Figure 1.
Dg is expressed by R cells during eye development.
Third instar larval (A–E) and pupal (F–H) eye discs stained with DG-C antibody. A–C, third instar eye disc with a small Df(2R)Dg248 clone, identified by lack of GFP (C, green), stained with anti-DG-C (A, red in C) and 24B10 (B, blue in C). Areas lacking GFP also lack DG-C staining demonstrating specificity of the antibody (A and C). Posterior to the morphogenetic furrow (left of arrow) DG-C is concentrated at the apical surface of R cells that are identified by 24B10 expression (B and C, blue). (D–E) whole mount wild type third instar disc stained with DiI to highlight membranes (D) and DG-C (E), viewed onto the basal surface of the disc. DG-C (E) localizes to the basal surface of the disc, in the anterior region, and at the morphogenetic furrow. (F–H) eye disc at 40% pupal development carrying a small Df(2R)Dg248clone, identified by lack of GFP (G and H, green). GFP positive wild type R cells, highlighted by the membrane bound GFP (G and H, green), localize DG-C (F and H, red) to the apical surface. In A–C the scale bar is 20 µm, D–E the scale bar is 25 µm and the arrows or asterisks indicates the position of the morphogenetic furrow with the posterior region to the left and anterior region to the right. Scale bar in F–H is 10 µm.
Figure 2.
(A, B) Transverse retinal sections of adult eyes stained with F-actin to highlight the R cell rhabdomeres. The retina of DgO55/DgO43 (B) is thinner than wild type (A) and R cells are obviously shorter. (C) Chart shows mean rhabdomere lengths in wild type and Dg mutants (wild type n = 30; DgO55/DgO55 n = 51; DgO38/DgO86 n = 23; DgO38/DgO43 n = 14). Scale bars in (A) represents 75 µm.
Figure 3.
Over-expression of Dg-C in R cells increases ommatidial size.
Over-expression of Dg-C was demonstrated with a concentration of anti-DGcyto antibody too low to detect wild type levels of DG (A) in third instar discs that revealed clear labeling of omatidia in transgenic flies (B). Cross sections of ommatidia (outlined) in wild type flies (C) and flies containing UAS- Dg-C/+ alone (D), elav-Gal4/+ alone (E) and elav-Gal4/UAS-Dg-C (F) showed that over-expression of Dg-C in R cells induces bigger ommatidia and expansion of R cells (F) as compared with the controls (C–E). The number of R cells remains unchanged (F). Quantification of these data shows that the ommatidia perimeters (G) are slightly but significantly increased when Dg-C is over-expressed in R cells driven by elav-Gal4 as are ommatidial areas (H). **, P<0.01. Bar, 10 µm.
Figure 4.
Large mosaic clones of Df(2R)Dg248 results in disruption of the adult eye.
Light and scanning EM micrographs of the wild type fly eye (A–F) show the classical ommatidial facets and bristles of the external eye. A′–F′, show the equivalent micrographs of fly eyes of Df(2R)Dg248 clones generated by the ey, FLP/FRT system. The Df(2R)Dg248 tissue in A′ is white while tissue with heterozygous expression of the wild type gene is red. The Df(2R)Dg248 (white) regions appear flattened and glossy (A′). Cross sections of the internal eye reveal that the normal (B) array of R-cells with central rhabdomeres is disrupted (B′ arrows) and there appears to be cell debris (B′ arrowhead) in these regions. Electron micrographs reveal regions where the external eye has collapsed and the facets have been obliterated (C′, D′). A transverse section through the eyes reveals the normal length (E, double arrow head) of the retina and its ordered array of ommatidia (arrow). The Df(2R)Dg248 (white) region appears disrupted (arrow) and the retinal thickness is shorter (double arrow head). The lenses, which normally (F) form a biconvex disc, are flattened on their external, but not internal, faces though the cuticle that forms the external boundary is visible within a flattened and partially “empty” lens (arrow, F′). (G) ERGs were recorded after a 5 min dark adaptation followed by a 2 sec bright-light pulses (top trace). The bottom trace shows representative ERGs of wild type (left) and Df(2R)Dg248 regions (white) of mosaic eyes. In Df(2R)Dg248 regions the 9-14 mV R cell depolarization (left) was greatly diminished and the early and late synaptic transients (left) completely abolished. Quantification of ERG parameters in wild type and Df(2R)Dg248 patches showed significant differences (P<0.01) between wild type and Df(2R)Dg248 eyes.
Figure 5.
Third instar development is not disrupted in Df(2R)Dg248 mosaics.
Immunohistochemistry with antibodies to 24B10 (A, D), Elav (B, E) and Boss (Bride of Sevenless) (C, F) identify R cells in wild type (A–C) and ey/FLP induced mosaic eye imaginal discs (D–F) in the late 3rd instar larvae. 24B10 (A and D) and Elav (B and E), localize normally (A, B) in Df(2R)Dg248 deficient eye discs (D, E). Similarly Boss, which is expressed on the apical surface of R8 and necessary for formation of R7, is localized normally (C) in Df(2R)Dg248 deficient eye discs (F). Bar represents 10 µm.
Figure 6.
Df(2R)Dg248 is required for photoreceptor organization, growth and survival during pupal development.
A) An antibody, 24B10, was used to identify R cells in eye discs of 20% pupal development, mosaic for wild type and DG-deficient cells generated by FLP/FRT system. A line in (A) separates a disrupted region from a normal region. In 20 such preparations there were many small R cells (arrows, A) adjacent to patches with normal ommatidia. (B) Quantification of the lengths of lateral (apical to basal axis) membranes and (circumferential) basal membranes of R cell showed that those in disrupted regions were significantly shorter (**, P<0.01). C–E) 20% pupal development eye disc with small clones of Df(2R)Dg248 cells. Individual R-cells labeled for 24B10 (C). Df(2R)Dg248 clones are identified by a lack of GFP labeling (D). The merge (E) reveals the morphology of the Df(2R)Dg248 photoreceptors. An ommatidium composed of only Df(2R)Dg248 R cells (arrowhead) shows disorganized R cells. A single wild type R cell (arrow) has a normal morphology within an otherwise disorganized Df(2R)Dg248 ommatidium.
Figure 7.
An mRpL34 transgene rescues external eye defects associated with Df(2R)Dg248.
SEM of two day old female adult eyes (A–E) and the corresponding histology (A′–E′). A wild type eye shows the ordered array of ommatidia both on the surface (A) and within the retina (A′). On the surface, a Dg043 eye (B) has a small region (arrow) of mispatterning of ommatidia. Within the Dg043 retina (B′) the patterning of the retina is normal. The surface of a Dg086 eye (C) as well as the within the retina (C′) shows a normal pattern of ommatidia. The Dg086 eye shape is more spherical relative to wild type. A Df(2R)Dg248 mosaic eye has a grossly disrupted array of ommatidia on its surface (D) as well as within (D′). Expression of an mRpL34 transgene in a Df(2R)Dg248 mosaic eye significantly rescues the external and internal eye disruption (E and E′). The scale bars are 100 µm (A–E); 10 µm (A′–E′). Line marks the retinal equator in A′ and C′.