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Drosophila enhancer-Gal4 lines show ectopic expression during development

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ABSTRACT

In Drosophila melanogaster the most widely used technique to drive gene expression is the binary UAS/Gal4 system. We show here that a set of nervous system specific enhancers (elav, D42/Toll-6, OK6/RapGAP1) display ectopic activity in epithelial tissues during development, which is seldom considered in experimental studies. This ectopic activity is variable, unstable and influenced by the primary sequence of the enhancer and the insertion site in the chromosome. In addition, the ectopic activity is independent of the protein expressed, Gal4, as it is reproduced also with the expression of Gal80. Another enhancer, LN2 from the sex lethal (Sxl) gene, shows sex-dependent features in its ectopic expression. Feminization of LN2 expressing males does not alter the male specific pattern indicating that the sexual dimorphism of LN2 expression is an intrinsic feature of this enhancer. Other X chromosome enhancers corresponding to genes not related to sex determination do not show sexual dimorphism in their ectopic expressions. Although variable and unstable, the ectopic activation of enhancer-Gal4 lines seems to be regulated in terms of tissue and intensity. To characterize the full domain of expression of enhancer-Gal4 constructs is relevant for the design of transgenic animal models and biotechnology tools, as well as for the correct interpretation of developmental and behavioural studies in which Gal4 lines are used.

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D42 enhancer is expressed in wing cells but its gene Toll-6 is not. (a–d) Caspase 3 (C3) (green) and Wingless expression (red) in wild-type (a,b) and D42-Gal4>Bsk (c,d). Note the extensive apoptosis (c) and distorted Wg pattern (d) caused by Bsk. (e,f) Resulting adult wings with morphological abnormalities due to the Bsk-elicited apoptosis. (g–j) Caspase 3 (C3) (green) and Wingless pattern (red) in D42>Toll-6 RNAi (g,h) wing disc. (i,j) Wingless pattern (red) in en>Toll-6 RNAi (GFP) wing disc. (k,l) Resulting adult wings. Note the lack of morphological effects after inactivating Toll-6 in the posterior wing. (m,n) Toll-6-GFP protein trap (Mi{MIC}Toll-6MI02127) shows signal in larval brain (m) but not in imaginal discs (n). (o,p) Control and Toll-6ex16 mutant adult wings. Cell nuclei are marked in blue (DAPI).
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RSOS170039F3: D42 enhancer is expressed in wing cells but its gene Toll-6 is not. (a–d) Caspase 3 (C3) (green) and Wingless expression (red) in wild-type (a,b) and D42-Gal4>Bsk (c,d). Note the extensive apoptosis (c) and distorted Wg pattern (d) caused by Bsk. (e,f) Resulting adult wings with morphological abnormalities due to the Bsk-elicited apoptosis. (g–j) Caspase 3 (C3) (green) and Wingless pattern (red) in D42>Toll-6 RNAi (g,h) wing disc. (i,j) Wingless pattern (red) in en>Toll-6 RNAi (GFP) wing disc. (k,l) Resulting adult wings. Note the lack of morphological effects after inactivating Toll-6 in the posterior wing. (m,n) Toll-6-GFP protein trap (Mi{MIC}Toll-6MI02127) shows signal in larval brain (m) but not in imaginal discs (n). (o,p) Control and Toll-6ex16 mutant adult wings. Cell nuclei are marked in blue (DAPI).

Mentions: To determine if the ectopic enhancer expression implies a transient expression of the corresponding native gene, we induced apoptosis by driving a core component of the JNK pathway, Basket (Bsk), [28] under D42-Gal4 control. Wing imaginal discs were stained with anti-caspase 3 (C3) to detect apoptosis, and with anti-Wingless (WG) to monitor wing disc development. Epithelial activation of D42-Gal4/Toll-6 leads to Bsk-dependent apoptosis, as revealed by C3 staining, and to abnormalities in the Wg immune pattern which resulted in defective adult wings (figure 3a–f). Further, to resolve if Toll-6 gene expression is necessary for the development of the wing disc, we expressed a Toll-6 specific RNAi under the control of D42-Gal4. We did not detect any apoptosis or morphological defect and the Wg expression pattern was normal (figure 3g,h). To further demonstrate if Toll-6 is necessary for wing development, we used an independent Gal4 (engrailed, en) to drive Toll-6 RNAi expression in the posterior compartment of the wing disc (GFP in figure 3i–l). The expression of this RNAi with this independent driver did not affect the wing in any noticeable feature. Next, we used a Toll-6-GFP protein trap (Mi{MIC}Toll-6MI02127) to detect endogenous Toll-6 protein expression. We observed the canonical Toll-6-GFP signal in the brain (figure 3m). However, no signal was detected in imaginal disc (figure 3n). These results are in line with reported data showing no detectable Toll-6 mRNA in wing imaginal discs [29]. To further determine that Toll-6 expression is dispensable for wing development, we compared wild-type and Toll-6ex13 mutant wings (figure 3o,p). Mutant wings do not display morphological defects indicating that Toll-6 expression is not necessary for wing development. Thus, even though the Toll-6 enhancer D42 is active in the wing disc during development, Toll-6 is not switched on in the wing, at least to the point of manifesting a visible phenotype in disc size, shape or Wg pattern.Figure 3.


