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doi: 10.1242/10.1242/dev.00379

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Dally regulates Dpp morphogen gradient formation in the Drosophila wing

Momoko Fujise1,*,{dagger}, Satomi Takeo1,*, Keisuke Kamimura1,2, Takashi Matsuo1, Toshiro Aigaki1, Susumu Izumi1 and Hiroshi Nakato2,{ddagger}

1 Department of Biology, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-0397, Japan
2 Department of Molecular and Cellular Biology, and Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA
{dagger} Present address: Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan



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  		Fig. 1. Dpp morphogen gradients and expression of Dpp signaling components in the developing wing. (A,B) Patterns of Dpp-GFP expressed in A/P border cells by dpp-GAL4 driver (A; dppd12/+; dpp-GAL4/UAS-Dpp-GFP) and Dpp activity gradient visualized by anti-pMad antibody staining (B). (C,D) Expression patterns of the Dpp type I receptor gene tkv (C) and a putative Dpp co-receptor gene dally (D), were followed by anti-ß-galactosidase antibody staining of wing discs of the enhancer trap lines tkvP906 and dallyP2, respectively. Brackets show positions of the A/P border cells. Posterior is towards the right and ventral is towards the top of all wing discs and in all subsequent figures.

 


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  		Fig. 2. Expression of dally is regulated by Hh and En. (A-C) Misexpression of the membrane-tethered form of Hh, HhCD2, induces ectopic expression of dally. dally::lacZ expression was followed using anti-ß-galactosidase antibody staining (A). Clones expressing hhCD2 are marked by GFP in (B). (C) A merged image of A and B. Levels of dally::lacZ are increased both in cells inside and adjacent to the clones located in the A compartment (arrows), while clones in the P compartment do not alter dally expression. (D-F) dally is repressed by en in the P compartment. (D) Expression pattern of dally::lacZ. (E) Positions of en-mutant clones are shown by loss of GFP signal. (F) A merged image of D and E. dally expression is significantly elevated in en-mutant clones induced in the P compartment (arrow), indicating that dally is normally downregulated by en. Arrowheads in A and D indicate the AP boundary.

 


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  		Fig. 3. Expression of dally is repressed by Dpp signaling. (A-F) Clonal analysis for a severe tkv allele, tkva12, resulted in loss of the clones homozygous for tkv, because of growth defects, and preferential growth of their sister clones carrying two copies of the wild-type tkv in a genetic background heterozygous for tkv (one copy of wild-type tkv). The sister clones are marked by two copies of GFP (+/+), giving brighter signals than the single copy of GFP (+/–) (B,E). Wild-type (+/+) clones at the A/P border (A-C) and peripheral to the border (D-F) autonomously decrease the expression of dally::lacZ (arrows). (G-I) Clonal analysis for a mild tkv allele. dally::lacZ expression (G) is elevated in clones of cells homozygous for a partial loss-of-function tkv allele, tkv6 (arrows). (J-L) Effects of tkva12 mutation on dally::lacZ expression in the notum. Levels of dally expression (J) were significantly increased in tkva12 clones (arrows). Positions of clones are shown by loss of the GFP signal (H,K). (M-O) Effects of ectopically activated Dpp signaling on dally::lacZ expression. Expression of dally was repressed in the FLP-OUT clones that express a constitutively active form of tkv, tkvQ253D (M). Positions of the FLP-OUT clones are marked by GFP (N). (C,F,I,L,O) Merged images of the two left-hand images on each row.

 


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  		Fig. 4. Ectopic expression of dally increases the sensitivity of cells to Dpp. Three examples are shown for anti-pMad antibody staining of wing discs bearing clones of cells overexpressing dally. (A,D,G) Patterns of pMad. (B,E,H) Position of dally-expressing clones marked by GFP expression. (C,F,I) Overlay images. The pMad levels are increased in the dally-expressing clones in the wing pouch (arrows) and the hinge region (arrowheads). Clones induced in the peripheral domain did not show the elevated pMad signals (asterisks).

 


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  		Fig. 5. dally affects the Dpp morphogen activity gradient. (A-D) Anti-pMad antibody staining of the wing discs from wild-type (A), dallygem/+ (B), dallygem/dallygem (C) and dpp-GAL4/UAS-dally (D) animals. Arrowheads indicate the AP boundary. (E-H) Graphs indicate intensity profiles for pMad levels shown in A-D.

 


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  		Fig. 6. Expression of tkv in dally mutant wing disc. (A,B) tkv-enhancer trap expression was monitored using anti-ß-galactosidase antibody staining in wild-type (A) and dallygem/dallygem (B) backgrounds. (C,D) Anti-Tkv antibody staining of wild-type (C) and dallygem/dallygem (D) wing discs. tkv expression is not affected by the combinations of dally hypomorphic alleles, dallygem/dallygem and dallygem/dallyDP-527 (data not shown), that alter the shape of the Dpp activity gradient (Fig. 5C).

 


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  		Fig. 7. Distribution of Dpp-GFP in dally mutant wing disc. Dpp-GFP was expressed in A/P border cells by dpp-GAL4 and its distribution was monitored. Wild-type for dally gene (A), dallygem/dallygem (B-D), and dpp-GAL4/UAS-dally (E) larvae. All discs were heterozygous for dppd12 (see Materials and Methods). (F) Intensity profiles for Dpp-GFP in the posterior half of discs with three different genotypes are shown. 10, 8 and 12 samples were used for wild-type (green), dally homozygote (red) and dpp-GAL4/UAS-dally (blue), respectively, to obtain the averaged profiles.

 


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  		Fig. 8. Expression patterns and function of dally contribute to shaping the Dpp morphogen gradient in wing disc. (A) Model for Dally function in Dpp signaling. Dally (red) forms a signaling complex with Dpp (green) and receptor molecules (yellow) on the cell surface (left). Purple arrows represent signaling activity. Increased levels of Dally can enhance Dpp signaling by stabilizing the signaling complex (middle). However, excess levels of Dally sequester Dpp protein and show an inhibitory effect on signaling (right). (B) Levels of tkv expression (yellow), dally expression (red) and Dpp signaling (purple). Expression of both Tkv (a Dpp receptor) and Dally (a Dpp co-receptor) is regulated by several common molecular pathways in the wing. (1) Hh signaling suppresses tkv and activates dally. (2) En induces tkv and represses dally. (3) Dpp signaling downregulates both genes. As the Tkv receptor and Dally co-receptor mediate Dpp signaling, this regulatory pathway forms a negative-feedback loop. At anterior and posterior edges of the wing pouch, lower levels of Dpp signaling result in high levels of Tkv and Dally, which sensitize cells to Dpp.

 

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