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An RNA interference screen for genes required to shape the anteroposterior compartment boundary in Drosophila identifies the Eph receptor.

Umetsu D, Dunst S, Dahmann C - PLoS ONE (2014)

Bottom Line: Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, we identified a single novel candidate that interfered with the formation of a straight anteroposterior compartment boundary.Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos.Our results identify a hitherto unknown role of the Eph receptor tyrosine kinase in Drosophila and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics, Technische Universit├Ąt Dresden, Dresden, Germany.

ABSTRACT
The formation of straight compartment boundaries separating groups of cells with distinct fates and functions is an evolutionarily conserved strategy during animal development. The physical mechanisms that shape compartment boundaries have recently been further elucidated, however, the molecular mechanisms that underlie compartment boundary formation and maintenance remain poorly understood. Here, we report on the outcome of an RNA interference screen aimed at identifying novel genes involved in maintaining the straight shape of the anteroposterior compartment boundary in Drosophila wing imaginal discs. Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, we identified a single novel candidate that interfered with the formation of a straight anteroposterior compartment boundary. Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos. Our results identify a hitherto unknown role of the Eph receptor tyrosine kinase in Drosophila and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development.

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An assay to identify genes required to maintain the straight shape of the AP boundary.A. Scheme of tester stock and cross to generate clones of cell expressing double-stranded RNA. The tester stock contains a Flp recombinase transgene under control of the heat-shock inducible promoter hsp70 (hs) on chromosome I [36] and the engrailed-Venus transgene on chromosome II (not shown). Chromosome III contains a transgene composed of the Act5C promoter followed by a FRT site, the coding sequence for the mouse transmembrane protein CD2 followed by a transcriptional terminator (Term), a second FRT site, and finally Gal4. We have recombined on the same chromosome a transgene expressing DsRed under control of UAS sequences (not shown). The tester stock is crossed to individual RNAi fly lines. The resulting progeny larvae are heat-shocked to express the Flp recombinase that in turn induces recombination between the two FRT sites on the tester stock. This recombination places Gal4 under the direct control of the Act5C promoter leading to Gal4 expression specifically in the clones of cells. Gal4 then binds to the UAS sequences in the RNAi line and directs expression of double-stranded RNA that leads to RNA interference. At the same time, Gal4 will induce expression of DsRed (not shown) in the same cells. B. Scheme of the assay. The central part of the wing imaginal disc is schematically drawn as an oval. A Venus fluorescent protein (green) is specifically expressed in all cells of the posterior compartment under control of the promoter sequences of the engrailed gene (en-Venus). Clones of cells expressing double stranded RNA are identified by co-expression of DsRed (red). Examples on the left depict wild-type clones located in the anterior (A) or posterior (P) compartments. Examples on the right depict a anterior clone mis-segregating into the posterior territory (top) and an posterior clone mis-segregating into the anterior territory (bottom) of the wing disc. Clones co-expressing DsRed and Venus are depicted in yellow.
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pone-0114340-g001: An assay to identify genes required to maintain the straight shape of the AP boundary.A. Scheme of tester stock and cross to generate clones of cell expressing double-stranded RNA. The tester stock contains a Flp recombinase transgene under control of the heat-shock inducible promoter hsp70 (hs) on chromosome I [36] and the engrailed-Venus transgene on chromosome II (not shown). Chromosome III contains a transgene composed of the Act5C promoter followed by a FRT site, the coding sequence for the mouse transmembrane protein CD2 followed by a transcriptional terminator (Term), a second FRT site, and finally Gal4. We have recombined on the same chromosome a transgene expressing DsRed under control of UAS sequences (not shown). The tester stock is crossed to individual RNAi fly lines. The resulting progeny larvae are heat-shocked to express the Flp recombinase that in turn induces recombination between the two FRT sites on the tester stock. This recombination places Gal4 under the direct control of the Act5C promoter leading to Gal4 expression specifically in the clones of cells. Gal4 then binds to the UAS sequences in the RNAi line and directs expression of double-stranded RNA that leads to RNA interference. At the same time, Gal4 will induce expression of DsRed (not shown) in the same cells. B. Scheme of the assay. The central part of the wing imaginal disc is schematically drawn as an oval. A Venus fluorescent protein (green) is specifically expressed in all cells of the posterior compartment under control of the promoter sequences of the engrailed gene (en-Venus). Clones of cells expressing double stranded RNA are identified by co-expression of DsRed (red). Examples on the left depict wild-type clones located in the anterior (A) or posterior (P) compartments. Examples on the right depict a anterior clone mis-segregating into the posterior territory (top) and an posterior clone mis-segregating into the anterior territory (bottom) of the wing disc. Clones co-expressing DsRed and Venus are depicted in yellow.