Drosophila enhancer-Gal4 lines show ectopic expression during development
D42 enhancer is expressed in wing cells but its gene Toll-6 is not. (a–d) Caspase 3 (C3) (green) and Wingless expression (red) in wild-type (a,b) and D42-Gal4>Bsk (c,d). Note the extensive apoptosis (c) and distorted Wg pattern (d) caused by Bsk. (e,f) Resulting adult wings with morphological abnormalities due to the Bsk-elicited apoptosis. (g–j) Caspase 3 (C3) (green) and Wingless pattern (red) in D42>Toll-6 RNAi (g,h) wing disc. (i,j) Wingless pattern (red) in en>Toll-6 RNAi (GFP) wing disc. (k,l) Resulting adult wings. Note the lack of morphological effects after inactivating Toll-6 in the posterior wing. (m,n) Toll-6-GFP protein trap (Mi{MIC}Toll-6MI02127) shows signal in larval brain (m) but not in imaginal discs (n). (o,p) Control and Toll-6ex16 mutant adult wings. Cell nuclei are marked in blue (DAPI).
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RSOS170039F3: D42 enhancer is expressed in wing cells but its gene Toll-6 is not. (a–d) Caspase 3 (C3) (green) and Wingless expression (red) in wild-type (a,b) and D42-Gal4>Bsk (c,d). Note the extensive apoptosis (c) and distorted Wg pattern (d) caused by Bsk. (e,f) Resulting adult wings with morphological abnormalities due to the Bsk-elicited apoptosis. (g–j) Caspase 3 (C3) (green) and Wingless pattern (red) in D42>Toll-6 RNAi (g,h) wing disc. (i,j) Wingless pattern (red) in en>Toll-6 RNAi (GFP) wing disc. (k,l) Resulting adult wings. Note the lack of morphological effects after inactivating Toll-6 in the posterior wing. (m,n) Toll-6-GFP protein trap (Mi{MIC}Toll-6MI02127) shows signal in larval brain (m) but not in imaginal discs (n). (o,p) Control and Toll-6ex16 mutant adult wings. Cell nuclei are marked in blue (DAPI).
Mentions: To determine if the ectopic enhancer expression implies a transient expression of the corresponding native gene, we induced apoptosis by driving a core component of the JNK pathway, Basket (Bsk), [28] under D42-Gal4 control. Wing imaginal discs were stained with anti-caspase 3 (C3) to detect apoptosis, and with anti-Wingless (WG) to monitor wing disc development. Epithelial activation of D42-Gal4/Toll-6 leads to Bsk-dependent apoptosis, as revealed by C3 staining, and to abnormalities in the Wg immune pattern which resulted in defective adult wings (figure 3a–f). Further, to resolve if Toll-6 gene expression is necessary for the development of the wing disc, we expressed a Toll-6 specific RNAi under the control of D42-Gal4. We did not detect any apoptosis or morphological defect and the Wg expression pattern was normal (figure 3g,h). To further demonstrate if Toll-6 is necessary for wing development, we used an independent Gal4 (engrailed, en) to drive Toll-6 RNAi expression in the posterior compartment of the wing disc (GFP in figure 3i–l). The expression of this RNAi with this independent driver did not affect the wing in any noticeable feature. Next, we used a Toll-6-GFP protein trap (Mi{MIC}Toll-6MI02127) to detect endogenous Toll-6 protein expression. We observed the canonical Toll-6-GFP signal in the brain (figure 3m). However, no signal was detected in imaginal disc (figure 3n). These results are in line with reported data showing no detectable Toll-6 mRNA in wing imaginal discs [29]. To further determine that Toll-6 expression is dispensable for wing development, we compared wild-type and Toll-6ex13 mutant wings (figure 3o,p). Mutant wings do not display morphological defects indicating that Toll-6 expression is not necessary for wing development. Thus, even though the Toll-6 enhancer D42 is active in the wing disc during development, Toll-6 is not switched on in the wing, at least to the point of manifesting a visible phenotype in disc size, shape or Wg pattern.Figure 3.

View Article: PubMed Central - PubMed

ABSTRACT

In Drosophila melanogaster the most widely used technique to drive gene expression is the binary UAS/Gal4 system. We show here that a set of nervous system specific enhancers (elav, D42/Toll-6, OK6/RapGAP1) display ectopic activity in epithelial tissues during development, which is seldom considered in experimental studies. This ectopic activity is variable, unstable and influenced by the primary sequence of the enhancer and the insertion site in the chromosome. In addition, the ectopic activity is independent of the protein expressed, Gal4, as it is reproduced also with the expression of Gal80. Another enhancer, LN2 from the sex lethal (Sxl) gene, shows sex-dependent features in its ectopic expression. Feminization of LN2 expressing males does not alter the male specific pattern indicating that the sexual dimorphism of LN2 expression is an intrinsic feature of this enhancer. Other X chromosome enhancers corresponding to genes not related to sex determination do not show sexual dimorphism in their ectopic expressions. Although variable and unstable, the ectopic activation of enhancer-Gal4 lines seems to be regulated in terms of tissue and intensity. To characterize the full domain of expression of enhancer-Gal4 constructs is relevant for the design of transgenic animal models and biotechnology tools, as well as for the correct interpretation of developmental and behavioural studies in which Gal4 lines are used.

No MeSH data available.


Related in: MedlinePlus