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Brief Communication 1247 Flamingo controls the planar polarity of sensory bristles and asymmetric division of sensory organ precursors in Drosophila Bingwei Lu*, Tadao Usui†, Tadashi Uemura‡, Lily Jan* and Yuh-Nung Jan* The sensory bristles of the fruit fly Drosophila are organized in a polarized fashion such that bristles on the thorax point posteriorly. These bristles are derived from asymmetric division of sensory organ precursors (SOPs). The Numb protein, which is localized asymmetrically in a cortical crescent in each SOP, segregates into only one of the two daughter cells during cell division, thereby conferring distinct fates to the daughter cells [1,2]. In neuroblasts, establishment of apical–basal polarity by the protein Inscuteable is crucial for orienting asymmetric division, but this is not the case for division of SOPs [3]. Instead, the Frizzled (Fz) protein mediates a planar polarity signal that controls the anteroposteriorly oriented first division (pI) of SOPs [4]. Here, we report that Flamingo (Fmi), a seven-transmembrane cadherin [5], controls the planar polarity of sensory bristles and the orientation of the SOP pI division. Both the loss of function and overexpression of fmi disrupted bristle polarity. During mitosis of the SOP, the axis of the pI division and the positioning of the Numb crescent were randomized in the absence of Fmi activity. Overexpression of Fmi and Fz caused similar effects. The dependence of proper Fmi localization on Fz activity suggests that Fmi functions downstream of Fz in controlling planar polarity. We also present evidence suggesting that Fz also functions in the Wingless pathway to pattern sensory organs. Addresses: *Howard Hughes Medical Institute, Departments of Physiology and Biochemistry, University of California, San Francisco, California 94143-0725, USA. †Department of Biophysics, School of Science, ‡Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. Correspondence: Yuh-Nung Jan E-mail: [email protected] Received: 25 August 1999 Revised: 24 September 1999 Accepted: 24 September 1999 Published: 25 October 1999 Current Biology 1999, 9:1247–1250 0960-9822/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved. Results and discussion Loss of fmi function disrupts the planar polarity of noninnervated hairs on the Drosophila wing [5]. To investigate the role of fmi in regulating the planar polarity of sensory bristles, we examined the bristles on the adult notum (dorsal thorax) in fmi mutants. Null mutants of fmi are embryonic lethal [5]. We found that a transheterozygous combination between fmiE59, a putative null allele carrying a nonsense mutation in the segment of the gene encoding the ectodomain of Fmi [5], and fmi71, for which the molecular lesion has not yet been determined [6], gives viable adult flies that display abnormal polarity in both types of sensory bristles (macrochaetes and microchaetes) (Figure 1b). Instead of pointing posteriorly within the epithelial plane as in wild-type flies (Figure 1a), these bristles pointed in different directions: posteriorly, laterally and, less frequently, anteriorly. Moreover, some bristles also pointed upwards. The macrochaetes tended to be less affected than the microchaetes, and bristles located at different positions on the notum were affected to different degrees (Figure 1e). Although only a small percentage of microchaetes located at the anterior (10%) and dorsocentral (13%) positions exhibited abnormal polarity, a higher percentage at the posterior (57%) and lateral (61%) positions were affected. A similar planar polarity phenotype was observed in fmiE59 mutant clones. As in fmiE59/fmi71 transheterozygotes, fmiE59 mutant clones generated at different positions on the notum exhibited position-dependent penetrance of bristle polarity phenotypes. Marking the mutant clones with the yellow marker allowed us to examine whether Fmi activity was cell autonomous. In most cases, bristles that displayed abnormal polarity were confined within fmi mutant clones (n = 50; Figure 1d). Infrequently, a wildtype bristle immediately adjacent to a mutant clone exhibited abnormal polarity. Thus, unlike fz, which has a long-range distal non-cell-autonomous effect on the wing [7], fmi acts largely cell autonomously on the notum to control the planar polarity of sensory bristles. A similar cell-autonomous function of Fmi is observed for the polarity of non-innervated hairs on the wing [5]. Not only did loss of fmi function disrupt bristle planar polarity, overexpression of fmi caused similar effects. We overexpressed fmi on the adult notum using the apterous gene to drive expression of the Gal4-encoded transcription factor and an fmi transgene driven by the upstream activator sequence (UAS) to which Gal4 binds. The apterous gene is expressed throughout the parts of the wing and haltere discs that give rise to the dorsal surface of wing blades and haltere, and in the regions that form the notum, scutellum and wing hinge. The microchaetes