Fig 1.
omb expression level influences eye size.
(A) w ombP1 (an enhancer trap insertion that does not affect omb expression and function, Sun et al., 1995), (B) w ombP1 l(1)omb3198/w ombbi. The expanded territory of ventral eye fate is clearly evident. Because of the increased size, the eye surface is more convex. Therefore, the unaffected dorsal pigmentation is not fully visible under this angle. (C) ombP3>omb-RNAi showed strong overgrowth in the eye. The overgrowth is stronger in the ventral than in the dorsal part of the eye. The eye is convoluted. (D-E) mirr-lacZ (anti-beta-galactosidase, red). Phalloidin staining (green). Elav (blue). (D-D”’) mirr-lacZ/+ eye disc showing the dorsal-specific expression of mirr-lacZ. D, D’ and D”, D”’ are two focal planes. The D”, D”’ focal plane shows the ventral flap. (E-E”’) l(1) ombD4/ ombP7 eye disc. E, E’ and E”, E”’ are two focal planes. The E”, E”’ focal plane shows the ventral flap. The dorsal and ventral eye regions were distinguished (separated by a white line) based on mirr-lacZ and the position of the optic stalk. Two different focal planes are acquired in each eye disc. The area of eye disc including the ventral flap, based on two focal planes, were measured by the software, Zeiss Zen 2009. The results are summarized in (F). The ventral area of l(1)ombD4/ombP7; mirr-lacZ/+ are significantly enlarged compared to that of mirr-lacZ/+. The dorsal area of l(1)ombD4/ombP7; mirr-lacZ/+ are not significant increased compared to that of mirr-lacZ/+. Differences (*) presented in (E) and (J) are significant (Student′s t-test, **, p<0.05; n.s., non-significant). In all panels anterior is left and dorsal up.
Fig 2.
Omb blocks cell proliferation in eye disc.
Cell proliferation was monitored by staining against the mitotic marker phospho-histone 3 (pH3), BrdU incorporation, and by comparing clone size in late third instar eye discs. (A-D) Arrowhead points to the position of the optic stalk. (A) Wild type eye disc showing the two mitotic waves (arrows) labeled by anti-pH3 (red). (B) The ombP7 mutant showed an increased number of pH3-positive nuclei in the ventral eye compared to wild type. (C, D) BrdU incorporation showed an increase of proliferating cells in the ventral flap (arrow) of the ombP7 mutant eye disc (D) compared to the wild type eye disc (C). (E) mirr-lacZ was used to mark the dorsal region. pH3 positive cells were scored in ombP7/Y; mirr-lacZ/+ and mirr-lacZ dorsal and ventral eyes. In order to include the ventral flap area, the images of several optical sections were merged. The quantification results are summarized in (E). The mitotic cells in ventral eye of ombP7 is significant increased compared to ventral eye in wild type (p<0.05). (F, G) A wild type eye disc with clones (marked by the absence of GFP) at two focal planes to show the central region (F) and the ventral and dorsal flap regions (G). The clones were of similar size in all regions (summarized in J). (H, I) An eye disc with l(1)ombD4 clones (marked by the absence of GFP) at two focal planes to show the central (H) and ventral and dorsal flap regions (I). The wild type clones and l(1)ombD4 mutant clones were induced at the same time. The l(1)ombD4 clones in the ventral flap were on average about 3.5 times larger than omb clones in the central region of the disc or than wild type clones (summarized in J). Differences (*) presented in (E) and (J) are significant (Student′s t-test, *** p<0.001; **, p<0.05).
Fig 3.
Ectopic omb expression can block morphogenetic furrow initiation, progression, and differentiation.
