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First published online 13 December 2006
doi: 10.1242/dev.02737

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Hox control of morphogen mobility and organ development through regulation of glypican expression

¹ Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
² Department of Biochemistry and Molecular Biophysics, Columbia University, HHSC 1104, 701 W. 168th Street, New York, NY 10032, USA.

View larger version (90K): [in this window] [in a new window]   Fig. 1. dally expression and Dpp signaling are reduced in the posterior haltere. (A-A'') Comparison of dally-lacZ and P-Mad staining in the wing and haltere. In the wing, dally-lacZ is expressed similarly on both sides of the AP organizer (A'). In the haltere, dally-lacZ expression is low in the P compartment, especially in P cells abutting the AP boundary (arrow). P-Mad staining in the wing is detected in two slopes that peak at either side of the AP organizer, whereas in the haltere, P-Mad is detected in a single stripe (A''). (B) dally-lacZ intensity traces for the wing and haltere discs shown in A. The approximate position of the AP boundary is indicated here (and in D,F) by an arrow. Note the symmetry in dally expression in the wing with respect to the AP organizer (just left of the arrow), as compared with the asymmetry in the haltere. Also note the generally lower dally levels in AP organizer cells and the hyper-repressed levels in the P cells abutting the AP boundary in the haltere. (C-C'') The repression of dally-lacZ in the haltere correlates with very low P-Mad staining in the P compartment. The yellow line marks the AP compartment boundary. Note that strong P-Mad labeling is detected for only one or two cell diameters posterior to the AP boundary, and that the basal level of P-Mad staining is higher in the A compartment (compare levels at asterisks in C''). (D) Intensity plots of P-Mad and dally-lacZ stainings from the pouch region of the wild-type haltere disc shown in C. In the P compartment, P-Mad staining drops off rapidly, coinciding with a sharp drop in dally-lacZ. P-Mad staining persists much further away from the AP organizer in the A compartment (arrow). The blue dotted line marks the low point of the P-Mad signal. (E-E'') ptc-Gal4 UAS-dpp::GFP wing and haltere discs stained for P-Mad (blue) and for GFP using an extracellular staining protocol (red). The green channel shows GFP autofluorescence. In the haltere (enlarged in E' and E''), extracellular Dpp::GFP and P-Mad detection stop a few cell boundaries posterior to the AP boundary (white arrow), whereas these signals decay more gradually in the A compartment (compare staining around asterisks). (F) Intensity plots of extracellular-GFP, P-Mad and GFP autofluorescence for the haltere disc shown in (E'). P-Mad and extracellular-GFP staining extend further anteriorly than posteriorly from the site of Dpp::GFP production (approximated by GFP autofluorescence). W, wing; H, haltere; A, anterior compartment; P, posterior compartment.

View larger version (72K): [in this window] [in a new window]   Fig. 2. tkv -independent Dpp signaling differences in the wing and haltere. (A) Wing and haltere discs in which tkv is uniformly expressed from the Actin 5C promoter (act>tkv). In the wing, the P-Mad gradient is compacted to a single stripe, similar to the pattern found in the wild-type haltere (Crickmore and Mann, 2006). (B) Intensity traces for P-Mad in the wing (blue) and haltere (red) shown in A. In the act>tkv wing, the P-Mad signal is symmetric along the AP axis relative to the AP organizer, which is approximated by peak P-Mad levels. In the act>tkv haltere, the P-Mad signal decays more gradually in the A compartment (blue arrow) than in the P compartment. The approximate position of the AP boundary is indicated by the arrow. (C,D) Enlargements of the wing and haltere discs shown in A. W, wing; H, haltere; A, anterior compartment; P, posterior compartment.

View larger version (54K): [in this window] [in a new window]   Fig. 3. Posterior Dally levels influence Dpp signaling and compartment size. (A,A') An en-Gal4; UAS-GFP haltere disc stained for P-Mad. The AP boundary is traced in yellow. Little P-Mad staining is found in the P compartment of the haltere. (B,B') An en-Gal4; UAS-lacZ, UAS-dallyweak haltere disc stained for P-Mad. Boosting dally levels in the P compartment of the haltere increases the intensity and extent of posterior P-Mad staining. This genotype also causes a reduction in P-Mad staining in the A cells abutting the AP compartment boundary (arrow). (C,C') Driving a stronger version of UAS-dally (UAS-dallystrong) ith en-Gal4 causes most of the P-Mad signal to shift into the P compartment, non-autonomously reducing P-Mad levels in adjacent A-compartment cells to near basal levels (arrow). (D) en-Gal4; UAS-dallyweak haltere discs have a larger P:A ratio (in the Nubbin-marked, appendage-forming domain) than control (en-Gal4; UAS-lacZ) haltere discs. (E) Expressing UAS-dallyweak in the haltere P compartment increases overall adult haltere size by 5% as compared with control (en-Gal4; UAS-lacZ) halteres. A, anterior compartment; P, posterior compartment.

