Broad is dynamically expressed in optic lobe NEs during larval stages

The expression of Broad protein in late-third instar larval brains was previously reported (Spokony and Restifo 2009; Zhou et al. 2009). We wanted to see how Broad is expressed in earlier larval stages, thus further examined Broad expression in the larval optic lobe using an antibody that recognizes the N-terminal common region of all Broad isoforms. Broad is not expressed in the optic lobe at late-second instar (48 hr ALH) (Figure 1, D and D’). At early-third instar (60 hr ALH), Broad is weakly expressed in the NEs of the OPC and IPC, and in the medulla (Figure 1, E and E’); Broad expression increases by midthird instar (72 hr ALH) (Figure 1, F and F’) and reaches a high level at late-third instar (96 hr ALH) (Figure 1, G, and G’). Broad is also detected in the developing lamina after midthird instar (Figure 1, F, F', G, G', and H). The timing of Broad expression parallels the NE-to-NB transition in the optic lobe, suggesting a possible role of Broad in NE differentiation into NBs.

To verify which Broad isoform is expressed in specific regions of the optic lobe, we used isoform-specific Broad antibodies. Br-Z1 becomes detectable at early-third instar in a few NEs and NBs at the medial edge of the OPC neuroepithelium (Figure 1, J, and J’, arrowheads), and accumulates in the OPC NEs to a relatively high level by late-third instar (Figure 1, K, K', L, L’ and M). Br-Z1 is also expressed in the IPC, in the developing lamina, and in young but not old medulla neurons (Figure 1, K, K', L, L’ and M). In contrast, Br-Z3 is not expressed in the OPC or IPC NEs during the entire larval stages, but is expressed in older medulla neurons at early-third instar (Figure 1O’, arrow) and accumulates to a high level by late-third instar (Figure 1, Q’ and R; Spokony and Restifo 2009; Zhou et al. 2009).

Loss of Broad function causes lamina and medulla defects in the optic lobe

To analyze the potential role of Broad in the optic lobe, we knocked down Broad activity using two br RNAi lines (RNAi1 and RNAi2 refer to BL#27272 and BL#33641, respectively) that target the common region of all Broad isoforms. The Gal4 drivers used, NP7340-Gal4 and c768-Gal4, are both active in the OPC and IPC NEs (Figure S1, A–B’ and E–F’; Yasugi et al. 2010; Wang et al. 2013). The progenies from these crosses were cultured at 31° until the wandering larval stage when the brains were examined. The RNAi knockdown was efficient as no Broad protein was detected in the OPC NEs of br RNAi brains (Figure S2, B’, C’, D’, and E’); interestingly, Broad expression in medulla neurons was also largely eliminated in these br RNAi brains (Figure S2, B’, C’, D’, and E’), even though the Gal4 drivers were not active in the medulla cortex (Figure S1, A, A’, E, and E’). br RNAi knockdown in the brain caused a delay of larval development, as it took 6–8 hr more for br mutant larvae to reach the wandering larval stage.

We next examined the potential defects of br RNAi brains using specific brain markers. The lamina in a wild-type brain is a crescent-shaped structure that can be visualized by staining for the nuclear protein Dachshund (Figure 2, A and A’). br RNAi brains had a much smaller lamina that often had an abnormal morphology (Figure 2, C, C’, E, and E’, 100%, n = 13). To analyze the medulla, we stained the brains for Elav protein to visualize the medulla cortex, which is dome-shaped in the wild-type brain at late-third instar (Figure 2, A, B, and B’). The medulla of br RNAi brains contained cortex areas that lacked Elav⁺ neurons (Figure 2, C, D, D’, E, F, and F’, arrows, 71%, n = 21), but were filled with cells expressing Armadillo, a marker strongly expressed in NEs (Figure 2, D and F, arrowheads). Thus, the loss of Broad function caused defects in both the lamina and the medulla. These defects could have resulted from a defect in NE proliferation and/or their differentiation to NBs. Below, we designed experiments to test these possibilities.

