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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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Transient expression of nervous system specific enhancers. GMR10B11-Gal4 (a–c) and GMR78G09-Gal4 (d–f) expression revealed by G-TRACE in larval CNS. c105-Gal4 (g–i) and GH298-Gal4 (j–l) expression in the adult brain. Note the differences between the historical (GFP) and current (RFP) expression domains in the four enhancers. Since all cells correspond to the CNS, this difference must be considered ‘transient’, rather than ‘ectopic’. Arrows indicate cells with active enhancer (RFP), arrowheads indicate historical enhancer activity (GFP) and asterisks indicate cells with coincident historical and current expressions (yellow). Bar in (a) = 50 µm.
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RSOS170039F4: Transient expression of nervous system specific enhancers. GMR10B11-Gal4 (a–c) and GMR78G09-Gal4 (d–f) expression revealed by G-TRACE in larval CNS. c105-Gal4 (g–i) and GH298-Gal4 (j–l) expression in the adult brain. Note the differences between the historical (GFP) and current (RFP) expression domains in the four enhancers. Since all cells correspond to the CNS, this difference must be considered ‘transient’, rather than ‘ectopic’. Arrows indicate cells with active enhancer (RFP), arrowheads indicate historical enhancer activity (GFP) and asterisks indicate cells with coincident historical and current expressions (yellow). Bar in (a) = 50 µm.

Mentions: We aimed to determine the contribution of the enhancer's sequence to its unstable early activation. To that end, we took advantage of the enhancer-Gal4 insertions directed to the exact same and insulated chromosomal site with no detectable Gal4 basal activity [30]. Under these conditions, promoters are subject to the same chromatin structural determinants except for the enhancer nucleotide sequence. We compared two same-site/different-sequence insertions: GMR10B11 and GMR78G09 (see electronic supplementary material, table S1). Both insertions are in the 3 L chromosome arm (68A4 polytene band) and show specific activity in the nervous system. G-TRACE experiments confirmed that both enhancer domains are restricted to the nervous system during development. Nevertheless, although GMR10B11 is active in larval brain cells, its historical (GFP) and current (RFP) expressions are not identical (figure 4a–c). This demonstrates that GMR10B11 expression is transient in certain larval brain cells. GMR78G09 is also expressed in the brain although in fewer cells (about 28 cells, RFP, in third instar larvae). In this case, a group of about 14 cells activated this enhancer in the ventral ganglion during development but not at third instar larvae (figure 4d–f, arrowheads). These results indicate that the nucleotide sequence of the enhancer is determinant to establish the time and number of cells that will display transient expression during development. In the cases of GMR10B11 and GMR78G09 the non-canonical expression domain is transient but not ectopic since it occurs within the same tissue.Figure 4.


Drosophila enhancer-Gal4 lines show ectopic expression during development
Transient expression of nervous system specific enhancers. GMR10B11-Gal4 (a–c) and GMR78G09-Gal4 (d–f) expression revealed by G-TRACE in larval CNS. c105-Gal4 (g–i) and GH298-Gal4 (j–l) expression in the adult brain. Note the differences between the historical (GFP) and current (RFP) expression domains in the four enhancers. Since all cells correspond to the CNS, this difference must be considered ‘transient’, rather than ‘ectopic’. Arrows indicate cells with active enhancer (RFP), arrowheads indicate historical enhancer activity (GFP) and asterisks indicate cells with coincident historical and current expressions (yellow). Bar in (a) = 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383858&req=5

RSOS170039F4: Transient expression of nervous system specific enhancers. GMR10B11-Gal4 (a–c) and GMR78G09-Gal4 (d–f) expression revealed by G-TRACE in larval CNS. c105-Gal4 (g–i) and GH298-Gal4 (j–l) expression in the adult brain. Note the differences between the historical (GFP) and current (RFP) expression domains in the four enhancers. Since all cells correspond to the CNS, this difference must be considered ‘transient’, rather than ‘ectopic’. Arrows indicate cells with active enhancer (RFP), arrowheads indicate historical enhancer activity (GFP) and asterisks indicate cells with coincident historical and current expressions (yellow). Bar in (a) = 50 µm.
Mentions: We aimed to determine the contribution of the enhancer's sequence to its unstable early activation. To that end, we took advantage of the enhancer-Gal4 insertions directed to the exact same and insulated chromosomal site with no detectable Gal4 basal activity [30]. Under these conditions, promoters are subject to the same chromatin structural determinants except for the enhancer nucleotide sequence. We compared two same-site/different-sequence insertions: GMR10B11 and GMR78G09 (see electronic supplementary material, table S1). Both insertions are in the 3 L chromosome arm (68A4 polytene band) and show specific activity in the nervous system. G-TRACE experiments confirmed that both enhancer domains are restricted to the nervous system during development. Nevertheless, although GMR10B11 is active in larval brain cells, its historical (GFP) and current (RFP) expressions are not identical (figure 4a–c). This demonstrates that GMR10B11 expression is transient in certain larval brain cells. GMR78G09 is also expressed in the brain although in fewer cells (about 28 cells, RFP, in third instar larvae). In this case, a group of about 14 cells activated this enhancer in the ventral ganglion during development but not at third instar larvae (figure 4d–f, arrowheads). These results indicate that the nucleotide sequence of the enhancer is determinant to establish the time and number of cells that will display transient expression during development. In the cases of GMR10B11 and GMR78G09 the non-canonical expression domain is transient but not ectopic since it occurs within the same tissue.Figure 4.

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