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First published online 29 March 2006
doi: 10.1242/dev.02320

Development 133, 1789-1797 (2006)
Published by The Company of Biologists 2006
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Twinstar, the Drosophila homolog of cofilin/ADF, is required for planar cell polarity patterning

Adrienne Blair1, Andrew Tomlinson2, Hung Pham1, Kristin C. Gunsalus3,*, Michael L. Goldberg3 and Frank A. Laski1,{dagger}

1 Department of Molecular Cell and Developmental Biology, and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA 90095, USA.
2 College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
3 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA.


Figure 1
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  		Fig. 1. Aspects of planar cell polarity. (A) In a wild-type eye, ommatidia are rotated uniformly above and below the dorsoventral equator. (B) Localization of the polarity cue. In wild-type wing cells, Fz is localized to the distal sides of cells, whereas Fmi is localized to both the proximal and distal sides of cells. (C) The basic PCP pathway that determines wing hair polarity. (D) Classes of PCP defects in the wing epithelium. In wild type, a single hair is secreted from the distal-most vertex of each cell. Mutations of the PCP core group genes (fz, fmi/stan, dsh, pk and vang/stmb) cause hairs to project from central locations and to have a non-distal orientation. RhoA and Rok mutants show multiple wing hairs projected from the distal side of a cell. The tsr mutants show a single wing hair that is projected in a non-distal orientation and is not centered through the distal-most vertex of a cell.

 

Figure 2
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  		Fig. 2. Temperature-sensitive tsr mutantions. (A) Sequence comparisons of yeast, human and Drosophila cofilin sequences (Lappalainen et al., 1997Go). The cof1-22 mutation that gave a temperature sensitive phenotype in yeast is in red and denoted above the sequence. This mutation was used to predict and engineer a similar temperature-sensitive mutation in Drosophila, tsr139, that is in red and denoted below the sequence. The position of the tsrV27Q mutation is also indicated. (B) The cross generating tsrV27Q/tsr139; tsr{Delta}96 progeny. The conditional alleles P[w+, tsr139] and P[w+, tsrV27Q] together rescued the lethality caused by the tsr{Delta}96 mutation at the permissive temperature and allowed analysis of PCP defects in different tissues.

 

Figure 3
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  		Fig. 3. tsr PCP defects in adult epithelia. tsr{Delta}96 flies rescued by tsr139/tsrV27Q or P[WHTG] have PCP defects in several epithelia. (A-C) The wing (anterior is upwards and distal is rightwards). (A) Wild-type wings always have a uniform and distally pointing hair orientation; (B) tsr139/tsrV27Q wing hairs have a non-distal orientation; and (C) a P[WHTG] wing has hairs oriented in swirls or non-distally (not shown). (D) The eye. A thin section through a heat-shocked P[WHTG] eye shows a field of ommatidia that have randomly adopted polarities. (E) The abdomen. A P[WHTG] cuticle shows a random orientation of fine hairs. A region of hairs with wild-type orientation that points posteriorly is shaded. (F-H) The leg. A wild-type leg (F) shows normal segmentation and bristle pattern. Blue, tarsal segment 3; yellow, the correct joint position and polarity. P[WHTG] legs (G,H) have aberrant tarsal segments. Blue, the equivalent of tarsal segment 3 (from tibia); yellow, aberrant joints with duplications. At higher magnification (I), some P[WHTG] leg bristles show reversed polarity as do the `bract-socket vectors' (Held et al., 1986Go). Bracts (purple) are fine hair-like structures at the base of each bristle: two bracts, one above the other, have the correct polarity: growing from the proximal side of the socket and point distally (to the right). The third bract has reversed polarity, growing from the distal side of the socket and points proximally. (J) The notum. A tsr139/tsrV27Q cuticle shows some of the fine hairs have lost the proper posterior-pointing orientation. Occasionally multiple hairs are observed (arrow).

 

Figure 4
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  		Fig. 4. Prehair emergence in pupal wings with reduced Tsr activity. Confocal images of wild-type and tsr mutant pupal wings stained with phalloidin. Anterior is upwards; distal is towards the left. (A) A wild-type wing shows prehairs initiating as accumulations of F-actin near or at the distal-most vertex of each cell. (B) A similarly staged tsr139/tsrV27Q wing shows prehairs initiated as F-actin accumulations located centrally (dot) or as elongated F-actin structures (asterisk). (C) An older wild-type wing shows prehairs centered over distal cell vertices. (D) A similarly staged tsr139/tsrV27Q wing shows prehairs with abnormal orientations that are similar to the pattern of hairs of the adult wing (Fig. 2B). tsr139/tsrV27Q prehairs are longer, thinner and extend non-distally.