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Figure 2

Loss of Broad function leads to lamina and medulla defects in the optic lobe. Late-third instar brains stained for Elav (blue), Dac (red), and Arm (green). (A–B’) WT brains have a crescent-shaped lamina (A and A’) and a dome-shaped medulla (A, B, and B’). (C–D’) NP7340-Gal4/UAS-br^RNAi1 brains have a very thin and defective lamina (C and C’), and a medulla with a cortex area that lacks Elav⁺ neurons (C, D, and D’, arrow) but contains Arm⁺ cells ((D) arrowhead). (E–F’) NP7340-Gal4/UAS-br^RNAi2 brains have a small and defective lamina (E and E’), and a medulla with cortex areas that lack Elav⁺ neurons (E, F, and F’, arrows) but contain Arm⁺ cells ((F) arrowhead). (A, A’, C, C’, E, and E’) are three-dimensional projection images; (B, B’, D, D’, F, and F’) are single confocal section images. Lateral is to the right. Bar, 20 μm in (A) for (A, A’, C, C’, E, and E’), and in (B) for (B, B’, D, D’, F, and F’). Arm, Armadillo; Dac, Dachshund; RNAi, RNA interference; WT, wild-type.

Loss of Broad function prolongs neuroepithelial growth and inhibits NE-to-NB transition

To understand how Broad affects optic lobe development, we followed the process of NE growth and differentiation in the OPC from early-to-late larval stages. We examined NE morphology and numbers, NE proliferation, and their differentiation into medulla NBs. The columnar NEs can be visualized by staining for adherens junction proteins DE-cadherin and Armadillo, or Dlg, which outlines all cell cortices; medulla NBs are quite large, rounded cells that express Dpn.

Compared with wild-type brains, br RNAi brains at early-third or midthird instar had normal numbers of OPC NEs that had normal cell morphology (Figure 3, A, B, D, and E; quantified in Figure 3, G and H). However, late-third instar br RNAi brains contained significantly more NEs as compared with wild-type brains (Figure 3, C and F; quantified in Figure 3G) and the mutant NEs were more elongated than wild-type NEs at this stage (Figure 3, C and F; quantified in Figure 3H), resembling wild-type OPC NEs at midthird instar (Figure 3F, compare with Figure 3B; quantified in Figure 3H).

