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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 Universität 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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Validation of the phenotype of Eph RNAi lines.A. Exon-intron structure of the Eph gene and regions targeted by the indicated RNAi lines. B–D. Wing imaginal discs displaying clones of cells expressing double-stranded RNA targeting Eph using the RNAi lines (B) 4771, (C) GL01189 or (D) GL00192. The clones of cells are identified by co-expression of DsRed (red); cells of the posterior compartment are labeled by expression of Venus under control of the engrailed gene (en-Venus, green). Two clones expressing EphdsRNA located in different compartments sharing a common interface along the AP boundary locally distort the shape of the AP boundary (asterisks). In (B), dcr2 is co-expressed to increase the efficiency of RNAi. E. Number of cases where two clones expressing double-stranded RNA targeting Eph located in different compartments sharing a common interface along the AP boundary distort the AP boundary towards the posterior side (A>P), towards the anterior side (P>A), or do not distort the AP boundary (no effect). Scale bar is 50 µm.
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pone-0114340-g006: Validation of the phenotype of Eph RNAi lines.A. Exon-intron structure of the Eph gene and regions targeted by the indicated RNAi lines. B–D. Wing imaginal discs displaying clones of cells expressing double-stranded RNA targeting Eph using the RNAi lines (B) 4771, (C) GL01189 or (D) GL00192. The clones of cells are identified by co-expression of DsRed (red); cells of the posterior compartment are labeled by expression of Venus under control of the engrailed gene (en-Venus, green). Two clones expressing EphdsRNA located in different compartments sharing a common interface along the AP boundary locally distort the shape of the AP boundary (asterisks). In (B), dcr2 is co-expressed to increase the efficiency of RNAi. E. Number of cases where two clones expressing double-stranded RNA targeting Eph located in different compartments sharing a common interface along the AP boundary distort the AP boundary towards the posterior side (A>P), towards the anterior side (P>A), or do not distort the AP boundary (no effect). Scale bar is 50 µm.

Mentions: Finally, we used six additional RNAi lines to test whether the distortion of the AP boundary was due to the targeting of Eph. The regions of the Eph gene targeted by these RNAi lines are shown in Fig. 6A. RNAi lines 1511R-1, GL01189, GL00192, and 4771 were targeting non-overlapping regions of the Eph gene (Fig. 6A). Clones expressing double-stranded RNAs from any of these four additional RNAi lines had smooth edges (Fig. 6B–D and data not shown). Moreover, clones interfacing from the anterior and posterior side did disturb the shape of the AP boundary, similar to the RNAi line originally used in the screen (Fig. 6B–D and data not shown). In the case of clones targeting Eph, the AP boundary could either be distorted towards the anterior or towards the posterior side, although distortions to the posterior side were more frequently observed (Fig. 6B–E). Clones targeting Eph did not distort the shape of the DV boundary (Fig. S1A, n = 15 pairs of clones interfacing from the dorsal and ventral side). These data indicate a specific role of the Eph receptor in shaping the AP boundary.


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)

Validation of the phenotype of Eph RNAi lines.A. Exon-intron structure of the Eph gene and regions targeted by the indicated RNAi lines. B–D. Wing imaginal discs displaying clones of cells expressing double-stranded RNA targeting Eph using the RNAi lines (B) 4771, (C) GL01189 or (D) GL00192. The clones of cells are identified by co-expression of DsRed (red); cells of the posterior compartment are labeled by expression of Venus under control of the engrailed gene (en-Venus, green). Two clones expressing EphdsRNA located in different compartments sharing a common interface along the AP boundary locally distort the shape of the AP boundary (asterisks). In (B), dcr2 is co-expressed to increase the efficiency of RNAi. E. Number of cases where two clones expressing double-stranded RNA targeting Eph located in different compartments sharing a common interface along the AP boundary distort the AP boundary towards the posterior side (A>P), towards the anterior side (P>A), or do not distort the AP boundary (no effect). Scale bar is 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114340-g006: Validation of the phenotype of Eph RNAi lines.A. Exon-intron structure of the Eph gene and regions targeted by the indicated RNAi lines. B–D. Wing imaginal discs displaying clones of cells expressing double-stranded RNA targeting Eph using the RNAi lines (B) 4771, (C) GL01189 or (D) GL00192. The clones of cells are identified by co-expression of DsRed (red); cells of the posterior compartment are labeled by expression of Venus under control of the engrailed gene (en-Venus, green). Two clones expressing EphdsRNA located in different compartments sharing a common interface along the AP boundary locally distort the shape of the AP boundary (asterisks). In (B), dcr2 is co-expressed to increase the efficiency of RNAi. E. Number of cases where two clones expressing double-stranded RNA targeting Eph located in different compartments sharing a common interface along the AP boundary distort the AP boundary towards the posterior side (A>P), towards the anterior side (P>A), or do not distort the AP boundary (no effect). Scale bar is 50 µm.
Mentions: Finally, we used six additional RNAi lines to test whether the distortion of the AP boundary was due to the targeting of Eph. The regions of the Eph gene targeted by these RNAi lines are shown in Fig. 6A. RNAi lines 1511R-1, GL01189, GL00192, and 4771 were targeting non-overlapping regions of the Eph gene (Fig. 6A). Clones expressing double-stranded RNAs from any of these four additional RNAi lines had smooth edges (Fig. 6B–D and data not shown). Moreover, clones interfacing from the anterior and posterior side did disturb the shape of the AP boundary, similar to the RNAi line originally used in the screen (Fig. 6B–D and data not shown). In the case of clones targeting Eph, the AP boundary could either be distorted towards the anterior or towards the posterior side, although distortions to the posterior side were more frequently observed (Fig. 6B–E). Clones targeting Eph did not distort the shape of the DV boundary (Fig. S1A, n = 15 pairs of clones interfacing from the dorsal and ventral side). These data indicate a specific role of the Eph receptor in shaping the AP boundary.

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 Universität 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