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, 203 (1), 219-40

Integration of Orthogonal Signaling by the Notch and Dpp Pathways in Drosophila

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Integration of Orthogonal Signaling by the Notch and Dpp Pathways in Drosophila

Elizabeth Stroebele et al. Genetics.

Abstract

The transcription factor Suppressor of Hairless and its coactivator, the Notch intracellular domain, are polyglutamine (pQ)-rich factors that target enhancer elements and interact with other locally bound pQ-rich factors. To understand the functional repertoire of such enhancers, we identify conserved regulatory belts with binding sites for the pQ-rich effectors of both Notch and BMP/Dpp signaling, and the pQ-deficient tissue selectors Apterous (Ap), Scalloped (Sd), and Vestigial (Vg). We find that the densest such binding site cluster in the genome is located in the BMP-inducible nab locus, a homolog of the vertebrate transcriptional cofactors NAB1/NAB2 We report three major findings. First, we find that this nab regulatory belt is a novel enhancer driving dorsal wing margin expression in regions of peak phosphorylated Mad in wing imaginal discs. Second, we show that Ap is developmentally required to license the nab dorsal wing margin enhancer (DWME) to read out Notch and Dpp signaling in the dorsal compartment. Third, we find that the nab DWME is embedded in a complex of intronic enhancers, including a wing quadrant enhancer, a proximal wing disc enhancer, and a larval brain enhancer. This enhancer complex coordinates global nab expression via both tissue-specific activation and interenhancer silencing. We suggest that DWME integration of BMP signaling maintains nab expression in proliferating margin descendants that have divided away from Notch-Delta boundary signaling. As such, uniform expression of genes like nab and vestigial in proliferating compartments would typically require both boundary and nonboundary lineage-specific enhancers.

Keywords: Apterous; Dpp/BMP; Notch; Su(H); developmental genetics; imaginal disc patterning; nab; pQ signal integration; polyglutamine.

