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First published online 23 June 2005
doi: 10.1242/dev.01920

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Lateral inhibition in proneural clusters: cis-regulatory logic and default repression by Suppressor of Hairless

Brian Castro, Scott Barolo^*, Adina M. Bailey and James W. Posakony

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093-0349, USA

View larger version (78K): [in a new window]   Fig. 1. The E(spl)m PNC enhancer drives strong expression in non-SOPs but is directly repressed by Su(H) in SOPs. (A) Diagram of reporter constructs in which wild-type and mutant versions of the E(spl)m PNC cis-regulatory module drive expression of GFP or RFP via a minimal Hsp70 promoter. Positions of the five Su(H)-binding sites (S) and single proneural (PN) protein-binding site (E box) are indicated; mutant sites are indicated by a red cross. (1) Wild-type module; (2) module with E box mutated (Em); (3) Su(H)-binding sites mutated (Sm); (4) E box and Su(H) sites mutated (EmSm). (B-D,F-H) Single wing disc from a late third-instar larva carrying one copy each of E(spl)m-RFP and E(spl)m Sm-GFP and stained with anti-Hindsight (Hnt) antibody (C) to mark SOPs. Insets show higher-magnification views of the boxed region of the wing disc. (B'-D',F'-H') Microchaete row (adjacent to the dorsal midline) on the notum of a single pupa of the same genotype 14 hours after puparium formation (APF), also stained with anti-Hnt (C'). (B,B') RFP signal driven by the wild-type module is strong in most non-SOP cells but minimal or absent in SOPs (B,D,B',D'). (E) Mutation of the E box (Em) causes severe loss of PNC expression; expression is retained at the wing margin and in a small subset of PNC cells. (F,G,F',G') Mutation of the Su(H)-binding sites (Sm) abolishes or severely lowers reporter expression in non-SOP cells of the PNCs but causes strong ectopic expression in SOPs. (H,H') Overlay of B (B') and F (F') highlights dramatic change in the pattern of reporter gene activity when Su(H) sites are mutated. (I) Mutation of both the E box and the Su(H) sites (EmSm) demonstrates that ectopic SOP expression of the Sm mutant (F) is dependent on direct input from the proneural proteins.

View larger version (44K): [in a new window]   Fig. 2. bHLH repressor-encoding genes of the E(spl)-C are also subject to direct transcriptional repression by Su(H) in SOPs. (A) Diagrams of rescue fragment and enhancer-reporter constructs for E(spl)m8. (1) Genomic DNA fragment including the E(spl)m8 gene, with protein coding sequence (blue) and positions of Su(H) (S) and proneural protein (E) binding sites marked. Arrow indicates transcription start site. (2,3) E(spl)m8 enhancer-GFP reporter constructs with Su(H)-binding sites intact (2) or mutated (Sm; mutant sites indicated by a red cross) (3). Minimal Hsp70 promoter is shown. (B-G) Wing discs from late third-instar larvae carrying one copy of either E(spl)m8-GFP (B-D) or E(spl)m8 Sm-GFP (E-G). (B,D,E,G) GFP expressed from E(spl)m8 enhancer-reporter transgenes (green). (C,D,F,G) Anti-Hnt antibody staining (magenta) marks SOPs. Insets in merged D and G show higher-magnification views of the boxed regions of the two wing discs. (B) E(spl)m8-GFP is strongly expressed in PNCs, but activity is restricted to non-SOPs (D; inset: SOPs in top and bottom clusters are obstructed by non-SOPs). (E) Mutation of Su(H)-binding sites causes severe or complete loss of reporter expression in non-SOPs, but strong ectopic expression in SOPs (G).

View larger version (58K): [in a new window]   Fig. 3. Conservation of sequence, organization and activity of the E(spl)m PNC cis-regulatory module. (A) Alignment of orthologous genomic DNA segments corresponding to the E(spl)m PNC enhancer from D. melanogaster, D. pseudoobscura, D. hydei and D. virilis. Orthologous Su(H) (S) and proneural protein (E)-binding sites are connected by gray lines. (B) Nucleotide sequence alignments showing strong conservation (highlighted in green and yellow) in regions of the enhancer that contain Su(H) and proneural protein-binding sites (boxed). With the exception of S4, the sequence of each binding site is precisely conserved, as is the spacing between S2 and the E box. (C) The D. virilis E(spl)m module drives strong GFP expression (green) in PNCs of the late third-instar wing disc of D. melanogaster that is excluded from SOPs, similar to the D. melanogaster module (see Fig. 1B-D). Boxed region in 1 is shown at higher magnification on right (2-4); anti-Hnt staining (magenta) marks SOPs (1,3,4).