in apterous–Gal4 > UAS–fmi flies were affected to similar degrees as in fmiE59/fmi71 transheterozygotes; the macrochaetes were more affected than those in fmiE59/fmi71 flies (Figure 2c). On the wing, overexpression of fmi with a 1248 Current Biology Vol 9 No 21 Figure 2 Figure 1 (a) (a) (b) (b) (c) A LL RL C P Current Biology (c) Overexpression of fmi and fz lead to different effects on polarity and patterning of sensory bristles on the notum. (a) Wild-type, (b) apterous–Gal4 > UAS–fz, and (c) apterous–Gal4 > UAS–fmi bristles. Overexpression of fz or fmi both disrupted the planar polarity of bristles, with the latter having a more pronounced effect on both microchaetes and macrochaetes. Note that the scutellar bristles (arrowheads) point upwards and appear short in (c). (b) In addition to the planar polarity phenotype, overexpression of fz also caused ectopic induction of scutellar bristles (arrows). (c) In contrast, apterous–Gal4 > UAS–fmi flies did not show ectopic formation of scutellar bristles. (d) by both overexpression and loss of function of fmi suggests that the planar polarity signaling pathway is sensitive to the level of Fmi activity. Bristles with abnormal polarity (%) (e) 80.0 fmi 70.0 Wild type 60.0 50.0 40.0 30.0 20.0 10.0 0.0 A C P Notum position RL LL Current Biology Loss of fmi activity disrupts planar polarity of bristles on the notum. (a) Normal anteroposterior polarity of sensory bristles on the notum of a wild-type fly. (b) The bristles on the notum of an fmiE59/fmi71 fly show disorganized polarity and point towards different directions. (c) A yellow-marked fmi+/fmi+ clone (outlined with a dotted red line) on the notum displays normal bristle polarity. (d) An fmiE59/fmiE59 mutant clone marked with the yellow marker, which causes the bristles to be paler (outlined with a dotted red line), shows disrupted planar polarity of bristles. (e) Histogram showing the percentage of bristles exhibiting abnormal polarity at different positions on the notum. The number of bristles with abnormal polarity and the total number of bristles were counted for each sector of the notum (A, anterior; P, posterior; C, dorsocentral; LL, left lateral; RL, right lateral; see (a) for locations of these sectors) in wild-type (n = 5) and fmiE59/fmi71 (n = 5) flies. patched–Gal4 driver alters the planar polarity of non-innervated wing hairs [5]. The disruption of bristle planar polarity The planar polarity pathway is also sensitive to the level of Fz activity. Overexpression of fz using UAS–fz and apterous–Gal4 also disrupted bristle planar polarity (Figure 2b), consistent with earlier experiments using fz constructs driven by the heat shock gene (hs) promoter [8]. Besides the planar polarity phenotype, overexpression of fz also led to the formation of ectopic macrochaetes. The apterous–Gal4 > UAS–fz flies exhibited frequent (~80%) ectopic induction of scutellar bristles, a phenotype not observed in apterous–Gal4 > UAS–fmi flies. Further overexpression studies with Drosophila Frizzled 2 (Dfz2) and dominant-negative forms of Fz and Dfz2 suggested that, in addition to its role in controlling planar polarity, Fz also functions together with Dfz2 in the Wingless pathway to pattern the sensory bristles (see the Supplementary material) [9–11]. The orientation of the SOP pI division axis and the positioning of Numb protein crescent on the cortex respond to polarity cues that are regulated by Fz signaling. In fz or dishevelled (dsh) loss-of-function backgrounds, the SOP pI division axis and the positioning of the Numb crescent are randomized [4]. We examined these two processes in the pupal notum in apterous–Gal4 > UAS–fz flies (n = 25), and found that both the spindle orientation and positioning of Numb crescent no longer followed the anteroposterior axis and became randomized (Figure 3e,f). Nevertheless, the Numb crescent still overlaid one pole of the misoriented spindles and the randomization of spindle orientation was Brief Communication Figure 3 (b) 0º A (i) (d) 270º 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 (a) (b) (c) (d) 90º P 180º fmi Wild type 0 45 90 13 5 18 0 22 5 27 0 31 5 (c) Figure 4 Percentage of mitotic SOPs (a) 1249 Position of numb crescent (e) (f) Current Biology (g) (h) Current Biology Overexpression of fz and fmi and loss of function of fmi affect the orientation of the SOP pI division and the positioning of Numb crescents. Dissected pupal nota from (a,b) wild-type, (c,d) fmiE59/fmi71, (e,f) apterous–Gal4 > UAS–fz and (g,h) apterous–Gal4 > UAS–fmi flies were stained with an antibody against the Asense protein (red in panels a,c,e,g; blue in panels b,d,f,h) to identify SOPs. The localization of Numb in SOPs was examined by staining with an anti-Numb antibody (green), and the spindle orientation by staining with an anti-α-tubulin antibody (red in panels b,d,f,h). Arrowheads, the Numb crescents; arrows, the two daughter cells arising from each SOP after the first division. Note that Numb is segregated to only one of the daughter cells. In all