Figures

Figure 1
Figure 1
Consideration of the polyglutamine (pQ) and polyasparagine (pN) content of factors in computational genomic searches for novel Notch-signal integrating enhancers. (A) Histogram of pQ tracts for the Dpp effectors, pMad (violet) and Medea (Med, blue); the Notch effectors, Su(H) (yellow) and its coactivators, the Notch intracellular domain (NICD, green) and Mastermind (Mam, red); and the temporal patterning factor Zelda (Zld, orange). Binding sites for these factors are enriched in the nab DWME. Also shown is the pQ content for activating TFs targeting the analogous NEEs: dorsal, Twist, Daughterless (Da), Zld, and Su(H). Snail (Sna) is a NEE-targeting transcriptional repressor and is devoid of pQ content. Each pQ tract, defined as a contiguous sequence of Qs ≥ 3, is represented by a single box in a bin corresponding to its length. In contrast to the patterning factors, the selector DNA binding factors (Ap, Dll, Sd, and Hth) and cofactors (Vg) are devoid of pQ tracts, likely indicating distinct modes of regulation separate from the signaling pathway effectors (see text). Similar trends are also seen with pN (B) or with mixed polycarboxamide (X) tracts (C).
Figure 2
Figure 2
A highly conserved regulatory block in the Drosophila nab locus is a robust dorsal wing margin enhancer (DWME). (A) Diagram of the nab locus from D. melanogaster showing sites matching binding motifs (color-coded boxes on separate tracks in large intronic box) within a 2.7-kb intronic block that contains several regulatory belts conserved across the genus. We cloned a predicted 764-bp region, the nab-A fragment (thick bold underline), for having one of the highest concentrations of sites for Su(H), Mad, Zelda, Apterous (Ap), and the Scalloped (Sd) and Vestigial (Vg) complex (Sd:Sd:Vg) without having any Tcf sites. (B and C) We cloned the nab-A fragment in front of two core promoter reporter genes: an hsp70 core promoter fused to an eGFP-nls reporter gene in a gypsy insulated construct (B), and an eve core promoter fused to the lacZ reporter gene (C). Independent P-element transgenic lines with each reporter cassette gave identical expression patterns despite differences in integration sites, core promoters, and enhancer orientation relative to the core promoter (arrows in enhancer box, and + or − signs in parentheses). The cloned 762-bp fragment is 2 bp shorter than the reference sequence due to single base pair contraction polymorphisms in two separate poly-A runs. (D–F) Expression from the nab-A (DWME) EGFP reporter along the dorsal wing margin. Different color channels indicate DAPI (D, cyan); GFP (E, green); and Wingless (Wg) (F, magenta), which mark the D–V compartment margin, a ring around the wing pouch, and a broad stripe across the proximal part of the wing disc. (G) Merged images of D–F. (H–J) Additional discs double-labeled with antibodies to Wg (H), and Ptc (I), and En (J), which mark the A–P margin and posterior compartment, respectively. The anterior DWME expression pattern is characteristically longer than the posterior compartment side, which in turn stretches deeper into the dorsal compartment than the anterior compartment expression pattern. (K–M) DWME-driven lacZ reporter activity in late third instar discs (K and L) or in germ-band extended embryos (M). DWME-driven expression was detected with overnight X-gal staining (K) or with a digU-labeled antisense lacZ RNA probe (L and M). Both wing (K and L) and haltere (L) discs show the characteristic twin spots of DWME-driven activity, while the embryonic expression is detected in a subset of lateral neuroblasts.
Figure 3
Figure 3
The Drosophila nab harbors several regulatory modules functioning as both enhancers and mutual silencers/attenuators. (A) We cloned and tested the indicated series of nab intronic fragments to understand the regulatory logic of nab’s expression in wing imaginal discs. The colored boxes follow the key in Figure 2 except Zelda sites are not shown for clarity. To refer to the different enhancers we identified, we defined four different intronic regions as the nab-C, nab-D, nab-A (DWME), and nab-B fragments. We also labeled the four best matches to Su(H) binding motifs (sites S1–S3) and mutated these sites in the indicated constructs (S1, S2, or S3 MUT and Xs in construct). These regions can be understood as having four major enhancer activities: the dorsal wing margin enhancer (DWME); a larval brain enhancer (BrE), which drives dense expression in neuronal lineages; a wing imaginal disc quadrant enhancer (QE), which complements the DWME activity to match the endogenous expression pattern; and a proximal wing enhancer (PWE). ++ indicates strong expression, + indicates weak expression, while maintaining the indicated pattern. * means there is a more nuanced description of the expression in the main text. Both the sizes and the direction of the cloned insert relative to the core promoter are shown for each construct. (B) Each cloned enhancer was found to drive a distinct expression activity associated with the endogenous nab locus, a distinct ectopic activity, or expression level that was not associated with the endogenous nab locus, and a distinct silencing or attenuation activity acting on ectopic expression patterns and levels (red repression symbols connecting one silencer/attenuation activity in one enhancer to the ectopic activity/level in another intronic enhancer).