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Figure 3

broad RNAi knockdown affects neuroepithelial growth and delays NE differentiation. (A–F) Neuroepithelial growth during the larval stages. Brains stained for Dlg (red), DE-cadherin (green), and Dpn (blue). Brackets indicate the region of NEs. (A–C) WT early-third instar (A), midthird instar (B), and late-third instar (C) brains. (D–F) c768-Gal4/UAS-br^RNAi2 early-third instar (D), midthird instar (E), and late-third instar (F) brains. (G) Quantification of NE numbers for experiments shown in (B, C, E, and F). NEs from a single confocal section of each brain at the level of maximal NE length were counted. Number (n) of brains indicated. Error bars indicate mean ± SD, **** P < 0.0001; Student’s t-test. (H) Quantification of NE lengths for experiments shown in (B, C, E, and F). The lengths of two-to-three NEs from a single confocal section of each brain at the level of maximal NE length were measured. Numbers (n) of NEs and brains indicated. Error bars indicate mean ± SD, **** P < 0.0001; one-way ANOVA with Tukey’s test. (I–L) BrdU labeling of NEs. Brains stained for BrdU (green) and Dlg (red). Dashed lines indicate the regions of NEs. (I and J) WT early-third instar (I) and late-third instar (J) brains. (K and L) c768-Gal4/UAS-br^RNAi2 early-third instar (K) and late-third instar (L) brains. (M) Quantification of BrdU-labeled NEs (%) for experiments shown in (I–L). BrdU-labeled and unlabeled NEs from a single confocal section of each brain at the level of maximal NE length were counted, and the percentages of BrdU-labeled NEs were calculated. Number (n) of brains indicated. Error bars indicate mean ± SD, ** P < 0.01; one-way ANOVA with Tukey’s test. (N–Q) PH3 labeling of NEs. Brains stained for PH3 (green) and Dlg (red). Brackets indicate the regions of NEs. (N and O) WT midthird instar (N) and late-third instar (O) brains. (P and Q) NP7340-Gal4/UAS-br^RNAi1 midthird instar (P) and late-third instar (Q) brains. (R) Quantification of PH3⁺ NEs (%) for experiments shown in (N–Q). PH3⁺ and PH3⁻ NEs from a single confocal section of each brain at the level of maximal NE length were counted, and the percentages of PH3⁺ NEs were calculated. Number (n) of brains indicated. Error bars indicate mean ± SD, ** P < 0.01; one-way ANOVA with Tukey’s test. (S-T’) WT (S and S’) and c768-Gal4/UAS-br^RNAi2 (T and T’) late-third instar brains stained for DE-cadherin (green) and Dpn (blue). Arrows indicate medulla NBs. (U) Quantification of NB numbers for experiments shown in (S-T’). Medulla NBs from a single confocal section of each brain at the level of the medulla neuropil being visible were counted. Number (n) of brains indicated. Error bars represent mean ± SD, **** P < 0.0001; Student’s t-test. Lateral is up, medial is down. Bar, 20 μm in (A) for (A–F), in (I) for (I–L), in (N) for (N–Q), and in (S) for (S–T’). BrdU, bromodeoxyuridine; Dlg, Discs large; Dpn, Deadpan; NB, neuroblast; NE, neuroepithelial cell; NS, not significant; PH3, phospho-histone H3; RNAi, RNA interference; WT/wt, wild-type.

We then performed BrdU-labeling assays to determine whether br RNAi knockdown affected NE proliferation. At early-third instar, wild-type and br RNAi NEs had similar rates of BrdU labeling (Figure 3, I and K; quantified in Figure 3M); however, at late-third instar, br RNAi NEs had a significantly higher rate of BrdU labeling than wild-type NEs (Figure 3, J and L; quantified in Figure 3M), and this high labeling rate of br mutant NEs was comparable to that of wild-type NEs at early-third instar (Figure 3M).

We further examined mitotic divisions by staining the NEs with anti-PH3 and found that the mitotic index of br mutant NEs at late-third instar was significantly higher than that of wild-type NEs (Figure 3, O and Q; quantified in Figure 3R), but was comparable to the mitotic index of wild-type NEs at midthird instar (Figure 3R). Thus, late-third instar br RNAi brains looked like younger wild-type brains, and the mutant NEs were highly proliferative even at the late-third instar stage.

Finally, we examined whether br RNAi knockdown affected the differentiation of NEs into NBs. We stained larval brains for the NB marker Dpn. In wild-type brains, the NEs begin to differentiate into medulla NBs at late-second/early-third instar, and by late-third instar many NEs had been converted to NBs. Late-third instar br RNAi brains had abundant NEs but had fewer medulla NBs than wild-type brains (Figure 3, S’ and T’; quantified in Figure 3U), indicating that the loss of Broad slowed down NE differentiation into medulla NBs.

Taken together, these results indicate that br RNAi knockdown resulted in highly proliferative NEs and a delay of NE differentiation into NBs.

Broad overexpression causes premature NE differentiation into NBs

To explore whether Broad is sufficient to drive NE differentiation, we ectopically expressed the different Broad isoforms (Z1–Z4) in the brain. Clones overexpressing Broad were induced by heat-shocking early-third instar larvae (60 hr ALH), which were then cultured at 31° until late-third instar when the brains were examined. Ectopic br-Z1 expression induced ectopic Dpn⁺ cells (Figure 4, B–D). In 16 NE clones examined, 11 clones contained one or two ectopic Dpn⁺ NEs (Figure 4B), while five clones did not. In clones comprising both NEs and medulla cells (n = 12), the NEs did not express Dpn, but one to several Dpn⁺ medulla cells were found immediately under the neuroepithelial layer (Figure 4, C and D). Clones in the medulla cortex also contained one to several Dpn⁺ cells (Figure 4C, arrow; 82/90 clones examined), these clones presumably originated from the NEs. In control clones, no ectopic Dpn⁺ cells were induced either in the NEs or in the medulla (Figure 4A; n = 31). Thus, we conclude that ectopic br-Z1 expression can drive premature NE differentiation into NBs.