 

Figure 5
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  		Fig. 5. Fz-GFP and Fmi are mislocalized in tsr mutant wings. Shown are: Fz-GFP (A,D,F); Fmi (B,E,G); and merged images of Fz-GFP in green and phalloidin-stained F-actin in red (C,F,I). (A-C) A wild-type wing, prior to prehair emergence aged greater than 48 hours APF at 18°C. Fz-GFP and Fmi are asymmetrically localized in a zigzag pattern at the PD boundary. The merged image shows Fz-GFP and Fmi colocalization. (D-F) A moderately affected tsr139/tsrV27Q wing of similar age shows an interrupted asymmetrical distribution of Fz-GFP. Fz-GFP is frequently missing from cell vertices and cell sides (asterisk). Similarly, Fmi shows an interrupted asymmetrical distribution. The merged image shows the extent of colocalization. (G-I) A severely affected tsr139/tsrV27Q wing shows that the recruitment of Fz-GFP to cell boundaries is largely lost, whereas Fmi is still enriched at cell boundaries where it shows an uneven and interrupted distribution. The merged image shows the zigzag pattern was interrupted.

 

Figure 6
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  		Fig. 6. Prehairs do not initiate from the correct location in tsr mutant wings. Fz-GFP (A,E,I); Fmi (B,F,J); F-actin (C,G,K); and merged images of Fz-GFP in green and phalloidin-stained F-actin in red (D,H,L). (A-D) A wild-type wing during prehair initiation aged ~64 hours APF at 18°C. Fz-GFP and Fmi show the characteristic asymmetrical distribution at the PD boundaries. F-actin accumulations show a single prehair centered at the distal-most vertex of each cell (C). The merged image shows the overlay of Fz-GFP (green) and F-actin (red) localization. (E-H) A moderately affected tsr139/tsrV27Q wing of a similar age shows a Fz-GFP distribution that was interrupted at the distal cell boundaries. (E) Fz-GFP accumulated unevenly and was missing from some cell boundaries. (F) Similarly, Fmi shows an uneven distribution at PD cell boundaries. (G) The F-actin accumulation shows prehairs were not centered through the distal vertices; few were extended. (H) The merged image. (I-L) tsr139/tsrV27Q, a severely affected wing shows that Fz-GFP is not enriched at PD boundaries and the Fmi distribution at PD boundaries is uneven showing puncti of strong staining alternating with gaps in the staining pattern. Phalloidin-stained F-actin (K) shows prehairs abnormally formed near cell centers (arrowhead). The merged image shows the extent of Fz-GFP delocalization.

 

Figure 7
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  		Fig. 7. Prehairs are oriented towards the highest accumulation of Fz-GFP and Fmi in tsr mutant wings. Shown are: Fz-GFP (A,E); Fmi (B,F); F-actin (C,G); and merged images (D,H; schematics of merged images D',H'). (A-D) Prehairs in a wild-type wing aged ~66 hours APF at 18°C. Fz-GFP and Fmi shows the characteristic asymmetrical distribution at the PD boundaries; the zigzag pattern has begun to lose polarity at this stage. F-actin accumulation shows a single prehair pointing distally and aligned with the distal-most vertex of each cell. The merged image shows the overlay of Fz-GFP and F-actin localization. (E-H) Shown is a similarly aged tsr139/tsrV27Q wing. Fz-GFP distribution is interrupted at the distal cell-cell boundaries and accumulates more densely at some cell boundaries (E, dot). Similarly, Fmi is unevenly distributed at PD boundaries. The F-actin accumulation shows prehairs have non-distal orientations and are not aligned through the distal-most vertices. Prehairs appear to have emerged through the PD boundary at the point of a dense accumulation of Fz-GFP and Fmi (H,H').

 

Figure 8
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  		Fig. 8. Overexpression of a constitutively active Limk results in wing PCP defects. The activated Limk protein was expressed using an ap-Gal4 promoter construct that only expresses on the dorsal wing blade. Transgenic flies grown at 23°C survive and show defects with wing hair polarity similar to those seen in fz class wings (A), or have wing hairs that show a more chaotic wing hair orientation pattern (B). (C) A pupal wing, treated with phalloidin, on which the ventral surface hairs are oriented correctly (left panel), but hairs on the dorsal surface show the PCP defect (right panel). (D) A pupal wing with prehairs emerging correctly from the distal-most vertices on the ventral blade (left panel), while the dorsal blade (right panel) shows a phenotype similar to the tsr139/tsrV27Q mutant (E).

 

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© The Company of Biologists Ltd 2006