Mirth and Shingleton, 2012 Mirth C.K.

Shingleton A.W. Integrating body and organ size in Drosophila: recent advances and outstanding problems.

Warren et al., 2006 Warren J.T.

Yerushalmi Y.

Shimell M.J.

O’Connor M.B.

Restifo L.L.

Gilbert L.I. Discrete pulses of molting hormone, 20-hydroxyecdysone, during late larval development of Drosophila melanogaster: correlations with changes in gene activity.

Brennan et al., 1998 Brennan C.A.

Ashburner M.

Moses K. Ecdysone pathway is required for furrow progression in the developing Drosophila eye.

Schwedes et al., 2011 Schwedes C.

Tulsiani S.

Carney G.E. Ecdysone receptor expression and activity in adult Drosophila melanogaster.

Holden et al., 1986 Holden J.J.A.

Walker V.K.

Maroy P.

Watson K.L.

White B.N.

Gausz J. Analysis of molting and metamorphosis in the ecdysteroid-deficient mutant L(3)3dts of Drosophila-melanogaster.

Brown et al., 2006 Brown H.L.

Cherbas L.

Cherbas P.

Truman J.W. Use of time-lapse imaging and dominant negative receptors to dissect the steroid receptor control of neuronal remodeling in Drosophila.

Mirth et al., 2009 Mirth C.K.

Truman J.W.

Riddiford L.M. The ecdysone receptor controls the post-critical weight switch to nutrition-independent differentiation in Drosophila wing imaginal discs.

Reddy et al., 2010 Reddy B.V.

Rauskolb C.

Irvine K.D. Influence of fat-hippo and notch signaling on the proliferation and differentiation of Drosophila optic neuroepithelia.

Yasugi et al., 2008 Yasugi T.

Umetsu D.

Murakami S.

Sato M.

Tabata T. Drosophila optic lobe neuroblasts triggered by a wave of proneural gene expression that is negatively regulated by JAK/STAT.

2010 Yasugi T.

Sugie A.

Umetsu D.

Tabata T. Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe.

Egger et al., 2010 Egger B.

Gold K.S.

Brand A.H. Notch regulates the switch from symmetric to asymmetric neural stem cell division in the Drosophila optic lobe.

Wang et al., 2011 Wang W.

Liu W.

Wang Y.

Zhou L.

Tang X.

Luo H. Notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in Drosophila optic lobe development.

Yasugi et al., 2010 Yasugi T.

Sugie A.

Umetsu D.

Tabata T. Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe.

Figure S3 Ecdysone Signaling Regulates Progenitor Pool Size via Delta, Related to Figure 4 Show full caption (A) Frontal section through the medulla of a 96 hr larvae showing that NE cells (E-cad in green), express EcR (in red). (B) The NE of early L3 CNSs explanted for 24 hr in a culture medium containing 1mg/mL of 20E (20-hydroxyecdysone) is almost entirely converted in neuroblasts. In the control medium (1μg/ml of 20E), the NE continues dividing. E-cad (green), Mira (red), PH3 (white). DTS3 is a temperature sensitive an allele of the zinc finger molting defective (mld) gene required for ecdysone biosynthesis in the prothoracic gland ( Neubueser et al., 2005 Neubueser D.

Warren J.T.

Gilbert L.I.

Cohen S.M. Molting defective is required for ecdysone biosynthesis. Holden et al., 1986 Holden J.J.A.

Walker V.K.

Maroy P.

Watson K.L.

White B.N.

Gausz J. Analysis of molting and metamorphosis in the ecdysteroid-deficient mutant L(3)3dts of Drosophila-melanogaster. DTS3 larvae, switched to restrictive temperatures for 6 days, is larger than the NE of wt late L3 larvae. Conversely, fewer NBs are produced in mldDTS3 mutants. E-cad (green), Mira (red), PH3 (white). The associated histogram depicts the average width of NE and medulla NB stripes in wt wandering L3 and mldDTS3 mutant larvae. The NE width is measured for the anterior half of the NE, and only NE cells located medial to the lamina furrow on the lateral side of the NE are taken in account. wt NE (n = 10, mean = 7.0, SD = 1.1), wt NB (n = 10, mean = 6.2, SD = 1); DTS3 NE (n = 11, mean = 10.6, SD = 2.2). DTS3 NB (n = 11, mean = 3.6 SD = 0.8). ∗∗∗p < 0.001. (C) mldis a temperature sensitive an allele of the zinc finger molting defective (mld) gene required for ecdysone biosynthesis in the prothoracic gland (). A shift to the restrictive temperature (29°C) at early L3 abrogates the late-larval ecdysone pulse, allowing DTS3 mutant larvae to wander without pupariating for up to 15 days (). The NE of mldlarvae, switched to restrictive temperatures for 6 days, is larger than the NE of wt late L3 larvae. Conversely, fewer NBs are produced in mldmutants. E-cad (green), Mira (red), PH3 (white). The associated histogram depicts the average width of NE and medulla NB stripes in wt wandering L3 and mldmutant larvae. The NE width is measured for the anterior half of the NE, and only NE cells located medial to the lamina furrow on the lateral side of the NE are taken in account. wt NE (n = 10, mean = 7.0, SD = 1.1), wt NB (n = 10, mean = 6.2, SD = 1); DTS3 NE (n = 11, mean = 10.6, SD = 2.2). DTS3 NB (n = 11, mean = 3.6 SD = 0.8).p < 0.001. (D) Plots showing that the number of NE cells is increased in EcRDN clones compared to wt. wt (mean = 14.7, n = 25, SD = 9); EcRDN (mean = 29.5, n = 14, SD = 18). ∗∗p < 0.001. Plots showing that the number of neurons cells decreases by ∼70% in EcRDN clones compared to wt. wt (mean = 170, n = 17, SD = 134); EcRDN (mean = 45, n = 12, SD = 48). ∗∗∗p < 0.001. (E) Plot depicting the signal intensity of Delta in early and late NEs along a medial-to-lateral axis (orange line). Note that for both early and late stages, immunostaining and image acquisition were performed under the same conditions. (F) Clones misexpressing Delta delays NE-to-NB conversion. E-cad (blue), Mira (red). (G) Loss of Delta in EcRDN clones abrogates the delay in the progression of the proneural wave. GFP (green), E-cad (red) and Mira (blue).