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Figure 4

Ectopic Broad expression leads to premature NE differentiation into medulla neuroblasts. (A–I) Clones expressing br-Z1, br-Z2, br-Z3, or br-Z4 were induced, and late-third instar brains were dissected and stained for Dlg (red) and Dpn (blue). GFP (green) marks the clone. (A) A WT control clone. (B–D) br-Z1-expressing clones. (B) A clone contains two Dpn⁺ NEs. (C) A clone contains one NE and three Dpn⁺ medulla cells. Arrow indicates a medulla clone with Dpn⁺ cells. (D) A clone contains two NEs and two Dpn⁺ medulla cells. (E and F) Clones expressing br-Z2. (E) A clone with several lamina precursor cells and two Dpn⁺ NEs. (F) A medulla clone containing one Dpn⁺ cell. (G and H) Clones expressing br-Z3. (G) A clone has two basal Dpn⁺ NEs. (H) A clone contains one Dpn⁺ NE and two Dpn⁺ medulla cells. (I) A br-Z4-expressing clone with NEs and three Dpn⁺ medulla cells. (J–N) Overexpression of br-Z1, br-Z2, br-Z3, or br-Z4 under c768-Gal4 control. Late-third instar brains stained for Dlg (red), Dpn (blue), and DE-cad (green). Compared with WT brains (J), brains overexpressing br-Z1 (K), br-Z2 (L), br-Z3 (M), or br-Z4 (N) had many precocious Dpn⁺ cells but were largely depleted of the outer proliferation center NEs. Lateral is to the left. Bar, 20 μm in (A) for (A–I), and in (J) for (J–N). DE-cad, DE-cadherin; Dlg, Discs large; Dpn, Deadpan; NE, neuroepithelial cell; WT, wild-type.

Interestingly, ectopic expression of br-Z3, which normally is not expressed in the NEs (Figure 1R), also caused premature NE differentiation into NBs (Figure 4, G and H). In eight NE clones examined, six clones contained at least one ectopic Dpn⁺ NE. Shown in Figure 4G is a clone that contained two ectopic Dpn⁺ NEs located on the basal side of the neuroepithelial layer, while another clone contained a Dpn⁺ NE and several Dpn⁺ medulla cells (Figure 4H). All medulla clones examined (n = 63) contained at least one Dpn⁺ cell (data not shown). Ectopic br-Z2 expression also induced ectopic Dpn⁺ NEs (Figure 4E, 7/18 NE clones), as well as ectopic Dpn⁺ cells in medulla clones (n = 53) (Figure 4F). br-Z4 expression in clones similarly led to premature NE differentiation into NBs (Figure 4I; 3/8 NE clones had ectopic Dpn⁺ NEs and 47/47 medulla clones had Dpn⁺ cells). These results suggest that the different Broad isoforms, though normally expressed in different regions of the optic lobe, might regulate similar sets of target genes that promote NE differentiation into NBs when overexpressed.

