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doi: 10.1242/10.1242/dev.00159

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The ubiquitin ligase Hyperplastic discs negatively regulates hedgehog and decapentaplegic expression by independent mechanisms

Jeffrey D. Lee¹, Kazuhito Amanai^2,*, Allen Shearn² and Jessica E. Treisman^1,

¹ Skirball Institute of Biomolecular Medicine and Department of Cell Biology, NYU School of Medicine, 540 First Avenue, New York, NY 10016, USA
² Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
^* Present address: Center for Developmental Biology and Department of Pharmacology, UT Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA

View larger version (102K): [in a new window]   Fig. 1. Loss of hyd leads to ectopic differentiation and overgrowth in the eye disc. (A-C) Adult eyes; (A) wild type; (B) hydK3.5 clones have induced outgrowths of wild-type (white+) tissue; (C) hydK3.5 mutant clones generated in a Minute background. All surviving photoreceptors are white- and therefore hyd mutant. (D-I) Third instar eye discs. Anterior is to the left and dorsal up in this and subsequent figures. (D-F) Anti-Elav staining in brown and X-gal staining, to show arm-lacZ expression, in blue. (D) Clones of wild-type tissue identified by lack of arm-lacZ expression. (E) Clones of hydK3.5 mutant cells identified by lack of arm-lacZ expression. Ectopic photoreceptors are visible in an anterior clone. (F) hydK3.5 clones generated in a Minute background. Remaining wild-type tissue is marked by arm-lacZ expression. (G,H) Anti-Ato staining in wild type (G) and in a disc containing an unmarked hydK3.5 clone (H), where a ring of ectopic Ato staining is visible anterior to the furrow. (I) An early third instar eye disc with hydK3.5 clones generated in a Minute background, stained with anti-Elav in red and anti-Ato in green. Ato is already restricted to single cells at the anterior of the disc.

View larger version (63K): [in a new window]   Fig. 3. Removal of hh function partially suppresses the hyd mutant phenotype. All panels show third instar eye discs. (A-D) Anti-Elav staining in brown and X-gal staining, reflecting arm-lacZ expression (wild-type tissue), in blue. (A) Ectopic differentiation in hydK3.5 mutant clones. (B) hhrJ413 clones; lack of photoreceptor differentiation is only apparent in clones at the posterior margin (arrow). (C) hydK3.5, hhrJ413 double mutant clones. No ectopic photoreceptors form, but some clones still induce overgrowth of wild-type tissue (arrow). (D) hydK3.5, hhrJ413 clones generated in a Minute background. The few photoreceptors that form are in wild-type tissue. (E) An adult eye containing hydK3.5, hhrJ413 clones and exhibiting some overgrowth (arrow). (F) Unmarked hydK3.5 clones stained with anti-Elav in brown and X-gal, reflecting dpp-lacZ expression, in blue. dpp is ectopically expressed anterior to the furrow. (G,H) A third instar eye disc containing hydK3.5, hhAC mutant clones (labeled by lack of GFP expression in H). Anti-ß-galactosidase (in red) is used to visualize dpp-lacZ expression; ectopic dpp is still visible in some clones (arrow in H).

View larger version (101K): [in a new window]   Fig. 2. Loss of hyd induces ectopic hh expression in the eye disc. All panels show third instar eye discs. (A-D) Anti-Elav staining in red, anti-ß-galactosidase staining, reflecting hh-lacZ expression, in blue, and GFP in green. (A-C) hydK7.19 clones (marked by lack of GFP expression in C); (D) wild type. hh is misexpressed anterior to its normal domain in hyd mutant clones. (E) hydK7.19 clones made in a Minute background, showing hh-lacZ expression in green. hh is misexpressed throughout the eye disc. The two portions of the eye-antennal disc are indicated: e, eye disc; a, antennal disc. (F) In situ hybridization with a hyd antisense probe. hyd is predominantly expressed anterior to the furrow. The sense probe showed only very faint non-specific staining (not shown). (G-I) hydK7.19, atoIH3 clones positively marked by GFP expression in (H,I) are stained with anti-Elav in red and anti-ß-galactosidase, reflecting hh-lacZ expression, in blue. Lack of ato prevents differentiation, but not hh expression, within the hyd clones (arrows); ectopic hh then leads to differentiation of surrounding wild-type tissue.

View larger version (79K): [in a new window]   Fig. 4. hyd regulates dpp but not hh through Ci. (A,B,D-F) Third instar eye discs with anti-Elav staining in red. hydK7.19 clones expressing UAS-ci76 with tub-GAL4 are positively marked by GFP expression in B,E,F. (A,B) Anti-ß-galactosidase staining reflecting dpp-lacZ expression in blue. (D,F) Anti-ß-galactosidase staining reflecting hh-lacZ expression in blue. Expression of Ci76 in hyd clones blocks dpp expression but not hh expression. (C) An adult eye with hydK7.19, hhrJ413 clones expressing UAS-ci76. No overgrowth is visible.

View larger version (147K): [in a new window]   Fig. 5. Ci, but not Smo, accumulates in the absence of hyd and hh. All panels show third instar eye discs with hydK3.5, hhAC mutant clones (marked by lack of GFP expression in B,C,E,F). (A,C) Staining with an antibody to the C-terminal region of Ci. (D,F) Staining with anti-Smo. Arrows indicate examples of double mutant clones that do not accumulate Ci (C) and do not accumulate Smo (F).

View larger version (103K): [in a new window]   Fig. 6. hyd affects hh expression and Ci accumulation in the wing disc. All panels show third instar wing discs with anterior to the left and dorsal up. (A,B) hydK7.19 mutant clones (marked by lack of GFP expression in B). hh-lacZ is shown in red and is increased in round posterior clones. (C-H) hydK3.5 mutant clones (marked by lack of GFP expression in D,F,H). Staining with anti-Smo is shown in red in C and D and staining with an antibody to the C-terminal region of Ci is shown in red in E,F. Anterior hyd mutant clones accumulate higher levels of full-length Ci, but do not accumulate Smo protein. (G,H) dpp-lacZ expression (in red). dpp expression is increased in clones near the AP boundary, but dpp is not activated in more anterior clones. (I-L) slmbB93 mutant clones (marked by lack of GFP expression in J,L). Staining with an antibody to the C-terminal region of Ci is shown in red in I,J and is stronger than in hyd mutant clones. (K,L) dpp-lacZ expression (in red). dpp is misexpressed only in anterior clones that lie outside the wing pouch.

View larger version (8K): [in a new window]   Fig. 7. Model for Hyd function. Arrows represent positive effects, and barred lines represent negative effects. Hyd represses hh transcription, probably indirectly. Hyd also blocks the accumulation of full-length Ci without affecting Smo. Since Slmb has been implicated in processing Ci to the repressor form Ci75, we suggest that Hyd may instead act by targeting Ci for degradation.