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Drosophila enhancer-Gal4 lines show ectopic expression during development

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.

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Activation of the neural elav enhancer in wing disc cells. (a) Schematic description of the G-TRACE technique. An enhancer controls the expression of Gal4 (blue) which results in a red reporter signal (RFP). This red signal will be maintained as long as the enhancer is active, thus, it reflects the current expression domain. On the other hand, the first time in development when the enhancer becomes active the flipase encoding construct is also activated (black box). The flipase, through the excision of a STOP cassette (black triangles), allows the expression of a GFP-encoding construct (green box). This reporter is now controlled by a ubiquitous p63 promoter, thus becoming independent from the original enhancer. This GFP reporter signal represents the historical expression domain. (b–e) G-TRACE data from elav enhancer in larval brains (b,c) and wing discs (d,e). Note that the enhancer is not CNS specific. (f–i) In vivo images of early developmental stages activation of elav enhancer in first instar (f,g) and second instar (h,i) larvae. (j–o) Quantification (j) of G-TRACE pattern of elav enhancer comparing two different elav-Gal4 insertions, chromosome II (k–m) and chromosome III (n,o). Note that the ectopic elav enhancer expression in the wing disc is not consistent between left and right sides of the same animal. This is evidence of the variable nature of the ectopic expression. (p) Gal4 mRNA quantitative RT-PCRs from chromosome II and chromosome III elav-Gal4 lines. (q,r) G-TRACE data for elav enhancer (q) and anti-ELAV staining (r) in larval brain. (s,t) G-TRACE data for elav enhancer (s) and anti-ELAV staining (t) in larval wing imaginal disc. Note that the ELAV protein is CNS specific. Scale bar, 50 µm. Statistics: t-test *p < 0,05. n = 5 wing discs/sample. Arrows in (e) indicate active enhancer cells (RFP).
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RSOS170039F1: Activation of the neural elav enhancer in wing disc cells. (a) Schematic description of the G-TRACE technique. An enhancer controls the expression of Gal4 (blue) which results in a red reporter signal (RFP). This red signal will be maintained as long as the enhancer is active, thus, it reflects the current expression domain. On the other hand, the first time in development when the enhancer becomes active the flipase encoding construct is also activated (black box). The flipase, through the excision of a STOP cassette (black triangles), allows the expression of a GFP-encoding construct (green box). This reporter is now controlled by a ubiquitous p63 promoter, thus becoming independent from the original enhancer. This GFP reporter signal represents the historical expression domain. (b–e) G-TRACE data from elav enhancer in larval brains (b,c) and wing discs (d,e). Note that the enhancer is not CNS specific. (f–i) In vivo images of early developmental stages activation of elav enhancer in first instar (f,g) and second instar (h,i) larvae. (j–o) Quantification (j) of G-TRACE pattern of elav enhancer comparing two different elav-Gal4 insertions, chromosome II (k–m) and chromosome III (n,o). Note that the ectopic elav enhancer expression in the wing disc is not consistent between left and right sides of the same animal. This is evidence of the variable nature of the ectopic expression. (p) Gal4 mRNA quantitative RT-PCRs from chromosome II and chromosome III elav-Gal4 lines. (q,r) G-TRACE data for elav enhancer (q) and anti-ELAV staining (r) in larval brain. (s,t) G-TRACE data for elav enhancer (s) and anti-ELAV staining (t) in larval wing imaginal disc. Note that the ELAV protein is CNS specific. Scale bar, 50 µm. Statistics: t-test *p < 0,05. n = 5 wing discs/sample. Arrows in (e) indicate active enhancer cells (RFP).

Mentions: The G-TRACE technique has been instrumental in this study [19]. Briefly summarized, it consists of three constructs that contain: UAS-RFP fluorescent protein, UAS-Flipase and Act-FRT-STOP-FRT-GFP, respectively. The system reports the temporal activation of the enhancer-Gal4 under study (figure 1a, modified from [19]). The enhancer-Gal4 activity induces the expression of the UAS-Flipase (Flp) and UAS-RFP (red) constructs. The Flp enzyme recognizes FRT sites and removes the STOP cassette, allowing the expression of Act>GFP (green) in these cells and their progeny. Thus, we can determine the current expression of an enhancer-Gal4 at the moment of dissection (red, RFP) and its historical expression during development (Act>GFP (green)). The inventors of this technique showed already that some enhancer-Gal4 lines exhibit divergence of activity at different stages of development within the same tissue [19].Figure 1.