Mentions: We sought to identify genes required to maintain the shape of the AP boundary by RNA interference. Expression of double-stranded hairpin RNA from a transgene can result in the RNA interference mediated knock-down of gene function in Drosophila[33]. We developed an assay to assess whether the knock-down of a particular gene influences the shape of the AP boundary. In this assay, double-stranded hairpin RNA is expressed in clones of cells using the Gal4-UAS [34] and FRT-flp [35], [36] systems (Fig. 1A). Clones of cells located along the AP boundary were tested whether or not they influenced the shape of the AP boundary (Fig. 1B). To identify the clones of cells and the AP boundary, we used two fluorescent reporters. One reporter (DsRed) identified the clone of cells that expressed the double-stranded RNA and the second reporter (en-Venus) labeled all cells of the P compartment and thus visualized the AP boundary (see Fig. 1B). Hand-dissected wing imaginal discs were then viewed under a fluorescence microscope. Segregation defects were recognized as en-Venus negative clones of cells located in the posterior territory of the wing imaginal disc or by en-Venus positive clones in the anterior territory of the wing imaginal disc (Fig. 1B).


An RNA interference screen for genes required to shape the anteroposterior compartment boundary in Drosophila identifies the Eph receptor.

Umetsu D, Dunst S, Dahmann C - PLoS ONE (2014)

An assay to identify genes required to maintain the straight shape of the AP boundary.A. Scheme of tester stock and cross to generate clones of cell expressing double-stranded RNA. The tester stock contains a Flp recombinase transgene under control of the heat-shock inducible promoter hsp70 (hs) on chromosome I [36] and the engrailed-Venus transgene on chromosome II (not shown). Chromosome III contains a transgene composed of the Act5C promoter followed by a FRT site, the coding sequence for the mouse transmembrane protein CD2 followed by a transcriptional terminator (Term), a second FRT site, and finally Gal4. We have recombined on the same chromosome a transgene expressing DsRed under control of UAS sequences (not shown). The tester stock is crossed to individual RNAi fly lines. The resulting progeny larvae are heat-shocked to express the Flp recombinase that in turn induces recombination between the two FRT sites on the tester stock. This recombination places Gal4 under the direct control of the Act5C promoter leading to Gal4 expression specifically in the clones of cells. Gal4 then binds to the UAS sequences in the RNAi line and directs expression of double-stranded RNA that leads to RNA interference. At the same time, Gal4 will induce expression of DsRed (not shown) in the same cells. B. Scheme of the assay. The central part of the wing imaginal disc is schematically drawn as an oval. A Venus fluorescent protein (green) is specifically expressed in all cells of the posterior compartment under control of the promoter sequences of the engrailed gene (en-Venus). Clones of cells expressing double stranded RNA are identified by co-expression of DsRed (red). Examples on the left depict wild-type clones located in the anterior (A) or posterior (P) compartments. Examples on the right depict a anterior clone mis-segregating into the posterior territory (top) and an posterior clone mis-segregating into the anterior territory (bottom) of the wing disc. Clones co-expressing DsRed and Venus are depicted in yellow.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4256218&req=5

