Limits...
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.

Show MeSH
Examples of phenotypes observed in the RNA interference screen.A–F. Wing imaginal discs displaying clones of cells expressing double-stranded RNA targeting (A) α-cat, (B) wts, (C) PlexA, (D) ph-p, (E) kto, and (F) mad. 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). Clones of cells distorting the AP boundary are marked by an asterisk. Scale bars are 50 µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256218&req=5

pone-0114340-g004: Examples of phenotypes observed in the RNA interference screen.A–F. Wing imaginal discs displaying clones of cells expressing double-stranded RNA targeting (A) α-cat, (B) wts, (C) PlexA, (D) ph-p, (E) kto, and (F) mad. 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). Clones of cells distorting the AP boundary are marked by an asterisk. Scale bars are 50 µm.

Mentions: 428 out of the 3114 RNAi lines resulted in clones of cells that in terms of size, shape, appearance or sorting at the AP boundary differed from control clones (Fig. 3B, Table S1). 68.7% of these 428 RNAi lines led to clones of cells that did not have a coherent appearance as control clones had, but in which cells were rather dispersed. An example for such an RNAi line is line 19182 (VDRC) (Fig. 4A), which targets the gene α-catenin. α-catenin encodes a component of adherens junctions that is essential for cell survival in wing imaginal discs [39]. We speculate that the dispersed clone appearance is due to cell death. 3.7% and 6.3% of the RNAi lines yielded clones that were smaller or larger than control clones, respectively. For example, RNAi line 9928 (VDRC), which targets the gene warts, resulted in larger clones of cells (Fig. 4B). These data are consistent with the known function of Warts as a tumor suppressor [40]. These differences in clone size are thus consistent with a role of the targeted genes in growth control. 15.9% of the 428 RNAi lines gave rise to clones that had a smooth clonal border, indicating that the targeted genes play a role in cell-cell recognition or cell-cell adhesion. Clones expressing these double-stranded RNAs, however, did not influence the shape of the AP boundary. An example of such an RNAi line is line 27240 (VDRC) (Fig. 4C), which targets the gene Plexin A. Plexin A encodes a transmembrane receptor protein tyrosine kinase required, for example, for axon guidance in the nervous system [41]. A further RNAi line that fell into this class was 10679 (VDRC), which targets ph-p, a member of the Polycomb group of genes. In contrast to the other lines in this category, expression of double-stranded RNA from this line resulted in the expression of the Venus marker in anterior cells (Fig. 4D), consistent with the function of Polycomb group genes to repress homeotic genes. Finally, nine out of the 428 RNAi lines (2.1%) resulted in clones of cells that did display sorting defects along the AP boundary and locally disturbed the shape of the AP boundary. Six of these RNAi lines targeted genes encoding components of signal transduction pathways or transcriptional regulators that were previously shown to be required to maintain the A/P boundary (ci, smo, punt, mad, kto (2x) [23], [24], [26], [27], [42]; Fig. 4E–F, Table S1). The ‘blind’ identification of these 6 RNAi lines furthermore confirms the feasibility of our screen.


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)

Examples of phenotypes observed in the RNA interference screen.A–F. Wing imaginal discs displaying clones of cells expressing double-stranded RNA targeting (A) α-cat, (B) wts, (C) PlexA, (D) ph-p, (E) kto, and (F) mad. 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). Clones of cells distorting the AP boundary are marked by an asterisk. Scale bars are 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114340-g004: Examples of phenotypes observed in the RNA interference screen.A–F. Wing imaginal discs displaying clones of cells expressing double-stranded RNA targeting (A) α-cat, (B) wts, (C) PlexA, (D) ph-p, (E) kto, and (F) mad. 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). Clones of cells distorting the AP boundary are marked by an asterisk. Scale bars are 50 µm.
Mentions: 428 out of the 3114 RNAi lines resulted in clones of cells that in terms of size, shape, appearance or sorting at the AP boundary differed from control clones (Fig. 3B, Table S1). 68.7% of these 428 RNAi lines led to clones of cells that did not have a coherent appearance as control clones had, but in which cells were rather dispersed. An example for such an RNAi line is line 19182 (VDRC) (Fig. 4A), which targets the gene α-catenin. α-catenin encodes a component of adherens junctions that is essential for cell survival in wing imaginal discs [39]. We speculate that the dispersed clone appearance is due to cell death. 3.7% and 6.3% of the RNAi lines yielded clones that were smaller or larger than control clones, respectively. For example, RNAi line 9928 (VDRC), which targets the gene warts, resulted in larger clones of cells (Fig. 4B). These data are consistent with the known function of Warts as a tumor suppressor [40]. These differences in clone size are thus consistent with a role of the targeted genes in growth control. 15.9% of the 428 RNAi lines gave rise to clones that had a smooth clonal border, indicating that the targeted genes play a role in cell-cell recognition or cell-cell adhesion. Clones expressing these double-stranded RNAs, however, did not influence the shape of the AP boundary. An example of such an RNAi line is line 27240 (VDRC) (Fig. 4C), which targets the gene Plexin A. Plexin A encodes a transmembrane receptor protein tyrosine kinase required, for example, for axon guidance in the nervous system [41]. A further RNAi line that fell into this class was 10679 (VDRC), which targets ph-p, a member of the Polycomb group of genes. In contrast to the other lines in this category, expression of double-stranded RNA from this line resulted in the expression of the Venus marker in anterior cells (Fig. 4D), consistent with the function of Polycomb group genes to repress homeotic genes. Finally, nine out of the 428 RNAi lines (2.1%) resulted in clones of cells that did display sorting defects along the AP boundary and locally disturbed the shape of the AP boundary. Six of these RNAi lines targeted genes encoding components of signal transduction pathways or transcriptional regulators that were previously shown to be required to maintain the A/P boundary (ci, smo, punt, mad, kto (2x) [23], [24], [26], [27], [42]; Fig. 4E–F, Table S1). The ‘blind’ identification of these 6 RNAi lines furthermore confirms the feasibility of our screen.

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