesulfonate (ANS), p-nitrophenyl-α-D-glucopyranoside (pNPG), and p-nitrophenol (pNP) were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA). All other chemicals used were of analytical grade or the equivalent.

Drosophila melanogaster flies were reared on standard agar-cornmeal media at 25 °C unless otherwise indicated^{8 (link), 50 (link)}. The detailed food compositions, except preservatives, are shown in Fig. 4B. Casein, peptone, and trehalose were obtained from Wako Chemical, BD Biosciences, and Hayashibara, Co., respectively. All experiments were conducted under non-crowded conditions. No yeast paste was added to the fly tubes for any of the experiments. The percentage of puparium formation and adult flies was determined by counting homozygotes and heterozygotes in the same vials as an internal control.

In situ hybridization was performed according to a previous report (Fujiwara et al., 2007 (link)). Briefly, each digoxigenin (DIG)-labeled cRNA probe was amplified by PCR using primer sets (Table 2) and labeled using the Roche DIG RNA labeling kit (Merck, Schwalbach, Germany). Embryos at E10.5 and the heads of mice at E14.5 were fixed using MEMFA (2 mM EGTA, 1 mM MgSO4, and 3.7% formaldehyde) in 100 mM MOPS (pH 7.5) overnight at 4°C, and this was followed by immersion in 30% trehalose (Wako) in 20 mM HEPES to cryoprotect the tissues. Cryosections (7 μm thickness) from the transverse or sagittal plane were hybridized with DIG-labeled cRNA probe and were visualized with alkaline phosphatase-conjugated anti-DIG antibody (Merck) using 4-nitroblue tetrazolium chloride (NBT; Merck) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Merck). The sections were observed under a BZ-X800 microscope (KEYENCE, Osaka, Japan).