Flip-out induced omb expression clones (Act5C>omb) marked by GFP coexpression repressed Elav (cyan) and Eya (red) expression. (A-A”) A clone at the posterior margin (arrow) inhibited MF initiation. (B-B”) A clone at the MF (arrow) inhibited MF progression (as indicated by CycB pattern, red) and neuronal differentiation (Elav, cyan). (C-C”) Omb expression level in Act5C>omb clones varied. Omb expression in a single ommatidial clusters (arrows) could autonomously block neuronal differentiation (Elav, cyan). The Z-section along the white line is shown in C-1 to C-1”. The relative level of Omb induction correlates to the signal of coexpressed GFP. (D) Tangential semi-thin sections through an adult eye of an hs-omb transgenic fly exposed to a single 1hr 37°C heat shock during mid-L3. Ommaditial patterning resumed normally beyond the dorso-ventral scar (arrow).
Fig 4.
Omb blocks Jak/STAT signaling.
10XSTAT-GFP is a reporter of Jak/STAT signaling [76]. We added a nuclear localizing signal (nls) to obtain 10XSTAT-GFP-nls. (A) 10XSTAT-GFP-nls expression pattern (GFP, green) in wild type third instar eye disc. (B) The 10XSTAT-GFP-nls was ectopically expressed in the ventral eye margin (arrow) in an ombP7 hypomorphic mutant eye disc. (A, B) The position of the MF, based on the DIC image, is marked by an arrowhead. (C) STAT-lacZ is repressed by Jak/STAT signaling. In wild type late third instar eye disc, its expression was strong in the lateral poles and weaker around the DV midline, as reported [57]. (D) In l(1)omb15/Y eye discs, STAT-lacZ expression was attenuated in the ventral region. (E-E”’) 10XSTAT-GFP-nls (green) was ectopically induced in l(1)ombD4 mutant clones (clone marked by loss of RFP (red) expression and by dashed line). (E’-E”’) Higher magnification of the square marked in (E). 10XSTAT-GFP-nls was non-autonomously induced by loss of omb in the ventral margin. (F) dpp>hop+GFP caused an enlargement of the eye disc (Elav, red; GFP, green). (G) Coexpression of hop with omb (dpp>omb+hop) could largely rescue the dpp>omb phenotype (dpp-lacZ, green; Elav, red). (H-H’) Reducing STAT dosage in ombP7/Y; STAT92E397/+ larvae reduced the size of the ventral retinal field compared to that in ombP7/Y (Fig. 2B). Different focal planes of ombP7/Y; STAT92E397/+ were shown in H and H’. The quantified eye areas are summarized in (I).
Fig 5.
omb can repress upd transcription.
(A-A”’) omb expression clone induced by Act5C-GAL4 suppressed upd-lacZ expression (Act5C>omb+GFP in upd-lacZ). (A’-A”’) are a higher magnification of the area marked in (A). GFP (green) marks the Omb expressing cells. Omb expressing cells suppressed upd-lacZ (red). (B-C) RNA in situ hybridization of third instar eye discs. (B) In late third instar, no signal was detected by upd anti-sense probe in wild type eye disc. (C) upd mRNA was ectopically expressed in the ventral eye margin of ombP7/Y. (D, E) Coexpression of omb with upd (in dpp>omb+upd) partially or fully rescued retinal development in the eye disc (D) and adult eye (E). (Elav, red; Wg, blue)
Fig 6.
Functional relationship between wg and omb.
(A) dpp>arm+GFP eye discs were reduced in size and showed no neuronal differentiation (Wg, red; Elav, cyan; GFP, Green). (B, D) dpp>arm+omb-RNAi caused partial rescue of eye disc size and neuronal differentiation (B) and adult eye (D). (C, E) dpp>omb-RNAi+GFP did not affect retinal development in eye disc (C) and adult eye (E). Misexpression of arm by ey-GAL4 (ey>arm) caused eye size reduction in adult eye (F) and in eye disc (J). Reduction of omb genetic dosage (l(1)ombD4/+) in the background of ey>arm partially rescued the eye size with full penetrance in adult (G-I) and in eye disc (K, L). 31% of these eye discs showed ventral expansion of retinal differentiation. Interestingly, 44~50% of l(1)ombD4/+; ey>arm flies have dorsal ectopic eyes in adult (I) and in eye discs (6L).