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First published online 30 May 2006
doi: 10.1242/dev.02427

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Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila

Yaopan Mao^1,*, Cordelia Rauskolb^2,*, Eunjoo Cho^1,, Wei-Li Hu^3,, Heather Hayter³, Ginny Minihan³, Flora N. Katz^3, and Kenneth D. Irvine^1,¶

¹ Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA.
² Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA.
³ Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA.

View larger version (103K): [in a new window]   Fig. 1. Requirements for dachs in the wing. Adult wings, all at the same magnification, from (A) wild type, (B) d1, (C) dGC2, (D) dGC13, (E) d1/d210, (F) d1/Df(2L)N22-5 and (G) dGC13/Df(2L)ED623. This wing came from exceptional individual that eclosed, and represents the mildest dachs-null phenotype observed. (H) dGC13/Df(2L)N22-5. (I) dGC13; tub-Gal4 UAS-d:V5[18-2] (J) tub-Gal4 UAS-d:V5[8-3]. Arrows in A,B indicate crossveins; arrows in E,F indicate ectopic crossvein material, whereas the anterior crossvein is missing. Wings in C,D,H are from pharate adults.

View larger version (106K): [in a new window]   Fig. 2. Requirements for dachs in the leg. Panels A-J show adult legs, all at the same magnification, from (A) wild type, (B) d1, (C) dGC2, (D) dGC13, (E) dGC13/d210, (F) dGC13/Df(2L)ED623, (G) dGC13; tub-Gal4 UAS-d:V5[18-2], (H) tub-Gal4 UAS-d:V5[8-3], (I) an animal with fat8 mutant clones and (J) an animal with fat8 dGC13 double mutant clones. In H and I, arrows indicate outgrowths and insets show internal cuticle vesicles. (K-L) Pupal legs from wild type and dGC2, the tip of the leg (asterisk) is mispositioned in L. (M) dGC13 pharate adult, with only one external leg and five that failed to evert (arrowheads).

View larger version (102K): [in a new window]   Fig. 3. Influence of fat and dachs on downstream target genes. Imaginal discs, with wild-type cells marked by GFP (green), stained for Ser (red) or fj-lacZ (magenta) expression. In this and subsequent figures, panels marked ' show individual channels of the panel to the left. Arrows indicate examples of ectopic or elevated expression, arrowheads indicate examples of reduced or absent expression. (A) Eye disc with reduced fj-lacZ within dGC13 mutant clones. (B) Eye disc with elevated fj-lacZ within fat8 clones. (C) Wing disc with reduced fj-lacZ within dGC13 clones. (D) Wing disc with elevated fj-lacZ within fat8 clones. (E) Eye disc with reduced fj-lacZ within fat8 dGC13 mutant clones (white arrowheads), and no ectopic fj-lacZ (yellow arrowheads). (F) Leg disc with normal Ser expression within large (Minute) dGC13 clones. (G) Leg disc with ectopic Ser within proximal fat8 clones (H) Leg disc with normal Ser expression (arrowhead) within large (Minute) proximal fat8 dGC13 clones. (I) Pupal leg disc with reduction of Ser expression within the second tarsal segment.

View larger version (131K): [in a new window]   Fig. 4. Influence of fat and dachs on cell affinity. Third instar wing imaginal discs, stained for expression of Wg (red). GFP (green) marks wild-type cells. (A) fat8 mutant clones (black) are abnormally large and round. Some elevation of Wg expression in the proximal wing is also visible (arrow), although it is mostly out of the plane focus. (B) d1 mutant clones have normal, elongated shapes. (C) fat8 d1 double mutant clones lack ectopic Wg expression (arrowhead), and are similar to d1 mutant clones in size and shape, and obviously distinct from fat8 mutant clones. See also Table S1 in the supplementary material.

View larger version (109K): [in a new window]   Fig. 5. Influence of dachs on polarity. (A-H) Regions of abdomens. (C,F,G) Mutant clones, marked by yellow bristles (asterisks). (A) Wild type; hairs point posteriorly. (B) fat8/fatG-rv; hair polarity is severely disturbed. (C) fat8 mutant clones; hair polarity is severely disturbed. (D) dGC13; hairs point posteriorly. (E) fat8 dGC13; hair polarity is moderately disturbed. (F) fat8 dGC13 mutant clones; hair polarity is moderately disturbed. (G) dGC13 mutant clones; hair polarity appears normal. (H) dGC13 ck13 double mutant clones (outlined); polarity can not be assessed within the clone because of ck, but polarity outside the clones appears normal. (I) High-magnification of a region of a dGC13/Df(2L)ED623 wing, most hairs point distally, as in wild type. (J-N) Eye discs, stained for Elav (red) and Prospero (green). Arrows indicate ommatidial orientation. (J) Wild type, (K) dGC13. (L-N) Mutant clones, marked by absence of GFP (blue in upper panels, white in lower panels). Ommatidia that are mis-rotated by more than 90° are marked by yellow arrows. To compensate for tissue curvature, some panels are composites of multiple focal planes.

