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First published online 10 May 2006
doi: 10.1242/dev.02401

Development 133, 2315-2324 (2006)
Published by The Company of Biologists 2006
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Separating the adhesive and signaling functions of the Fat and Dachsous protocadherins

Hitoshi Matakatsu and Seth S. Blair*

Department of Zoology, University of Wisconsin, 250 North Mills Street, Madison, WI 53706, USA.


Figure 1
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  		Fig. 1. Structures of full-length and deleted Ft and Ds proteins. Shown are the cleaved signal sequences (black), cadherin domains (purple), EGF-like domains (green), Laminin-G domains (yellow-green), transmembrane domains (light blue) and the HA tag (dark blue). Sites mutated in ftfd and ftG-rv are also shown. ftfd contains a stop codon in the 9th cadherin domain. ftG-rv contains a mdg3 retrotransposon insertion (the beginning of its sequence is underlined) that introduces a stop codon in the 27th cadherin domain; it also contains additional rearrangements in the first intron and a previously described insertion in the 33rd cadherin domain (not shown) (Mahoney et al., 1991Go).

 

Figure 2
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  		Fig. 2. Expression of full-length and deleted ft and ds constructs. (A-F) Expression of full-length and deleted ft and ds constructs in the posterior compartments (right) of ftG-rv/ftfd (A-C), ds05142 (D,E) or wild type (F) wing imaginal discs with en-gal4. (A) UAS-ft. (B) UAS-ft{Delta}ICD. In both A and B, Ft or the HA-tagged Ft{Delta}ICD (green and center panels) is concentrated at the cell cortex, and leads to stronger anti-Ds staining (purple and right panels) in the region of misexpression. (C) UAS-ft{Delta}ECD; anti-Ft staining (green and center panel) is diffuse and not concentrated at the cell cortex, and does not lead to stronger anti-Ds staining (purple and right panel) in the region of misexpression. There appears to be a slight decrease in Ds levels. (D) UAS-ds. (E) UAS-ds{Delta}ICD. In both D and E, anti-Ds staining (green and center panels) is concentrated at the cell cortex, and leads to stronger anti-Ft staining (purple and right panel) in the region of misexpression. (F) UAS-ds{Delta}ECD; anti-Ds staining (white) is diffuse and not concentrated at the cell cortex. (G) AyGal4 FLPout clone expressing UAS-ds{Delta}ECD and UAS-GFP (green) in a wild-type wing imaginal disc. The clone does not lead to stronger anti-Ft staining (purple and right panel).

 

Figure 3
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  		Fig. 3. Aggregation of S2 cells induced by transfection with full-length and deleted ft and ds constructs. Expression in cells was induced by co-transfection with act-gal4. Cells in A-E were counterstained with rhodamine-labeled phalloidin (purple). (A) Control cells transfected with UAS-GFP (green) did not aggregate. (B,C) Cells co-transfected with UAS-ft, UAS-GFP and either UAS-ds (B) or UAS-ds{Delta}ICD (C) aggregated. (D,E) Cells co-transfected with UAS-GFP and either UAS-ft{Delta}ECD and UAS-ds (D) or UAS-ds{Delta}ECD and UAS-ft (E) did not aggregate. (F,G) Mixture of cells transfected with UAS-ds and UAS-GFP (green), and cells transfected with either UAS-ft (F, red) or UAS-ft {Delta}ICD (G, red) aggregated. Ft (anti-Ft, red and center panel) or Ft{Delta}ICD (anti-HA, red and center panel) concentrated with Ds (blue and right panels) at the sites of cell contact.

 

Figure 4
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  		Fig. 4. Comparison of overgrowth of wing imaginal discs. Typical wing discs for each genotype are shown at the same magnification. ft- is ftG-rv/ftfd, ds- is dsUAO71, and ds- ft- is dsUAO71 ftG-rv/dsUAO71ftfd. (A) Wild type. (B) Overgrowth in ft-. (C) Rescue of ft- overgrowth with UAS-ft and act-gal4. (D) Rescue of ft- overgrowth with UAS-ft{Delta}ECD and act-gal4. (E) Potentiation of ft- overgrowth with UAS-ft{Delta}ICD and act-gal4. (F) Mild overgrowth in ds-. (G) Potentiation of ft- overgrowth by ds-.