Figure 4
Figure 4
Activities of dissected regulatory modules from the nab intronic enhancer/silencer complex. (A–I) GFP reporter expression driven by nab-GAL4>UAS-eGFP (A) or by different nab enhancer modules driving eGFP-nls (B–I). The first row depicts third instar wing imaginal discs; the middle row (single prime) depicts additional third instar discs labeled for halteres (h), leg (l), and wing (w) discs; and the third row (double prime) depicts expression patterns in third instar larval brains, if any. (A–I) The endogenous nab expression pattern in wing imaginal discs (A) appears to be the result of discrete activities from: the nab-A fragment, which contains the dorsal wing margin enhancer (B); the nab-C fragment, which contains a wing pouch/quadrant enhancer (C); a nondescript proximal wing disc enhancer seen with most intronic fragments containing the nab-D region (triple arrows in D and E); and the nab-B fragment, which contains a hinge enhancer that functions in the absence of the nab-C fragment (arrowheads in F–I). Note that the nab-C fragment is missing expression at the margin and has more uniform levels of expression throughout the pouch relative to either the nab-GAL4 enhancer trap (A) or fragments containing both the nab-C and nab-A regions (empty arrow in C). (A′–I′) Fragments with activities from both the nab-A and nab-C fragments recapitulate in endogenous nab expression in the haltere disc. (A′′–I′′) Most of the endogenous expression of nab in the larval brain and ventral nerve cord (A′′) is recapitulated by nab intronic fragments carrying the nab-B fragment (E′′–H′′) but it is noticeably weaker by itself (F′′). This pattern corresponds to dense expression in neuronal lineages and four cells in the posterior tip of the ventral nerve cord. The nab-C fragment has ectopic brain activity that is completely silenced by activities present in the nab-DA fragment (see entire brain in D′′ and optic lobes and posterior ventral nerve cord regions in E′′). The nab-AB fragment also drives a strong ectopic leg disc ring pattern (see G′ and H′) that is repressed in the presence of the nab-C fragment (see E′). Similarly, the dorsal wing margin activity of nab-A (B) is silenced in the presence of nab-B (see empty arrows pointing to margin in nab-AB disc in G) except when nab-D is also present (see nab-DAB disc in H). The S3 Su(H) binding site in nab-B appears to mediate repression of the wing hinge activity inherent to nab-B and the dorsal wing margin activity inherent to nab-A because there is augmentation of both of these patterns in the nab-AB S3 mutated construct (I, arrows point to twin spots along the dorsal margin of the compartment boundary, and arrowheads point to expanded hinge pattern). This same site is not absolutely required for the nab-B brain activity (I′′). Neither the nab-DA nor the nab-A are able to drive any brain expression (representative blank nab-DA disc is shown in B′′), strongly suggesting that the nab-B fragment is necessary and sufficient for the overall gross expression pattern of nab in the brain. (B′′′ and C′′′) Shown are blown-up images of the nab-A and nab-C activities in the wing disc (green) double labeled for Wg (magenta).
Figure 5
Figure 5
The nab DWME is induced by Notch signaling via its Su(H) sites. (A) The DWME contains two Su(H) sites, S1 and S2, and drives a unique stereotypical pattern. To test the role of these sites, we mutated each individually in the minimalized nab-A enhancer fragment. (B) The S1 mutated nab-A fragment exhibits no expression in wing imaginal discs. (C) The S2 mutated nab-A fragment results in discs with either no expression or weak dorsal margin expression as shown in these representative discs. (D) The nab-CDA fragment with an S1 mutated Su(H) exhibits reduced expression at the D–V compartment margin (arrow). (E) Quantitative comparison of wild-type, S1 mutated, and S2 mutated discs according to levels of expression. (F) Quantitative comparison of nab-A DWME activity in wild-type or mutant Notch backgrounds. Also shown is representative DWME reporter activity from discs in wild-type (G) or mutant Notch (H) backgrounds.
Figure 6
Figure 6
Apterous licenses the nab DWME to be receptive to Notch and Dpp signaling in the dorsal compartments of wing imaginal discs. (A) Cartoon of Ap expression (blue) in the dorsal compartment of a third instar wing imaginal disc. Wingless (Wg) expression (magenta) is found at the D–V compartment boundary in the wing pouch. (B–D) Wing imaginal discs stained for Wg (magenta), Gal4 (cyan), and GFP (green) in a line carrying the nab-A (DWME) eGFP reporter in a wild-type Ap background (B), in a heterozygous ap-GAL4 hypomorph caused by a GAL4 element integration (C), and a homozygous ap-GAL4 hypomorph (D). Note as the intensity of the Gal4 signal increases, the nab DWME-driven GFP signal decreases, which is consistent with its many Ap binding sites. (E and E′) The same ap-GAL4 hypomorph was used to drive nab RNAi to knock down expression in the dorsal compartment via both RNA and enhancer interference. Defects include an overly creased wing at wing vein II (arrows in E), a delta-like patterning defect where wing veins I and II intersect (arrow in E′), and occasionally missing or diminished anterior cross-veins (*acv). (F) Wild-type wing for comparison to E. (G) Wild-type notum (n) and scutellum (sc) with macrochaetes circled. (H) One balanced copy of the ap-GAL4 does not affect macrochaete patterning nor development of the scuttelum. (I–K) When ap-GAL4 drives nab-RNAi, adult flies develop grossly misshapen scutellums with severe macrochaete patterning defects. Three representative thoraxes are shown.
