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First published online 28 September 2005
doi: 10.1242/dev.02048

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DNA-binding domain mutations in SMAD genes yield dominant-negative proteins or a neomorphic protein that can activate WG target genes in Drosophila

Norma T. Takaesu¹, Eric Herbig^1,*, David Zhitomersky², Michael B. O'Connor² and Stuart J. Newfeld^1,

¹ School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA
² Howard Hughes Medical Institute, Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA

View larger version (91K): [in a new window]   Fig. 1. Mad1 and Med7 are dominant-negative alleles. (A) dpps6/dpphr4 wing with normal veins. Longitudinal veins 1-5, the anterior crossvein (acv) and the posterior crossvein (pcv) are shown. (B) dpps6/dpphr4 Df(2L)C28 with truncated L4/L5. (C) dpps6/dpphr4 Mad1 with abnormal L2/L3/L4, no crossveins and two margin notches. (D) dpps6/dpphr4 Med7 with a truncated L5 and two margin notches.

View larger version (139K): [in a new window]   Fig. 2. Expression of MAD1 and SMAD4130S generates phenotypes that resemble Mad loss-of-function phenotypes. (A) Wild-type wing. (B) UAS.Mad/ptc.Gal4 with extra vein tissue between L3/L4/L5. (C) UAS.Mad12/69B.Gal4 appears wild type. (D) Mad11/Mad12 with no L2, abnormal L4/L5, no crossveins and a large margin notch (not to scale). (E) UAS.Mad1/69B.Gal4 with truncated L2/L3/L4/L5, abnormal crossveins and a moderate margin notch. (F) UAS.SMAD4130S/A9.Gal4 with truncated L2/L3/L4/L5 and a small margin notch.

View larger version (121K): [in a new window]   Fig. 3. Expression of MAD1 results in expanded Drosophila SRF expression as a result of reduced rhomboid transcription. (A) Wild-type pupal wing disc stained for Drosophila SRF expression. Anterior is towards the top and distal to the left. L1-L5 primordia are indicated. (B) UAS.Mad/ptc.Gal4 has expanded regions without Drosophila SRF corresponding to L3/L4. (C) UAS.Mad12/69B.Gal4 appears wild type. (D) UAS.Mad1/69B.Gal4 with expanded regions of Drosophila SRF that limit the extent of L3/L4. (E) Wild-type pupal wing disc stained for rhomboid (rho) transcripts. L3/L4/L5 primordia are indicated. rho expression in L1/L2 is not yet fully developed at this stage; note the difference in the visibility of L1/L2 versus L3/L4/L5 in A. (F) UAS.Mad1/69B.Gal4 with reduced rho expression. Expression in L4 is severely reduced and distal truncations in L3/L5 are visible.

View larger version (135K): [in a new window]   Fig. 4. Expression of SMAD4100T induces ectopic mechanosensory bristles on the wing blade. (A) UAS.SMAD4100T/MS1096.Gal4 wing with six ectopic mechanosensory bristles (open arrowheads). This image is a composite of three wings such that all positions for ectopic bristles are shown (the three bristles on medial L3 never appear together). (B) Dorsal L1 of wild type with two campaniform sensilla (arrowheads). (C) Dorsal L1 of UAS.SMAD4100T/MS1096.Gal4 with a stout mechanosensory bristle replacing Twin Sensillum One (open arrowhead; black arrowhead indicates Twin Sensillum Two). (D) Ventral L3 of wild type with a campaniform sensillum (arrowhead). (E) Ventral L3 of UAS.SMAD4100T/MS1096.Gal4 with a stout mechanosensory bristle replacing the sensillum (open arrowhead). (F) Dorsal L3 of wild type with three campaniform sensilla (arrowheads). (G) Dorsal L3 of UAS.SMAD4100T/MS1096.Gal4 with a thin mechanosensory bristle replacing the proximal sensillum (open arrowhead). (H) Dorsal L3 of UAS.SMAD4100T/MS1096.Gal4 with a thin mechanosensory bristle replacing the middle sensillum (open arrowhead). (I) Ectopic thin mechanosensory bristles are seen on a wing with small, unmarked clones of cells homozygous for zw3M11 (open arrowheads).

