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doi: 10.1242/10.1242/dev.00206

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Two modes of recruitment of E(spl) repressors onto target genes

Nikolaos Giagtzoglou1,2, Pavlos Alifragis1,2,*, Konstantinos A. Koumbanakis1,2 and Christos Delidakis1,2,{dagger}

1 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Heraklion, Greece
2 Department of Biology, University of Crete, Heraklion, Greece
* Present address: Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK



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  		Fig. 1. Response of T5-0.9wt/luc and T5-0.9mut/luc reporters to bHLH effectors E(spl) m7, m{gamma} and m{delta} transiently transfected into S2 cultured cells. Amounts of transfected plasmids per well are shown in ng. Graphs show averages of four replicates with standard deviations as error bars. Luciferase activity obtained in the da+sc transfection (no repressors) is arbitrarily defined as 100%.

 


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  		Fig. 8. E(spl)-mediated repression requires Gro. (A-A'') Wing pouch of omb-Gal4/hsFLP; UAS-sc UAS-E(spl)m7/EE4-lacZ; FRT82B groE48/FRT82B {pi}Myc. Homozygous clones for groE48 are marked by the absence of {pi}Myc (green) and ß-galactosidase is visualized in red. Intense EE4-lacZ expression is autonomously induced within gro mutant cells, indicating lack of repression. Arrows indicate wild-type cells that express low levels of EE4-lacZ. This low-level patchy expression in wild type is expected, as the overview of an omb-Gal4; UAS-sc UAS-E(spl)m7/EE4-lacZ (non-mosaic) disk stained by X-gal shows in B. It indicates that E(spl)m7-mediated repression is strong, yet not complete; compare with Fig. 2E-H. (C) hsFLP; FRT82B groE48/FRT82B {pi}Myc mosaic notum. Even though the clones are unmarked, we presume that they correspond to patches exhibiting bristle tufting. (D) hsFLP; ap-Gal4/UAS-E(spl)m7; FRT82B groE48/FRT82B {pi}Myc mosaic notum. Similar bristle tufts are observed, presumably corresponding to groE48 homozygous territories, within a bald background because of the overall expression of E(spl)m7, which suppresses bristles within the gro+ territories.

 


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  		Fig. 2. Response of EE4-lacZ to bHLH transgene expression and E(spl) loss of function in third instar wing disks. (A-H) UAS transgenes (as noted on each panel) were driven by pnr-Gal4, which expresses in the proximal notum (region shaded green in A; note SC and DC proneural clusters, white and black arrowheads, respectively). (A) Wild-type pattern, which corresponds to the proneural clusters present at this stage (compare with Ac accumulation in I). (B) EE4-lacZ was abolished by UAS-E(spl)m7 expression. (C) EE4-lacZ was significantly reduced by UAS-E(spl)m{delta} expression. (D) UAS-E(spl)m7KNEQ also repressed strongly. (E) EE4-lacZ was activated when UAS-sc is present, but severely diminished when E(spl)m7 (F) or E(spl)m7KNEQ (H) were co-expressed. Note that weak patchy expression remains. (G) Co-expression of UAS-E(spl)m{delta} did not suppress EE4-lacZ activation by UAS-sc. (I) Wild-type and (J) UAS-E(spl)m7; pnr-GAL4 disk immunostained for Ac. Accumulation in proneural clusters was seen in both cases — despite E(spl)m7 expression. Over-accumulation in SOPs of the dorsocentral cluster was abolished by E(spl)m7 (arrow). Insets show the boxed region of the notum at twice the magnification. (K,L) EE4-lacZ disks developed lightly with X-gal to compare levels of expression between wild-type disks (K — compare with wild-type disk developed longer in A) and disks null for E(spl)m8 and m7 (L). Although the E(spl) mutation does not affect expression pattern, it results in a more intense signal in all proneural clusters. (M,M') A mitotic clone null for the entire E(spl)-C is visualized by absence of green nuclear {pi}Myc staining. ß-galactosidase (EE4-lacZ) is visualized in red. The clone (outlined in M'), which overlaps the distal wing margin, shows more intense EE4-lacZ staining, consistent with loss of repression due to the absence of E(spl) function. (M') Red channel only.