pone-0114340-g001: An assay to identify genes required to maintain the straight shape of the AP boundary.A. Scheme of tester stock and cross to generate clones of cell expressing double-stranded RNA. The tester stock contains a Flp recombinase transgene under control of the heat-shock inducible promoter hsp70 (hs) on chromosome I [36] and the engrailed-Venus transgene on chromosome II (not shown). Chromosome III contains a transgene composed of the Act5C promoter followed by a FRT site, the coding sequence for the mouse transmembrane protein CD2 followed by a transcriptional terminator (Term), a second FRT site, and finally Gal4. We have recombined on the same chromosome a transgene expressing DsRed under control of UAS sequences (not shown). The tester stock is crossed to individual RNAi fly lines. The resulting progeny larvae are heat-shocked to express the Flp recombinase that in turn induces recombination between the two FRT sites on the tester stock. This recombination places Gal4 under the direct control of the Act5C promoter leading to Gal4 expression specifically in the clones of cells. Gal4 then binds to the UAS sequences in the RNAi line and directs expression of double-stranded RNA that leads to RNA interference. At the same time, Gal4 will induce expression of DsRed (not shown) in the same cells. B. Scheme of the assay. The central part of the wing imaginal disc is schematically drawn as an oval. A Venus fluorescent protein (green) is specifically expressed in all cells of the posterior compartment under control of the promoter sequences of the engrailed gene (en-Venus). Clones of cells expressing double stranded RNA are identified by co-expression of DsRed (red). Examples on the left depict wild-type clones located in the anterior (A) or posterior (P) compartments. Examples on the right depict a anterior clone mis-segregating into the posterior territory (top) and an posterior clone mis-segregating into the anterior territory (bottom) of the wing disc. Clones co-expressing DsRed and Venus are depicted in yellow.
Mentions: We sought to identify genes required to maintain the shape of the AP boundary by RNA interference. Expression of double-stranded hairpin RNA from a transgene can result in the RNA interference mediated knock-down of gene function in Drosophila[33]. We developed an assay to assess whether the knock-down of a particular gene influences the shape of the AP boundary. In this assay, double-stranded hairpin RNA is expressed in clones of cells using the Gal4-UAS [34] and FRT-flp [35], [36] systems (Fig. 1A). Clones of cells located along the AP boundary were tested whether or not they influenced the shape of the AP boundary (Fig. 1B). To identify the clones of cells and the AP boundary, we used two fluorescent reporters. One reporter (DsRed) identified the clone of cells that expressed the double-stranded RNA and the second reporter (en-Venus) labeled all cells of the P compartment and thus visualized the AP boundary (see Fig. 1B). Hand-dissected wing imaginal discs were then viewed under a fluorescence microscope. Segregation defects were recognized as en-Venus negative clones of cells located in the posterior territory of the wing imaginal disc or by en-Venus positive clones in the anterior territory of the wing imaginal disc (Fig. 1B).

Bottom Line: Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, we identified a single novel candidate that interfered with the formation of a straight anteroposterior compartment boundary.Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos.Our results identify a hitherto unknown role of the Eph receptor tyrosine kinase in Drosophila and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics, Technische Universit├Ąt Dresden, Dresden, Germany.

ABSTRACT
The formation of straight compartment boundaries separating groups of cells with distinct fates and functions is an evolutionarily conserved strategy during animal development. The physical mechanisms that shape compartment boundaries have recently been further elucidated, however, the molecular mechanisms that underlie compartment boundary formation and maintenance remain poorly understood. Here, we report on the outcome of an RNA interference screen aimed at identifying novel genes involved in maintaining the straight shape of the anteroposterior compartment boundary in Drosophila wing imaginal discs. Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, we identified a single novel candidate that interfered with the formation of a straight anteroposterior compartment boundary. Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos. Our results identify a hitherto unknown role of the Eph receptor tyrosine kinase in Drosophila and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development.

Show MeSH