 

Figure 5
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  		Fig. 5. Assay for rescue of ftG-rv/ftfd overgrowth in posterior of imaginal wing discs. en-gal4 (anti-En, green) was used to drive expressions of full-length and deleted ft constructs. All are shown at the same magnification. (A) Wild type. (B). Overgrowth of anterior and posterior wing pouch in ftG-rv/ftfd. (C,D) Rescue of overgrowth in posterior wing pouch by UAS-ft (C) or UAS-ft{Delta}ECD (D). (E) Failure to rescue overgrowth in posterior wing pouch with UAS-ft{Delta}ICD. (A-E) There is no effect on the expression of the wing blade Wg target Vg (purple and right panels).

 

Figure 6
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  		Fig. 6. Overgrowth phenotype induced by expression of UAS-ft{Delta}ICD in wild-type wing discs. (A) Control ap-gal4 UAS-GFP wing disc showing dorsal region of expression (green). (B) Overgrowth induced in dorsal cells by ap-gal4 UAS-GFP UAS-ft{Delta}ICD. Ventral regions lacking expression in A and B are similar in size. (C,D) Overgrowth induced by hh-gal4 UAS-GFP UAS-ft{Delta}ICD in posterior compartment (green) of wing pouch and prospective hinge region. There is similar expression of the wing pouch Wg target Dll (purple and right panel) in anterior and posterior cells in C. The distal ring of wg-lacZ expression (purple and right panel) is similar in width in cells inside (arrowhead) and just anterior to (arrow) the region of misexpression in D. (E,F) Expression of UAS-ft{Delta}ICD in the posterior of wing imaginal discs using en-gal4 (E) or hh-gal4 (F). Discs were stained using an antiserum generated against the intracellular domain of Ft (Yang et al., 2002Go). (E) Expression in ftG-rv/ftfd discs. The region of misexpression was identified by the stabilization of Ds (green, left panel); the anti-Ft (purple and right panel) did not crossreact with Ft{Delta}ICD. (F) Expression in wild-type discs. Endogenous Ft (purple and right panel) was stabilized in the region of Ft{Delta}ICD expression, identified by the HA tag on Ft{Delta}ICD (green, left panel). (G) Anti-DE-cadherin (purple and right panel) staining after dorsal expression of UAS-GFP (green) and UAS-ft{Delta}ICD using ap-gal4. Shg levels were slightly decreased dorsally.

 

Figure 7
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  		Fig. 7. Effects of full-length and deleted ft constructs on PCP and crossvein spacing. (A-H) Polarity of hairs in adult wings; normal polarity is indicated by blue arrows and abnormal polarity by red arrows. Crossvein spacing is reduced by all constructs. (A) Wild type; positions of the longitudinal (L1-L5) and crossveins (ACV, PCV) are marked. (B) Defects in viable ft18 mutant. (C) Defects induced by large homozygous ftG-rv clone (y-) in the distal region of the wing blade between L2 and L3. The clone occupies the entire region shown. (D,E) Apparent partial rescue of PCP defects of ftG-rv/ftfd wing with act-gal4 and UAS-ft (D) or UAS-ft{Delta}ECD (E). (F-H) Proximal PCP defects induced in wild-type wing with act-gal4 and UAS-ft (F), UAS-ft{Delta}ECD (G) or UAS-ft{Delta}ICD (H). Defects were milder with UAS-ft{Delta}ECD. (I-L) Polarity of abdominal hairs. (I) Wild type. (J) Disruption in ftG-rv/ftfd. (K,L) Partial rescue of ftG-rv/ftfd by act-gal4 and UAS-ft (K) or UAS-ft{Delta}ECD (L).

 

Figure 8
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  		Fig. 8. Effects of full-length and deleted ds constructs on wing PCP and crossvein spacing. Normal polarity is indicated by blue arrows and abnormal polarity by red arrows. Proximodistal crossvein spacing in wild type (A) is indicated by a horizontal black double arrow, and is compared with abnormal spacing (red double arrow) in E-H. (A) Wild type. (B) ds05142. (C,D) Partial rescue of ds05142 PCP defects, especially in distal wing blade, by tub-gal4 and UAS-ds (C) or UAS-ds{Delta}ICD (D). (E-G) Expression of UAS-ds (E), UAS-ds{Delta}ICD (F) or UAS-ds{Delta}ECD (G) with tub-gal4 did not induce PCP defects, but reduced crossvein spacing. (H) The crossvein spacing defect induced by UAS-ds{Delta}ECD was stronger using en-gal4, but there were no PCP defects at the sharp boundary of misexpression between L3 and L4. (I-L) PCP in the posterior wing. (I) Wild type. (J,K) Expression of UAS-ds (J) or UAS-ds{Delta}ICD (K) in the central region of the wing using sal-gal4 results in the orientation of hairs away from the region of misexpression. (L) sal-gal4 and UAS-ds{Delta}ECD has no effect on PCP.

 




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