Figure 7
Figure 7
Distinct levels of nab expression are required for normal developmental patterning of the notum, scutellum, and wing veins. Wing vein patterning in female (A–D) and male (E–H) fly wings carrying a UAS-nab-RNAi transgene without a GAL4 driver (A, E, and the red wing outline in G), a nab locus GAL4 enhancer trap driver (B, C, F, and blue wing outline in G), or an en locus GAL4 enhancer trap driver augmented with additional UAS-dicer expression (D and H). Posterior cross-veins are often lost or incomplete in female wings with nab-RNAi knockdown arrows (B–D). The fourth wing vein is often lost in male wings (H). Thus, normal patterns of nab expression in both the dorsal and ventral compartments are crucial to wing vein patterning.
Figure 8
Figure 8
The nab DWME is stably maintained in off-margin clonal descendants. (A–L) GFP reporter activity driven by the nab DWME (nab-A fragment) starting during late second instar to late third instar wing imaginal discs are shown arranged according to the number of expressing cells based on nuclear GFP intensity. (A′–D′, H′, and I′) Zoomed-in images. Inspection of recently divided daughter cells (e.g., ovals in A′–D′ and I′) demonstrate that GFP activity is equally maintained in both cells even when the direction of cytokinesis places one of the daughter cells farther away from the dorsal compartment boundary. Cells far removed from the D–V boundary but still near the A–P compartment boundary display robust GFP expression (large arrows in H′). However, cells farther away from the A–P compartment boundary display decreasing levels of GFP expression even when they are on the dorsal margin itself (smaller arrows in H′ and I′). During early–late third instar (J and K) and late–late third instar/prepupal (L) discs, the DWME activity is maintained in clonal descendants of dorsal margin cells that are now located deep into the wing pouch. We thus propose that the DWME functions as a lineage-specific margin enhancer that maintains expression in dorsal off-margin clonal descendants. The disc in J is doubled stained for β-gal (magenta) driven by a 3-kb dpp enhancer (see Material and Methods). (M) The nab QE (nab-B fragment) is noticeably weaker or not active in dorsal compartment off-margin cells (arrowheads) from late third instar/prepupal discs. (N) DWME fragment in which all three Mad:Medea binding sequences have been mutated (5′-CG to 5′-TT mutations at each site) drives diminished expression in both margin and off-margin cells. Two representative discs are shown.
Figure 9
Figure 9
An enhancer model featuring selector licensing for pQ-mediated signal integration. (A) Shown are the trascription factor binding site motif distributions for the nab DWME (left) and the cut WME (right), both of which are induced by Notch signaling and Su(H) binding sites. As much as possible, matches to the indicated motifs are shown on separate tracks for ease of visualization. Tick marks represent 100-bp intervals. Boxes represent the minimalized enhancers, but in both cases, augmented expression is seen with larger fragments. (B and C) Below each enhancer are models of how each enhancer works in the context of both homeotic licensing and graded pQ signal integration or lack thereof. In both examples, selectors are envisioned as allowing certain cells (cell without Xs) to be receptive to transcriptional effectors of signaling pathways (Notch, Dpp, and Wg). The pQ/pN-rich Mad:Medea complexes are envisioned as stabilizing the active DWME in daughter cells of margin cells (B). In contrast, no such stabilizing effect is envisioned for the cut WME, which lacks both Mad:Medea sites (purple boxes) and instead has only Tcf (green boxes) and Su(H) (red boxes) binding sites (C). Thus, this enhancer would become inactive in margin daughter cells that divide away from the margin border, where there is active Notch–Delta signaling.

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References

    1. Akam M., 1998. Hox genes, homeosis and the evolution of segment identity: no need for hopeless monsters. Int. J. Dev. Biol. 42: 445–451. - PubMed
    1. Anderson J. D., Widom J., 2001. Poly(dA-dT) promoter elements increase the equilibrium accessibility of nucleosomal DNA target sites. Mol. Cell. Biol. 21: 3830–3839. - PMC - PubMed
    1. Archbold H. C., Broussard C., Chang M. V., Cadigan K. M., 2014. Bipartite recognition of DNA by TCF/Pangolin is remarkably flexible and contributes to transcriptional responsiveness and tissue specificity of wingless signaling. PLoS Genet. 10: e1004591. - PMC - PubMed
    1. Arnett K. L., Hass M., McArthur D. G., Ilagan M. X. G., Aster J. C., et al. , 2010. Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes. Nat. Struct. Mol. Biol. 17: 1312–1317. - PMC - PubMed
    1. Artavanis-Tsakonas S., 1999. Notch signaling: cell fate control and signal integration in development. Science 284: 770–776. - PubMed

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