Figure 4 Ecdysone Triggers NE → NB Conversion through the Downregulation of Delta and Is Required Cell Autonomously to Complete NE Elimination Show full caption (A) X-gal staining demonstrates that EcRE-lacZ is specifically activated in the NE of late L3, but not in early L3. (B) In late L3, wild-type MARCM clones span the NE (E-cad, red) and NB (Mira, blue) populations. Clones misexpressing EcRDN exhibit a delayed proneural wave, as shown by the systematic presence of more medial E-cad staining inside clones compared to surrounding tissue. (C) Delta (red) is upregulated in EcRDN clones throughout the NE (blue). (D) EcRDN GFP+ clones in the pharate adult retain NE cells and NBs (E-cad, red; Mira, blue; see higher magnifications) that are still proliferating (Mira, red; PH3, white). See also Figure S3

We then sought to identify the NR-resistant signal that initiates neurogenesis during phase 2. The end of phase 1 correlates with the first of a series of three L3 ecdysone bursts from the prothoracic gland (). Thus, ecdysone could be responsible for promoting the phase-1-to-phase-2 transition. We find that the common isoform of ecdysone receptor (EcR) is expressed in NE cells of late larvae and that an EcRE-lacZ transgenic reporter of ecdysone signaling () is activated in the NE of late L3 larvae ( Figures S3 A and 4 A ). Thus, the onset of EcR signaling in the NE temporally correlates with the major period of NE-to-NB conversion. To investigate further the relationship between ecdysone and NE-to-NB conversion, we first performed ex vivo experiments. CNSs from early L3 larvae (phase 1) explanted to a high concentration of ecdysone undergo precocious NE depletion, thus limiting the final number of medulla NBs that are generated ( Figure S3 B). Moreover, the NE of molting defective (mld) mutant larvae, in which the ecdysone pulses are abrogated (), continues expanding for several days while NE-to-NB conversion is reduced ( Figure S3 C). Together, these results demonstrate that EcR signaling during L3 is both necessary and sufficient to stimulate the NE-to-NB conversion with a concomitant reduction in the pool of NE cells. We then investigated if the requirement for EcR signaling was autonomous to NE cells. An efficient way to suppress both the activation and derepression functions of the ecdysone response is to express a dominant-negative EcR (). Control and EcR-expressing clones were induced in the medulla NE during early larval stages (24 hr) and examined at 96 hr. Control clones span the NE and NB regions, separated by a sharp and linear E-Cadherin (E-cad)/Mira boundary ( Figure 4 B). In contrast, EcRclones display a medially displaced boundary ( Figure 4 B), a phenotype that has been attributed to a delayed proneural wave (). This interpretation is supported by the increase in NE cells and the reduction in neurons observed in EcRclones relative to control clones ( Figure S3 D). We then sought to identify the downstream targets of EcR signaling relevant to the NE-to-NB conversion in the medulla. The Notch pathway has been shown to regulate the NE-to-NB conversion (). We detect strong expression of the Notch ligand, Delta, in the NE during phase 1, with particularly high levels at or close to the NE/NB boundary. Interestingly, during phase 2, Delta in the NE becomes strongly downregulated ( Figure S3 E). In EcRclones, however, there is a striking failure to downregulate Delta, which is most pronounced at the NE/NB boundary ( Figure 4 C). Moreover, overexpression of Delta in medulla clones is sufficient to shift the NE/NB border more medially, thus phenocopying EcRexpression ( Figure S3 F), whereas loss of Delta in EcRclones abrogates the delay ( Figure S3 G). Strikingly, in EcRclones, the NE remains present in pharate adults, continuing to proliferate and to generate NBs and neurons long after neurogenesis in the surrounding wild-type tissue has terminated ( Figure 4 D). Thus, EcR signaling is required in the NE for the timely termination of neurogenesis. In summary, these experiments demonstrate that ecdysone induces the symmetric-to-asymmetric switch through the repression of the Delta/Notch pathway in the NE. This late developmental event limits the neural progenitor pool and schedules the neurogenic phase to the diet-insensitive period.