Drosophila enhancer-Gal4 lines show ectopic expression during development
Activation of the neural elav enhancer in wing disc cells. (a) Schematic description of the G-TRACE technique. An enhancer controls the expression of Gal4 (blue) which results in a red reporter signal (RFP). This red signal will be maintained as long as the enhancer is active, thus, it reflects the current expression domain. On the other hand, the first time in development when the enhancer becomes active the flipase encoding construct is also activated (black box). The flipase, through the excision of a STOP cassette (black triangles), allows the expression of a GFP-encoding construct (green box). This reporter is now controlled by a ubiquitous p63 promoter, thus becoming independent from the original enhancer. This GFP reporter signal represents the historical expression domain. (b–e) G-TRACE data from elav enhancer in larval brains (b,c) and wing discs (d,e). Note that the enhancer is not CNS specific. (f–i) In vivo images of early developmental stages activation of elav enhancer in first instar (f,g) and second instar (h,i) larvae. (j–o) Quantification (j) of G-TRACE pattern of elav enhancer comparing two different elav-Gal4 insertions, chromosome II (k–m) and chromosome III (n,o). Note that the ectopic elav enhancer expression in the wing disc is not consistent between left and right sides of the same animal. This is evidence of the variable nature of the ectopic expression. (p) Gal4 mRNA quantitative RT-PCRs from chromosome II and chromosome III elav-Gal4 lines. (q,r) G-TRACE data for elav enhancer (q) and anti-ELAV staining (r) in larval brain. (s,t) G-TRACE data for elav enhancer (s) and anti-ELAV staining (t) in larval wing imaginal disc. Note that the ELAV protein is CNS specific. Scale bar, 50 µm. Statistics: t-test *p < 0,05. n = 5 wing discs/sample. Arrows in (e) indicate active enhancer cells (RFP).
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RSOS170039F1: Activation of the neural elav enhancer in wing disc cells. (a) Schematic description of the G-TRACE technique. An enhancer controls the expression of Gal4 (blue) which results in a red reporter signal (RFP). This red signal will be maintained as long as the enhancer is active, thus, it reflects the current expression domain. On the other hand, the first time in development when the enhancer becomes active the flipase encoding construct is also activated (black box). The flipase, through the excision of a STOP cassette (black triangles), allows the expression of a GFP-encoding construct (green box). This reporter is now controlled by a ubiquitous p63 promoter, thus becoming independent from the original enhancer. This GFP reporter signal represents the historical expression domain. (b–e) G-TRACE data from elav enhancer in larval brains (b,c) and wing discs (d,e). Note that the enhancer is not CNS specific. (f–i) In vivo images of early developmental stages activation of elav enhancer in first instar (f,g) and second instar (h,i) larvae. (j–o) Quantification (j) of G-TRACE pattern of elav enhancer comparing two different elav-Gal4 insertions, chromosome II (k–m) and chromosome III (n,o). Note that the ectopic elav enhancer expression in the wing disc is not consistent between left and right sides of the same animal. This is evidence of the variable nature of the ectopic expression. (p) Gal4 mRNA quantitative RT-PCRs from chromosome II and chromosome III elav-Gal4 lines. (q,r) G-TRACE data for elav enhancer (q) and anti-ELAV staining (r) in larval brain. (s,t) G-TRACE data for elav enhancer (s) and anti-ELAV staining (t) in larval wing imaginal disc. Note that the ELAV protein is CNS specific. Scale bar, 50 µm. Statistics: t-test *p < 0,05. n = 5 wing discs/sample. Arrows in (e) indicate active enhancer cells (RFP).
Mentions: The G-TRACE technique has been instrumental in this study [19]. Briefly summarized, it consists of three constructs that contain: UAS-RFP fluorescent protein, UAS-Flipase and Act-FRT-STOP-FRT-GFP, respectively. The system reports the temporal activation of the enhancer-Gal4 under study (figure 1a, modified from [19]). The enhancer-Gal4 activity induces the expression of the UAS-Flipase (Flp) and UAS-RFP (red) constructs. The Flp enzyme recognizes FRT sites and removes the STOP cassette, allowing the expression of Act>GFP (green) in these cells and their progeny. Thus, we can determine the current expression of an enhancer-Gal4 at the moment of dissection (red, RFP) and its historical expression during development (Act>GFP (green)). The inventors of this technique showed already that some enhancer-Gal4 lines exhibit divergence of activity at different stages of development within the same tissue [19].Figure 1.

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