Fig 1.
Two neuralized SOP enhancers contain conserved binding sites for both proneural proteins and bHLH repressors.
(A) Diagram of the neur locus and flanking genes, showing the locations of the neur4D and neur1B SOP enhancers [13]. (B) Expanded diagram of the neur4D enhancer, marking the positions of proneural and bHLH-R binding motifs, along with other conserved sequences. (C-H) GFP expression (green) driven by a wild-type (WT) neur4D reporter construct (C, C’, E, and G) or by a proneural motif mutant (PSm) version (D, D’, F, and H) in representative third-instar wing imaginal discs (C-D’), 12 h APF nota (E and F), and 24 h APF nota (G and H); C’ and D’ show the scutellar and dorsocentral regions of the wing disc (see boxes in C and D). SOPs are marked by Sens protein (magenta). Caret (<) in (E) identifies two small, adjacent GFP-positive, Sens-negative nuclei. (I) Expanded diagram of the neur1B enhancer, showing the positions of proneural and bHLH-R binding motifs, along with other conserved sequence blocks; refer to (B) for symbol definitions. (J-Q) GFP expression driven a wild-type (WT) neur1B reporter construct (J, L, N, and P), a construct in which the the single PS-type proneural motif is mutated (PSm; K and M), and a construct in which both the PS- and PA-type proneural motifs are mutated (PS+Am; O and Q) in third-instar wing imaginal discs (J, K, N, and O), 24 h APF nota (L and M), and third-instar leg imaginal discs (P and Q). In panels L and M, GFP is in green, Sens protein in magenta. See also S1 and S2 Figs.
Fig 2.
Mutation of bHLH repressor binding motifs in the neur4D and neur1B enhancers causes proneural motif-dependent ectopic reporter gene expression in non-SOPs.
(A-B’, D, and E) Comparison of neur4DRm and neur4DPSRm GFP reporter activities in third-instar wing imaginal discs (A-B’) and 12 h APF nota (D and E). (C) Quantification of ectopic GFP-expressing cells in the scutellar and dorsocentral macrochaete clusters in wing discs from larvae carrying the indicated reporter constructs. The proportion of discs exhibiting ectopic GFP is indicated for each genotype, and the graph reflects the average number of ectopic GFP cells over all discs. (F and G) Comparison of neur1BRm and neur1BPSRm GFP reporter activities in wing discs. (H) Quantification of ectopic GFP-expressing cells adjacent to the posterior dorsocentral (pDC) macrochaete SOP cell in wing discs from larvae carrying the indicated reporter constructs. Graph presented as described for C. A’ and B’ show the scutellar and dorsocentral regions of the wing disc (see boxes in A and B); insets in F and G show only the region surrounding the pDC SOP. F and G show only the GFP signal; in the remaining images, GFP is in green and Sens protein is in magenta. Caret in A’ points to GFP-positive, Sens-negative cells; see text for details. Error bars in C and H represent standard error of the mean (SEM).
Fig 3.
Mutation of bHLH repressor binding motifs in the 4D and 1B enhancer segments within a neur rescue construct causes ectopic expression of neur.
(A) Diagram of the region surrounding the neur locus. Shown are the boundaries of neur4D and neur1B, the extent of the neur rescue constructs, the locations of bHLH-R binding motifs (those mutated in the rescue constructs are indicated by X’s), and the location of the GFP coding sequence in the tagged rescue constructs. (B-G) Comparison of neur transcript accumulation in wing imaginal discs from neurRC-WT (B and D) and neurRC-4D,1BRm (C and E) larvae. Boxes in B and C surround the developing chordotonal organ of the tegula, shown under higher magnification in D and E. (F) Quantification of the area of neur probe in situ hybridization signal over the chordotonal organ of the tegula [17340±2888 SEM (n = 9) vs. 24040±1575 SEM (n = 21)]. (G) Quantification of the white intensity over the same region, which is inversely proportional to the darkness of staining [112±6.68 SEM vs. 89.2±2.8 SEM]. (H-Q) Comparison of GFP signal in wing imaginal discs from neurRC-WT-GFP (H, J, L, N, and P) and neurRC-4D,1BRm-GFP (I, K, M, O, and Q) larvae. Boxes in H and I denote regions shown at higher magnification in the indicated panels. J and K show GFP signal alone; L and M shown Sens protein signal alone; N and O show the merged signals (GFP in green, Sens in magenta). P and Q are likewise merged images. aDC, pDC: anterior and posterior dorsocentral macrochaetes; aSC, pSC: anterior and posterior scutellar macrochaetes; Ch. Or.: chordotonal organ.