View larger version (68K): [in this window] [in a new window]   Fig. 4. Consequences of asymmetric dally expression in the wing. All wing discs in this figure were stained and imaged together and can therefore be directly compared. (A-A'') Control ci>GFP wing disc stained for GFP, P-Mad and Nubbin (Nub, a marker for the appendage-forming region of the disc). The white and red arrows indicate expression of P-Mad in the anterior and posterior compartments, respectively. (B,B') When UAS-dallyRNAi is expressed with en-Gal4, the extent of P-Mad staining in the P compartment (red arrow) is reduced as compared with control wing discs (A'). (C) Driving UAS-dallyRNAi in the P compartment reduces the P:A ratio (0.65 compared with 1.04 in control discs). (D,E) Driving UAS-dallyRNAi with en-Gal4 decreases the overall size of the adult wing by 19% as compared with control (en-Gal4; UAS-GFP) wings. The decreased P:A ratio is evident in the posterior shift of vein L4 of en>dallyRNAi wings (magenta arrowhead) compared with control wings (black arrowhead). (F,F') Driving UAS-dallyRNAi in the A compartment with ci-Gal4 increases the extent of P-Mad staining in the P compartment (red arrow) compared with control wing discs (A'). The levels of P-Mad staining in the AP organizers of these discs is increased compared with control wing discs (A'; see main text). (G) Driving UAS-dallyRNAi in the A compartment increases the P:A ratio (1.36 compared with 0.94 in control discs). (H,I) Although the increased P:A ratio is evident in the anterior shifting of vein L4 (H, arrowheads), the overall size of ci-Gal4 UAS UAS-dallyRNAi wings is the same as in control wings (I). A, anterior compartment; P, posterior compartment.

View larger version (115K): [in this window] [in a new window]   Fig. 5. Ubx control of dally expression in the haltere. (A,A') Wild-type dally-lacZ patterns in wing and haltere discs. AP organizers are indicated by arrows to highlight high dally-lacZ levels in the wing and moderate levels in the haltere. (B,B') Clones in which the Dpp pathway is activated with UAS-tkvQD (marked by GFP) downregulate dally-lacZ in both the wing and haltere (arrows). (C,C') Driving high tkv levels in the wing converts the wing-like pattern of dally-lacZ into a haltere-like pattern in the A compartment. The moderate dally-lacZ expression level in AP organizer cells is indicated by the arrow. (D,D') dally-lacZ is derepressed in Ubx- haltere clones (identified by the absence of GFP) in the P compartment, especially within the domain of dally hyper-repression (arrow). (E,E') Clones of cells mutant for en (absence of GFP) in the P compartment derepress dally-lacZ in the haltere (arrow). (F,F') Clones expressing Daughters against Dpp (Dad; marked by GFP), which inhibits Dpp signaling, derepress dally-lacZ in the AP organizer (arrow) and dally hyper-repression (arrowhead) domains. Thus, Dpp represses dally in the medial haltere, and is required for the very low dally levels in the anterior-most cells of the P compartment. (G) Diagrams summarizing the locations and consequences of positive and negative inputs into dally expression (broken red line) in the haltere and wing. In the wing, because peak Dpp and Hh signaling occur in neighboring cells, dally levels are high in the AP organizer owing to Hh activation, but are lower in neighboring cells owing to Dpp repression. In the haltere, because peak Dpp and Hh signaling coincide in the same cells, these two conflicting inputs result in intermediate dally levels in the AP organizer. In addition, dally is hyper-repressed in P cells next to the AP boundary because of Ubx, en and Dpp signaling. Similar effects are also observed for dpp expression (blue dotted line), which, like dally, is activated by Hh and repressed by Dpp (see Crickmore and Mann, 2006). A, anterior compartment; P, posterior compartment.