 


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  		Fig. 5. Response of Gbe-B1-lacZ to bHLH activators. All panels show wing disks carrying one copy of the Gbe-B1-lacZ reporter. (A) Wild-type expression pattern, developed for 5 hours. (B) omb-Gal4; UAS-sc, developed for 5 hours. (C) omb-Gal4; UAS-E(spl)m7VP16, developed for 10 minutes. (D) omb-Gal4; UAS-E(spl)m{delta}VP16, developed for 10 minutes. (E) omb-Gal4; UAS-E(spl)m{gamma}VP16, developed for 30 minutes. F: omb-Gal4; UAS-E(spl)m7KNEQ-VP16, developed for 5 hours.

 


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  		Fig. 3. Adult phenotypes caused by bHLH transgene expression via pnr-Gal4. The UAS transgenes were as follows: (A) none, (B) UAS-sc, (C) UAS-E(spl)m7, (D) UAS-sc, UAS-E(spl)m7 co-expression, (E) UAS-E(spl)m{delta}, (F) UAS-sc, UAS-E(spl)m{delta} co-expression, (G) UAS-E(spl)m7KNEQ and (H) UAS-sc, UAS-E(spl)m7 KNEQ co-expression.

 


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  		Fig. 4. Response of EE4-lacZ to E(spl)VP16 variants. All panels show wing disks carrying one copy of the EE4-lacZ reporter. (A) Wild type. (B) omb-Gal4; UAS-E(spl)m7VP16. Note the intense staining in three proneural clusters: dorsal radius (white arrow), wing margin (arrowhead) and ventral radius (black arrow). The extent of the omb-Gal4 expression domain is visualized in Fig. 5C-E. (C) omb-Gal4; UAS-E(spl)m{delta}VP16. No activation is observed. (D) UAS-E(spl)m7VP16; pnr-Gal4. In addition to intense staining in the SC (white arrowhead) and DC (black arrowhead) proneural clusters, dispersed individual cells are expressing EE4-lacZ. (E) sc10-1/Y background essentially abolishes EE4-lacZ activity. (F) sc10-1/Y; UAS-E(spl)m7VP16; pnr-Gal4. The intense response of EE4-lacZ observed in D is absent due to absence of Ac and Sc. (G) omb-Gal4; UAS-sc. Note weak and somewhat patchy response of EE4-lacZ to uniform expression of Sc. (H,I) omb-Gal4; UAS-sc UAS-E(spl)m7VP16. Note a more uniform and much more intense response. H is under-stained, whereas I is stained to the same extent as G. The notum proneural clusters (top of each panel), where omb-Gal4 is not expressed, can be used to judge the extent of color development in A-C and G-I.

 


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  		Fig. 6. Comparison of E(spl)m7VP16 with E(spl)m7KNEQ-VP16 in the activation of EE4-lacZ and Ac expression. (A-F) X-gal staining of EE4-lacZ wing disks. (G-I) immunostaining for Ac protein. (A,G) Wild type. (B,H) UAS-E(spl)m7VP16; pnr-GAL4. (C,I) UAS-E(spl)m7KNEQ-VP16; pnr-GAL4. Reporter activation at the SC (white arrowhead) and DC (black arrowhead) proneural clusters is seen at equally high levels in B and C. (D) UAS-sc; pnr-GAL4. (E) UAS-sc UAS-E(spl)m7VP16; pnr-GAL4. F: UAS-sc UAS-E(spl)m7KNEQ-VP16; pnr-GAL4. X-gal development in D-F is at equivalent levels, as judged by the wing-margin proneural clusters, where pnr-Gal4 is not expressed. Note the increased transcriptional response of the EE4-lacZ reporter in E and F compared with D.

 


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  		Fig. 7. Effect of bHLH activator transgene expression on bristle production in the absence of endogenous ac and sc. Adult nota of flies carrying pnr-Gal4. (A-E) Wild type for the X chromosome. (F-J) sc10-1/Y results in a bald notum except for the central pnr-Gal4 domain, whenever sensory organ-inducing bHLH proteins were expressed. Effector transgenes expressed are as follows. (A,F) none. (B,G) UAS-sc. Sc expression induces bristles within the pnr-Gal4 domain. (C,H) UAS-E(spl)m7VP16. Bristles are induced even in the absence of ac and sc. (D,I) UAS-E(spl)m7KNEQ-VP16. Excess bristles are induced only in the ac+ sc+ background. (E,J) UAS-E(spl)m{delta}VP16. Bristles are induced even in the absence of ac and sc.

 




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