Fig 4.
Expression of neur in PNC cells prior to SOP specification.
(A) Diagram of heterochronic PNCs in the wing imaginal disc. Within such clusters (blue), one SOP (green) is specified before the other, which forms at a stereotypic position (yellow) a few cells away. (B) Multiplex fluorescent in situ hybridization with intron probes against CG32150 (green only, marks a specified SOP), sca (blue, marks cells of the PNC), and neur (overlap of green and red). Hoescht stain, marking nuclei, is represented by inverted gray. Six adjacent 1-μm sections are shown from the dorsocentral (DC) macrochaete cluster of a wing imaginal disc. The CG32150-, neur-positive pDC SOP nucleus is marked with a white broken circle in panels 3–6. neur-positive nuclei in the nearby aDC domain are marked with yellow broken circles when the neur signal is present and gray broken circles when a different nucleus has neur signal. (C-H) Wing imaginal disc from a neurRC-WT-GFP larva showing GFP signal (C and E-H; green in C, E, and H) and Sens protein signal (C, D, E, and H; magenta in C, E, and H) in the heterochronic scutellar (SC) macrochaete cluster (region boxed in C, magnified in D-H). (D) Maximum projection of Sens signal. (E) Cross-section through the pSC nucleus, showing the locations of individual sections in the remaining panels. (F-H) GFP signal in individual confocal sections, showing at least four GFP-positive cells in the aSC domain, where Sens signal has yet to be strongly activated.
Fig 5.
Forcing persistent non-SOP expression of neur causes both loss and gain of SOPs.
(A) Quantification of macrochaete gain and loss on the dorsal head and thorax of flies of the genotypes indicated at right. Error bars represent SEM. (B and C) Scutellar bristle positions in 24 hr APF nota of the indicated genotypes, stained with anti-Cut antibody, show loss of the SOP with neur misexpression. (D-I) Uniform expression of UAS-neur driven by tub-GAL4 in neur mutant clones using the MARCM system in either a wing imaginal disc (D-F) or a 12 hr APF notum (G-I). GFP (green in F and I) marks the territories of tub>neur expression; anti-Sens antibody signal (magenta in F and I) marks SOPs. Brackets in D and F mark SOP loss at the region of overlap between tub>neur activity and the wing margin. Sens-positive cells boxed in D are in a different focal plane from the GFP-expressing cells.
Table 1.
neurRC bristle counts.
Table 2.
Dorsocentral and scutellar bristle counts.
Fig 6.
Inhibition of Neur function in the SOP causes ectopic neur transcript accumulation and neur4D enhancer activity.
Comparison of the expression of neur mRNA (A and C) and of a neur4D-WT-DsRed reporter transgene (B and D) in wing imaginal discs from w1118 (A-B’) and neur-GAL4, UAS-Tom, UAS-m4 flies (C-D”). Insets in A and C show higher-magnification views of the dorsocentral (DC) macrochaete cluster (boxed regions). B’ and D’ show higher-magnification views within the anterior wing pouch (regions boxed in B and D). D” shows a higher-magnification view of the dorsocentral and scutellar (SC) clusters (region boxed in D); compare with Fig 2.
Fig 7.
Through N signaling, proneural-dependent SOP expression of Neur promotes the inhibition of both neur transcription and Neur function in non-SOP cells.
Proneural proteins activate neur transcription both directly, via binding sites in the neur4D and neur1B enhancers, and indirectly by activating expression of other positive regulators of neur in the SOP. neur-dependent N signaling, combined with proneural factor activity, non-autonomously promotes expression of both E(spl) bHLH-Rs and BFMs in non-SOP cells. The bHLH-Rs repress further transcription of neur directly, through binding motifs in neur4D and neur1B, and similarly inhibit the expression of other SOP-specific targets. The BFMs bind Neur and block its interaction with Dl, preventing non-SOP cells from sending an effective N signal back to the SOP.