Consistent with the clonal expression results, overexpression of Broad in the OPC NEs under the control of c768-Gal4 caused premature NE differentiation into NBs (Figure 4, J–N). In these experiments, the direct overexpression of UAS-br-Z1, UAS-br-Z2, UAS-br-Z3, or UAS-br-Z4 driven by c768-Gal4 caused early larval lethality; we thus used a temperature-sensitive Gal4 repressor Gal80^ts to temporally control Broad overexpression. The progenies were first cultured at 18° until second instar, then shifted to 31°, which inactivated the Gal80^ts repressor allowing Broad overexpression. Under these conditions, we obtained late-third instar larval optic lobes that had numerous Dpn⁺ NBs and a depletion of NEs (Figure 4, K–N); these phenotypes were observed in 83.3% of br-Z1-expressing brains (Figure 4K, n = 12), 77.8% of br-Z2-expressing brains (Figure 4L, n = 9), 93.3% of br-Z3-expressing brains (Figure 4M, n = 15), and 87.5% of br-Z4-expressing brains (Figure 4N, n = 8), whereas none of the wild-type brains examined (Figure 4J, n = 15) displayed these abnormalities. We conclude that ectopic Broad expression is sufficient to drive NE differentiation into NBs.

EcR is required for neuroepithelial growth and maintenance, and represses Broad expression

Broad is activated by the EcR signaling pathway during metamorphosis. To test whether Broad is also activated by EcR signaling in the larval optic lobe, we examined EcR expression and its possible role in the optic lobe. An antibody that recognizes all EcR isoforms was used to detect EcR expression in the optic lobe. EcR is expressed in the OPC NEs at late-second instar (Figure 5, A and A’), its expression in the NEs reaches a high level at midthird instar (Figure 5, B and B’), then declines through midlate third instar (84 hr ALH) (Figure 5, C and C’) and is low by late-third instar (Figure 5, D and D’). EcR is also expressed in the IPC NEs, though at a lower level than in the OPC NEs (Figure 5, A’ and C’). In the medulla cortex, EcR is expressed at a low level from second instar to late-third instar (Figure 5, A–D’).

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Figure 5

EcR is required for neuroepithelial growth and maintenance. (A–D’) Larval brains stained for EcR (red) and Arm (green). EcR expression at late-second instar (A and A’), midthird instar (B and B’), midlate third instar (C and C’), and late-third instar (D and D’). (E–E’’) An NP7340-Gal4/UAS-EcR^RNAi brain showing efficient EcR knockdown in the optic lobe (E’’) and disorganized NEs [(E’) white arrow]. (F–N’) Late-third instar wild-type, NP7340-Gal4/UAS-EcR^RNAi or ogre-Gal4/UAS-EcR^DN brains stained for the markers indicated. (F–H’) WT brains. (I–K’) NP7340/UAS-EcR^RNAi brains had a depleted OPC neuroepithelium, but had a number of Dpn⁺ cells (I), a misshapen lamina (J), and/or a medulla with a large cortex area that lacked Elav⁺ neurons but contained Arm⁺ cells (K and K’). (L–N’) ogre-Gal4/UAS-EcR^DN brains with abundant NEs (L), but no lamina [(M) yellow arrow indicates the lack of lamina] and very thin medulla (N and N’). Lateral is up for (A–E’’), to the left for (F, H, H’, I, K, K’, L, N, and N’); anterior is to the left for (G, J, and M). Bar, 20 μm in (A) for (A–D’), in (E) for (E and E’), and in (F) for (F–N’). Arm, Armadillo; Dac, Dachshund; Dlg, Discs large; Dpn, Deadpan; EcR, ecdysone receptor; NE, neuroepithelial cell; OPC, outer proliferation center; RNAi, RNA interference; WT, wild-type.

To analyze EcR function in the optic lobe, we knocked down EcR by expressing a UAS-EcR^RNAi construct using NP7340-Gal4. The RNAi knockdown was efficient as EcR protein was essentially eliminated in the optic lobe (Figure 5, E and E’’). In the EcR^RNAi brains, the lamina was underdeveloped and adopted an abnormal shape (Figure 5J, 86% of the brains had a defective lamina, n = 21). The medulla had large cortex areas that lacked Elav⁺ neurons but were filled with rounded Arm⁺ cells (Figure 5, K and K’, 100%, n = 21). At late-third instar, the OPC NEs were largely depleted, probably due to transformation of the NEs into more rounded, Dpn⁺ cells (Figure 5I, 100%, n = 18). Even at midthird instar, the OPC neuroepithelia in EcR^RNAi brains were disorganized and largely transformed (Figure 5, E and E’, 100%, n = 16). These observations indicate that EcR is required for neuroepithelial maintenance and growth in the optic lobe.

