Table 1.
Primer sequences for Quantitative RT-PCR.
Figure 1.
Generation of transgenic flies carrying hGBA variants.
(A) Sequence of hGBA. Blue and red fonts show R120W and RecNciI mutations, respectively. (B) Expression levels of hGBA mRNA confirmed by quantitative RT-PCR (n = about 30 fly heads per transgenic combination) with dRpL32 as internal control. Error bars represent SE. (C) Levels of hGBA protein confirmed by Western blotting (n = about 100 fly heads per transgenic combination). Total amounts of hGBA protein were decreased in hGBAR120W, and significantly decreased in hGBARecNciI transgenic combinations, compared with hGBAWT transgenic combination.
Figure 2.
Neurodevelopmental defects in the Drosophila eye caused by expression of hGBA carrying the RecNciI mutation.
We investigated the effects of overexpression to mutated hGBAs in fly eyes. (A) Phenotype of eyes overexpressing hGBAWT transgenic combination do not significantly differ from those of GMR control. Phenotype of eyes overexpressing hGBAR120W transgenic combinations occasionally differed in terms of morphology in some flies compared with control. Eye morphology is obviously affected in hGBARecNciI transgenic combinations compared with control. (B) Size histograms of ocelli in transgenic combinations (n = 3–5 flies each, about 100 ocelli each). Dispersion analysis showed obvious differences in variance of the sizes of ocelli between the hGBARecNciI transgenic combinations and the GMR control (F = 29.50–37.19; P<0.001; Levene's test).
Figure 3.
Endoplasmic reticulum (ER) stress detected in the mutated hGBA induced Drosophila eye.
We used xbp1-EGFP as an ER stress marker in which EGFP is expressed in frame only after ER stress [31]. (A) Weak fluorescence is generated in eye imaginal discs expressing the hGBAWT construct. The eye imaginal discs of hGBAR120W transgenic combinations emitted more fluorescence than that of hGBAWT transgenic combination. The eye imaginal discs of hGBARecNciI transgenic combinations emitted the most intense fluorescence. (B) Values generated by different transgenic combinations with fixed quantities of fluorescence intensity (n = 7–15 eye imaginal discs from third instar larvae per transgenic combination). Error bars represent SE. *Significant difference compared with values from GMR control (***P<0.001; Student's t test). (C) Endoplasmic reticulum stress marker gene, dBiP (major ER chaperone) mRNA expression in hGBAR120W and hGBARecNciI transgenic combinations was upregulated (n = about 30 fly heads per transgenic combination). Internal control was dRpL32. Error bars represent SE. *Significant difference compared with GMR control (*P<0.05; Student's t test). (D) High levels of hGBAs are expressed in whole bodies of heat-shocked flies. Expression levels of dBiP mRNA of hGBAR120W and hGBARecNciI transgenic combinations were also upregulated (n = about 30 flies per transgenic combination). Internal control was dRpL32. Error bars represent SE. *Significant difference compared with hs control (*P<0.05; **P<0.01; ***P<0.001; Student's t test).
Figure 4.
Feeding of ambroxol ameliorates neurodevelopmental defects and ER stress in the mutated hGBA induced Drosophila eye.
Ambroxol can recover morphological defects and decrease ER stress in transgenic flies. (A) Less fluorescence emitted by the eye imaginal discs of hGBARecNciI transgenic combinations treated with, than without 1 mM Ambroxol. (B) Values generated by different transgenic combinations at fixed quantities of fluorescence intensity (n = 12–43 eye imaginal discs of third instar larvae per transgenic combination). Error bars represent SE. *Significant difference compared with controls (all without Ambroxol) (***P<0.001; Student's t test). (C) Ambroxol (1 mM) decreases expression levels of dBiP mRNA in the heads of hGBARecNciI transgenic combinations (n = about 30 fly heads per transgenic combination). Internal control was dRpL32. Error bars represent SE. (D) Eye phenotypes of hGBARecNciI transgenic combinations incubated without or with 1 mM Ambroxol. Size and shape of ocelli were uniform, and layout uniformity was more similar to that of normal fly eyes treated with 1 mM Ambroxol. (E) Size histograms of ocelli in hGBARecNciI transgenic combinations treated with or without 1 mM Ambroxol. (n = 6–10 flies per transgenic combination; about 400 ocelli each). Dispersion analysis showed significant differences from hGBARecNciI transgenic combinations treated with and without 1 mM Ambroxol (F = 2.07–3.35; P<0.001; Levene's test).