View larger version (42K): [in a new window]   Fig. 5. Expression of GBB does not induce ectopic anterior margin bristles on the wing blade. (A) Ventral view of a UAS.Gbb/ap.Gal4 wing with all bristles missing from the proximal anterior wing margin (arrowhead). A distal portion of the anterior wing margin, indicated by the black bar, is shown at higher magnification in B,C. (B) Dorsal view: the row of stout mechanosensory bristles appears in a roughly wild-type pattern with occasional gaps. The row of widely spaced chemosensory bristles shows considerable irregularity in spacing, with some bristles displaced dorsally. (C) Ventral view: the row of alternating chemosensory and thin mechanosensory bristles is disorganized and numerous thin mechanosensory bristles are present in a region ventral to their normal location.

View larger version (97K): [in a new window]   Fig. 6. Expression of SMAD4100T suppresses wing phenotypes generated by dominant-negative Drosophila TCF. (A) UAS.DN-TCF/MS1096.Gal4 wing is only 10% of the size of a wild-type wing and has no margin. (B) UAS.DN-TCF/MS1096.Gal4 wing with two copies of UAS.SMAD4100T is roughly three-fold larger than the wing in A, and the anterior margin is restored. Transformation of Twin Sensillum One into a bristle is noted (arrowhead). (C) UAS.DN-TCF/MS1096.Gal4 wing with three copies of UAS.SMAD4100T is roughly four-fold larger than the wing in A, and the anterior margin is restored. Sensillum-to-bristle transformation is noted (arrowhead). (D) UAS.SMAD4100T/MS1096.Gal4 wing has a normal margin and two sensilla-to-bristle transformations (arrowheads). (E) Wing with MS1096.Gal4 driving two copies of UAS.SMAD4100T has a normal margin, sensilla-to-bristle transformation (arrowhead) and vein defects. (F) Wing with MS1096.Gal4 driving three copies of UAS.SMAD4100T has a reduced size, a normal margin, sensilla-to-bristle transformation (arrowhead) and vein defects.

View larger version (147K): [in a new window]   Fig. 7. Expression of SMAD4100T mimics the expression of constitutively active ARM. (A) Wild-type six-row denticle pattern shown with anterior to the left. The small, anteriorly pointed denticles of row one are indicated (arrowhead). (B) UAS.lacZ/en.Gal4 six-row denticle pattern; lacZ expression is coincident with row one (arrowhead). (C) UAS.CA-ARM/en.Gal4 five-row denticle pattern. The transformation of row one denticles into naked cuticle is indicated (arrowhead). (D) UAS.SMAD4100T/en.Gal4 six-row denticle pattern. The transformation of a subset of row one denticles into naked cuticle is indicated (arrowhead).

View larger version (43K): [in a new window]   Fig. 8. Expression of SMAD4100T activates the expression of the WG target gene achaete. (A) Wild-type third instar wing disc stained for Achaete (AC). a, anterior; m, proximal margin; v, ventral surface; dis, distal margin. (B) zw3M11 clones produce ectopic AC expression on the presumptive blade (arrowheads). (C) UAS.SMAD4100T/C765.Gal4 with ectopic AC expression on the presumptive blade (arrowheads). (D) UAS.Dpp/C765.Gal4. This is a lethal genotype. The disc is overgrown (image shown at a reduced magnification) but it has wild-type AC expression. (E) UAS.Gbb/C765.Gal4 with reduced AC expression in the proximal anterior margin primordia (arrowhead).

View larger version (36K): [in a new window]   Fig. 9. SMAD4100T may cause tumors via an `APC-like' mechanism not previously associated with defective TGFß signaling. Model for the mechanism of tumorigenesis used by SMAD4100T based upon its ability to activate WG target genes in flies. (Left) In the model, SMAD4100T functions in a way that mimics the repression of the ARM-destruction complex (ZW3, APC and Axin) in the WG pathway. In this illustration, we show SMAD4100T actively inhibiting the destruction complex, but SMAD4100T may interact with the WG pathway at other points, such as target promoters. SMAD4100T and proteins potentially affected by its activity are shown in red. (Right) The mechanism of tumorigenesis associated with loss-of-function mutations in APC. Loss of APC activity inhibits the ß-catenin destruction complex (GSK3ß, APC and Axin) leading to the overexpression of WNT target genes and Familial Adenomatous Polyposis. APC and proteins affected by the loss of APC function are shown in red.