To complement the EcR RNAi knockdown approach, we further accessed the requirement of EcR in the optic lobe neuroepithelia by expressing a dominant negative EcR (EcR-B1^W650A) (Cherbas et al. 2003), which is a truncated EcR protein that could not bind to its ligand ecdysone. Expression of UAS-EcR^DN driven by NP7340-Gal4 caused early larval lethality; we thus used another Gal4 driver, ogre-Gal4, that is expressed in the OPC NEs but not in IPC NEs (Figure S1, I–J’; Dillard et al. 2018). The expression of UAS-EcR^DN under ogre-Gal4 control caused a significant delay of larval development; the mutant animals needed a further 2–3 days to reach the wandering larval stage. The optic lobes from the mutant wandering larvae had abundant NEs, but had a very small medulla and virtually no lamina (Figure 5, L–N’, 100%, n = 33). These results seem to be the opposite to the EcR RNAi knockdown results (Figure 5, I–K’). However, the EcR^DN protein is thought to act as a constitutive repressor of target gene expression (Cherbas et al. 2003; Schubiger et al. 2005; Brown et al. 2006); the results of EcR^DN overexpression could be explained by a model in which EcR^DN constantly represses Broad expression to block NE differentiation into NBs (see below).

We tested whether Broad expression in the optic lobe is indeed regulated by EcR. We found that Broad protein levels increased in EcR^RNAi mutant NEs (Figure 6C’, arrowhead indicates a group of mutant NEs that had an increased level of Broad, compare with the expression in wild-type NEs in Figure 6A’; quantified in Figure 6E); more specifically, Br-Z1 showed a significant increase in the mutant NEs of EcR^RNAi brains (Figure 6D’, arrow, compare with Br-Z1 expression in wild-type NEs in Figure 6B’; quantified in Figure 6E). To confirm these results, we examined Broad expression in clones expressing EcR^RNAi. The clones were induced at 24 hr ALH, and the larvae were then cultured at 31° until late-third instar when the brains were analyzed. Indeed, Br-Z1 expression was cell-autonomously increased in the clones, as compared with neighboring wild-type NEs (Figure 6, F, F', G and G'; quantified in Figure 6H).