View larger version (106K): [in a new window]   Fig. 4. Failure of direct repression of E(spl)m8 by Su(H) can extinguish the SOP cell fate. (A) Part of notum region of an adult fly carrying two copies of an E(spl)m8 genomic DNA transgene (see Fig. 2A, construct 1), showing a normal pattern of adult mechanosensory bristles. Labeled macrochaetes: ASA, anterior supraalar; PSA, posterior supraalar; APA, anterior postalar. (B) Same region from a fly carrying two copies of the E(spl)m8 Sm transgene [Su(H) binding sites mutated], showing loss of the PSA bristle (broken circle). (C,C',D,D') The cellular basis of bristle loss caused by the E(spl)m8 Sm transgene is failure of SOP specification. (C,C') Same genotype as A; (D,D') same genotype as B. (C,D) Late third-instar wing discs stained with anti-Sens antibody to mark SOPs. (C',D') Higher-magnification views of the regions boxed in C and D, respectively. Labeled SOPs: PNP, posterior notopleural macrochaete; ANWP, campaniform sensilla of the anterior notal wing process. Broken circle in D' indicates position of missing PSA SOP. The presence of both ANWP SOPs indicates that both discs are at a stage late enough to observe the PSA SOP in wild-type discs. (E) Frequency of bristle loss is significantly greater in E(spl)m8 Sm homozygous flies (red) than in flies homozygous for the wild-type E(spl)m8 transgene (blue). Mann-Whitney U test: U=2, P=0.002.

View larger version (77K): [in a new window]   Fig. 5. Reducing the dose of H and gro de-represses expression of the E(spl)m PNC reporter in SOPs. (A-H) Wing discs from late third-instar larvae carrying one copy of E(spl)m-GFP (green) in either a w1118; A101/+ + (A,C-E) or w1118; A101/HE31 groE48 (B,F-H) background. (A,B,D,E,G,H) Antibody staining of ß-galactosidase expressed from the A101 enhancer trap insertion (magenta) marks SOPs. (C-E) Higher magnification of boxed region in A, showing exclusion of GFP signal from SOPs (arrows). ANWP, campaniform sensilla of the anterior notal wing process; APA, anterior postalar; PSA, osterior supraalar; PNP, posterior notopleural macrochaete. (F-H) Higher magnification of boxed region in B, showing ectopic GFP expression in SOPs (arrows). Reporter expression in non-SOPs is often reduced in this background. (I) Ectopic E(spl)m-GFP expression in SOPs is observed at much greater frequencies and intensities in w1118; A101/HE31 groE48 discs (dark green) than in w1118; A101/+ + discs (light green). ANP, anterior notopleural; PDC, posterior dorsocentral; PSC, posterior scutellar. Only six discs (instead of 16) could be scored for GFP expression at the PSA position in the HE31 groE48/+ + background, as the PSA SOP is often missing in this genotype (broken circle in F-H).

View larger version (33K): [in a new window]   Fig. 6. The Gro- and CtBP-binding motifs of H are each required for its full activity in promoting the SOP cell fate. (A) Half of a wild-type fly head showing the three macrochaete bristles of the orbital region (boxed; magnified in inset). (B) Expressing wild-type H (via UAS-H) in non-SOPs with an E(spl)m-Gal4 driver converts some of these cells to SOPs, producing ectopic bristles that are either normal (white arrow) or display a `double shaft' phenotype (black arrow) due to a cell fate conversion in the bristle lineage (Barolo et al., 2002) (black arrowhead indicates another double shaft broken during manipulation). (C) Frequency of supernumerary orbital bristles observed in adult flies carrying one copy of E(spl)m-Gal4 driving expression of wild-type H (one copy of UAS-H), H lacking its CtBP-binding motif (UAS-HC), H with its Gro-binding motif mutated (UAS-H[Gm]) or H lacking both motifs (UAS-H[Gm]C). Two different insertions of each UAS construct were tested (#1, #2).

View larger version (20K): [in a new window]   Fig. 7. Model of cell fate specification during lateral inhibition in PNCs. (A) In wild-type SOPs (right), which do not respond to the Dl/N signal, Su(H) is in its default repressive state, linked via H to the co-repressors Gro and CtBP. It is bound directly to high-affinity sites in SOP-inhibitory target genes in the E(spl)-C, and prevents activation of these genes by the high proneural protein levels prevailing in that cell. The proneural proteins are thus free to activate genes that promote the SOP fate. In wild-type non-SOPs (left), activation of the N receptor associates Su(H) with a transcriptional activation complex that includes NIC and Mam. In this activated state, Su(H) synergizes with the proneural proteins to directly activate expression of the same SOP-inhibitory genes, committing the cell to an epidermal fate. (B) In the absence of direct repression by Su(H) [e.g. by mutation of Su(H)-binding sites (Sm)], SOP-inhibitory genes of the E(spl)-C are ectopically activated in the SOP by the high levels of proneural proteins, which can drive the cell to adopt an inappropriate epidermal fate. In this model, three key regulators – the proneural proteins, Su(H) and Gro – each have transcriptional regulatory activities that are oppositely directed, with respect to cell fate, in SOPs versus non-SOPs. All three function both to promote and to inhibit the SOP fate during lateral inhibition.

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