View larger version (63K): [in a new window]   Fig. 6. Cloning and sequence of Dachs. (A) Localization of dachs on 2L. P elements used for male recombination are indicated above the chromosome. Horizontal arrows indicate the result of recombination, placing dachs distal to each insertion. Deficiencies used to map dachs are shown below. (B) Protein domains, transcript and gene structure of dachs. Exons are indicated as boxes and are numbered. Intervening introns appear as black lines. The locations of mutations in dachs alleles are indicated. (C) Amino acid sequence of Dachs. The conserved myosin head domain is in bold. Underlining identifies motifs discussed in the text: 1, coiled-coil domain; 2, ATP-binding domain; 3, unique insert in myosin head domain; 4, actin-binding domain; 5, active thiol domain; 6, IQ camodulin-binding domain; 7, putative transmembrane domain. The locations of mutations are indicated above: dGC2, inframe deletion indicated by broken line; dGC13, 11 bp deletion predicted to truncate at the indicated aa; d210, point mutation introducing a stop codon; d1, insertion of a blood retrotransposon.

View larger version (107K): [in a new window]   Fig. 7. dachs mRNA expression. dachs mRNA expression, visualized by in situ hybridization. (A) Stage 6 embryo, (B) stage 9 embryo, (C) stage 11 embryo, (D) wing disc and (E) leg disc.

View larger version (81K): [in a new window]   Fig. 8. Dachs protein localization. High magnifications of wing imaginal discs, stained for Fat (red, C-F) or E-cadherin (red, A,B,G) and Dachs:V5 (green), with GFP expression in blue. For experiments depicted in A and B, discs were treated in parallel, and images of clones were captured with identical confocal settings. (A) Dachs:V5-expressing clone in AyGal4 UAS-dachs:V5[9F] UAS-GFP. Arrows indicate distal clone edges with higher Dachs:V5; arrowheads indicate proximal clone edges with lower Dachs:V5. (B) Fat and Dachs:V5-expressing clone in AyGal4 UAS-fat UAS-dachs:V5[9F] UAS-GFP. (C) Fj and Dachs:V5-expressing clone in AyGal4 UAS-dachs:V5[9F] UAS-fj[6a2] UAS-GFP. Arrowhead indicates elevation of Fat, and coincident absence of Dachs:V5, at the edge of the clone. (D) Fj-expressing clone in AyGal4UAS-fj[6a2] UAS-GFP. Arrowhead indicates elevation of Fat at the edge of the clone. (E) Ds-expressing clone in AyGal4 GS-ds UAS-GFP. Arrow indicates elevation of Fat at the edge of the clone. (F) Ds and Dachs:V5-expressing clone in AyGal4 UAS-dachs:V5[9F] GS-ds UAS-GFP. Arrow indicates elevation of Fat, and coincident elevation of Dachs:V5, at the edge of the clone. (G) fat mutant clone (arrow), marked by absence of GFP, in tub-Gal4 UAS-dachs:V5[18-2]. Dachs staining is slightly elevated within the clone. (H) Vertical section through the apical-most 4.5 µm of the clone depicted in A. Most Dachs:V5 staining overlaps E-cadherin at the apical junctions, but there are discrete puncta (arrow), as well as more diffuse staining, in the cytoplasm. (I) Western blot, with anti-V5 or ant-Actin (control) on discs from: (1) fat8/fatG-rv; tub-Gal4/UAS-dachs:V5[18-2], (2) fat8/+; tub-Gal4/UAS-dachs:V5[18-2] and (3) wild type.

View larger version (38K): [in a new window]   Fig. 9. Working models for Fat regulation and signaling. (A,B) Schematics of a cross-section of two cells. Genetic and protein localization studies position Dachs downstream of Fat in a pathway that influences growth and gene expression in imaginal discs. (A) When Ds and Fat are engaged, Fat accumulates at the membrane, where it downregulates Dachs, thereby preventing Dachs-dependent growth and gene expression. (B) Four-jointed is proposed to modify Ds and thereby prevent it from engaging Fat. This allows accumulation of Dachs, and promotion of growth, affinity and gene expression. (C,D) Show schematics of a horizontal section of a field of cells, with localization of Ds (blue), Fat (red) and Dachs (green) schematized. (C) Expression of Fj in a clone of cells (gray) is proposed to antagonize Ds (i) (broken lines). The absence of a good Ds partner within the clone causes Fat in cells at the edge of the clone to localize to the clone perimeter (ii), where it downregulates Dachs (iii). (D) Expression of Ds in a clone of cells (i) (gray) is proposed to recruit Fat in neighboring cells to the edge of the clone (ii). This results in elevated Dachs at the edge of the clone (iii). See text for discussion of possible mechanisms.