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Figure 6

Regulation of Broad expression by EcR. (A–D’) Late-third instar WT and NP7340-Gal4/UAS-EcR^RNAi brains stained for Broad (Br-C) or Br-Z1 (red), Arm (green), and Phall (green). (A–B’) Broad (A and A’) or Br-Z1 (B and B’) expression in WT brains. (C–D’) Broad (C and C’) or Br-Z1 (D and D’) expression in NP7340-Gal4/UAS-EcR^RNAi brains. Arrowhead in (C’) indicates a group of EcR^RNAi mutant NEs that had increased Broad expression, as compared with WT NEs (A’). Arrow in (D’) indicates that EcR^RNAi mutant NEs had increased Br-Z1 expression as compared with WT NEs (B’). (E) Quantification of Broad and Br-Z1 expression in the NEs for experiments shown in (A–D’). Numbers of NE nuclei and brains (in parentheses) indicated inside the bars. Error bars indicate mean ± SD, **** P < 0.0001; Student’s t-test. (F–G’) EcR^RNAi-expressing clones stained for Br-Z1 (red), DE-cad (blue), and marked by GFP (green). The NEs in the clones [marked with green lines in (F’ and G’)] had increased Br-Z1 expression as compared with surrounding WT NEs. (H) Quantification of Br-Z1 expression in EcR^RNAi-expressing clones. Numbers of NE nuclei and GFP⁺ clones (in parentheses), or selected GFP⁻ areas (in parentheses) indicated inside the bars. Error bars indicate mean ± SD, **** P < 0.0001; Student’s t-test. (I–J’) Late-third instar ogre-Gal4/UAS-EcR^DN brains stained for Broad or Br-Z1 (red), Arm (green), and Phall (green). ogre-Gal4/UAS-EcR^DN brains had much reduced Broad expression in the NEs [(I’) dashed line, compare with WT NEs in (A’)], or completely lost Br-Z1 expression in the NEs [(J’) dashed line]. (K) Quantification of Broad expression in the NEs of ogre-Gal4/UAS-EcR^DN brains. Numbers of NE nuclei and brains (in parentheses) indicated inside the bars. Error bars indicate mean ± SD, **** P < 0.0001; Student’s t-test. (L–N’) Clones expressing UAS-EcR^DN or WT control stained for Broad (blue), Arm (red), and marked with GFP (green). (L and L’) Control clones. (M–N’) EcR^DN-expressing clones lost Broad expression cell autonomously. (O–O’’) An ecd¹ mutant brain stained for Arm (green), Dac (red), and Elav (blue) had a small me (O’) and very few la cells (O’’), but had a number of NEs (O). (P and P’) An ecd¹ mutant brain stained for Broad (red) and Phall (green) had reduced expression of Broad in the NEs (P’) as compared with WT NEs (A’). (Q) Quantification of Broad expression in the NEs of ecd¹ mutant brains. Numbers of NE nuclei and brains (in parentheses) indicated inside the bars. Error bars indicate mean ± SD, **** P-value < 0.0001; Student’s t-test. Lateral is up for (A–D’) and (I–J’), and is to the left for (F–G’), (L–N’), and (O–P’). Bar, 20 μm in (A) for (A–D’), in (F) for (F–G’), in (I) for (I–J’), in (L) for (L–N’), and in (O) for (O–P’). Arm, Armadillo; Dac, Dachshund; DE-cad, DE-cadherin; EcR, ecdysone receptor; la, lamina; me, medulla; NE, neuroepithelial cell; Phall, Phalloidin; RNAi, RNA interference; WT, wild-type.

We also examined the expression of Broad in larval brains expressing EcR^DN. The expression of EcR^DN under ogre-Gal4 control caused a strong reduction of Broad in the OPC NEs (Figure 6I’, compare with Figure 6A’; quantified in Figure 6K) and a complete loss of Br-Z1 in the NEs (Figure 6J’; 26/26 brains). Consistent with this result, clonal EcR^DN expression led to a cell-autonomous loss of Broad expression in both NE clones and medulla clones (Figure 6, M, M', N and N', 47/50 clones). Given the repressor nature of EcR^DN, these results were anticipated and were consistent with the EcR RNAi knockdown data.

Since EcR is activated by ecdysone, we asked whether ecdysone might regulate Broad expression in the optic lobe NEs. We tested this using ecd¹, a temperature-sensitive allele of the ecdysoneless (ecd) gene, the loss-of-function of which affects ecdysone synthesis (Garen et al. 1977). We cultured ecd¹ animals at 18° to allow them to reach the late-second instar stage, and then shifted the culture temperature to 31°. The loss of Ecd caused a severe delay of development, as the mutant larvae were still in the third-instar larval stages after 8 days of culture at 31°. These ecd¹ mutant animals had very small medulla and lamina (Figure 6, O’ and O’’, n = 3), but had abundant NEs in the optic lobe (Figure 6O), which expressed Broad protein at lower levels compared with wild-type NEs (Figure 6, P and P’, compare with Figure 6A’; quantified in Figure 6Q). Thus, the loss of Ecd function lead to a phenotype similar to that of EcR^DN expression, suggesting that the activity of ecdysone may be required to remove the repressor function of EcR to promote Broad expression.

Taken together, these results suggest that, contrary to the activation of Broad by ecdysone signaling during metamorphosis, Broad, particularly Br-Z1, expression in the optic lobe NEs is repressed by EcR signaling in the larval stages.