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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.

No MeSH data available.


Toll-6 enhancer in brain and wing cells during development. (a–d) G-TRACE signal from third instar larval brain (a,b) and wing disc (c,d) of the D42-Gal4 line inserted in Toll-6. (e,f) Historical expression of D42 enhancer (GFP) versus LacZ-reported Dpp expression (Dpp-LacZ, magenta). (g,h) elav-Gal80 suppresses D42-Gal4 activity in the brain (g) and wing disc (h). (i–o) Temporal expression experiments in D42-Gal4>G-TRACE>tub-Gal80TS brain (i,j) and wing discs (k–o) maintained at 17°C ‘OFF’ (Gal4 silenced), 48 h, 24 h or 12 h OFF or maintained at 29°C ‘ON’ (Gal4 active). (p) Quantification of GFP positive cells per time point. Student's t-test *p < 0.05. n = 5 wing discs/time point. Bar in (c) = 50 µm.
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RSOS170039F2: Toll-6 enhancer in brain and wing cells during development. (a–d) G-TRACE signal from third instar larval brain (a,b) and wing disc (c,d) of the D42-Gal4 line inserted in Toll-6. (e,f) Historical expression of D42 enhancer (GFP) versus LacZ-reported Dpp expression (Dpp-LacZ, magenta). (g,h) elav-Gal80 suppresses D42-Gal4 activity in the brain (g) and wing disc (h). (i–o) Temporal expression experiments in D42-Gal4>G-TRACE>tub-Gal80TS brain (i,j) and wing discs (k–o) maintained at 17°C ‘OFF’ (Gal4 silenced), 48 h, 24 h or 12 h OFF or maintained at 29°C ‘ON’ (Gal4 active). (p) Quantification of GFP positive cells per time point. Student's t-test *p < 0.05. n = 5 wing discs/time point. Bar in (c) = 50 µm.

Mentions: To assess if the phenomenon observed with the elav enhancer is general to other enhancers, we selected additional cases also described as CNS specific. Line D42-Gal4 corresponds to a Toll-6 gene enhancer [27]. As described, neurons from the third instar larval brain had activated D42 during early development (historical GFP, figure 2a, compared to current RFP, figure 2b). Similar to elav, D42 is also activated in wing disc cells during early development but not in third instar larvae (no RFP cells) (figure 2c,d). In addition, D42-Gal4 domain in the wing disc is also different between left and right discs within the same animal (figure 2c,d). The historical GFP positive cells in the vicinity of the anterior--posterior (A/P) border of the wing disc appear to align with the A/P compartment border. To determine if this is the case, we stained for β-galactosidase in dpp-LacZ expressing discs that mark this boundary (figure 2e,f). The GFP cells distribution does not correspond with the dpp enhancer signal.Figure 2.


Drosophila enhancer-Gal4 lines show ectopic expression during development
Toll-6 enhancer in brain and wing cells during development. (a–d) G-TRACE signal from third instar larval brain (a,b) and wing disc (c,d) of the D42-Gal4 line inserted in Toll-6. (e,f) Historical expression of D42 enhancer (GFP) versus LacZ-reported Dpp expression (Dpp-LacZ, magenta). (g,h) elav-Gal80 suppresses D42-Gal4 activity in the brain (g) and wing disc (h). (i–o) Temporal expression experiments in D42-Gal4>G-TRACE>tub-Gal80TS brain (i,j) and wing discs (k–o) maintained at 17°C ‘OFF’ (Gal4 silenced), 48 h, 24 h or 12 h OFF or maintained at 29°C ‘ON’ (Gal4 active). (p) Quantification of GFP positive cells per time point. Student's t-test *p < 0.05. n = 5 wing discs/time point. Bar in (c) = 50 µm.
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RSOS170039F2: Toll-6 enhancer in brain and wing cells during development. (a–d) G-TRACE signal from third instar larval brain (a,b) and wing disc (c,d) of the D42-Gal4 line inserted in Toll-6. (e,f) Historical expression of D42 enhancer (GFP) versus LacZ-reported Dpp expression (Dpp-LacZ, magenta). (g,h) elav-Gal80 suppresses D42-Gal4 activity in the brain (g) and wing disc (h). (i–o) Temporal expression experiments in D42-Gal4>G-TRACE>tub-Gal80TS brain (i,j) and wing discs (k–o) maintained at 17°C ‘OFF’ (Gal4 silenced), 48 h, 24 h or 12 h OFF or maintained at 29°C ‘ON’ (Gal4 active). (p) Quantification of GFP positive cells per time point. Student's t-test *p < 0.05. n = 5 wing discs/time point. Bar in (c) = 50 µm.
Mentions: To assess if the phenomenon observed with the elav enhancer is general to other enhancers, we selected additional cases also described as CNS specific. Line D42-Gal4 corresponds to a Toll-6 gene enhancer [27]. As described, neurons from the third instar larval brain had activated D42 during early development (historical GFP, figure 2a, compared to current RFP, figure 2b). Similar to elav, D42 is also activated in wing disc cells during early development but not in third instar larvae (no RFP cells) (figure 2c,d). In addition, D42-Gal4 domain in the wing disc is also different between left and right discs within the same animal (figure 2c,d). The historical GFP positive cells in the vicinity of the anterior--posterior (A/P) border of the wing disc appear to align with the A/P compartment border. To determine if this is the case, we stained for β-galactosidase in dpp-LacZ expressing discs that mark this boundary (figure 2e,f). The GFP cells distribution does not correspond with the dpp enhancer signal.Figure 2.

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.