panels, anterior is uppermost. (i) Histogram showing the position of the Numb crescent relative to the anteroposterior axis in dividing SOPs from wild-type (n = 62) and fmiE59/fmi71 (n = 58) flies. Anterior (A) is taken as 0°, posterior (P) as 180°. restricted two-dimensionally within the epithelial plane, as is observed in fz or dsh loss-of-function mutants [4]. To test the function of Fmi in regulating spindle orientation and Numb protein localization during the SOP pI division, we analyzed the effects of the loss of function and overexpression of fmi on the pupal notum. In fmiE59/fmi71 transheterozygous mutant pupae (n = 30), the Numb crescent was randomly positioned within the epithelial plane during the SOP pI division (Figure 3c,i). The adapter protein Partner of Numb (Pon), which controls Numb localization during SOP division [12], was also mispositioned, but the two proteins remained colocalized (data not shown). Staining for tubulin revealed that spindle orientation and Numb crescent positioning were still tightly coupled (Figure 3d). During the SOP pI division in Loss of fz function affects the subcellular localization of Fmi. Dissected nota from (a,b) wild-type and (c,d) fzr54/fzKD4a pupae were stained with (a,c) anti-Asense antibody and (b,d) anti-Fmi antibody. (b) In wildtype SOPs (arrowhead), Fmi staining was localized mostly to the cell–cell boundary, with residual punctate staining in the cytoplasm. Note that the SOP shows stronger Fmi staining than the surrounding epithelial cells. (d) In fz mutant SOPs (arrowheads), Fmi staining at the cell–cell boundary was reduced and there was a corresponding increase in cytoplasmic staining. Anterior is to the left in all panels. apterous–Gal4 > UAS–fmi pupae (n = 26), the Numb crescent was also mispositioned and the mitotic spindle misoriented within the epithelial plane, but they remained aligned with each other (Figure 3g,h). Therefore, loss of function and overexpression of fmi both disrupted the cellular process that regulates mitotic spindle orientation and protein localization during the SOP pI division. To gain a better insight into the mechanism of Fmi function, we examined its subcellular localization in SOP cells using an antibody against the ectodomain of Fmi [5]. Consistent with Fmi being a seven-transmembrane celladhesion molecule, it was localized to cell–cell boundaries in both the SOPs and their surrounding epithelial cells (Figure 4b). There was also a low level of punctate cytoplasmic staining, suggesting that cytoplasmic Fmi may be associated with intracellular vesicles. The Fmi staining in SOP cells was stronger than that in the surrounding epithelial cells, indicating elevated expression or stability of Fmi in the SOPs. No apparent polarized distribution of Fmi was observed in mitotic SOPs. In nota of apterous–Gal4 > UAS–fz flies, the localization of Fmi in the SOPs was similar to that in wild-type flies (data not shown). In nota of fzr54/fzKD4a mutant flies, we observed that Fmi staining at the cell–cell boundary was reduced, whereas cytoplasmic Fmi staining was increased (Figure 4d). A similar effect was observed in the notum of dsh1 mutant flies (data not shown). In the wing epithelia, 1250 Current Biology Vol 9 No 21 localization of Fmi at the cell–cell boundary was also affected in fz and dsh mutant backgrounds [5]. Therefore, the proper recruitment of Fmi from the cytoplasm to the cell–cell boundary depends on Fz signaling. It has been shown that Dsh can be selectively recruited to the membrane by Fz but not by Dfz2 [13]. Whether a similar mechanism is involved in recruiting Fmi and Dsh to the membrane remains to be determined. Our data indicate that Fmi functions largely in a cellautonomous manner to control the planar polarity of sensory bristles on the Drosophila notum. The similar polarity phenotypes caused by overexpression and loss of function of fmi or fz, together with the dependence of proper Fmi localization on Fz activity, suggest that Fmi functions downstream of Fz in the planar polarity pathway. It should be noted that, although loss of fmi function affected both bristle polarity and the positioning of Numb crescents, the correlation between these two phenotypes was not strict. Although the orientation of the Numb crescent was largely random in the fmi mutant, this was not the case for bristle polarity. It is possible that there exists some other mechanical constraints in the developing imaginal epithelia that influence the orientation of bristles or that Fz-independent cues can direct bristle orientation. The tight coupling of misoriented mitotic spindle and mislocalized Numb crescent during SOP pI divisions suggests that a downstream activity which coordinates these two processes is still intact when fmi or fz are overexpressed or inactive. During neuroblast division, Inscuteable acts downstream of Bazooka to coordinate spindle orientation and Numb localization [3,14]. It is unlikely, however, that Inscuteable is the activity required to couple these two processes during the SOP pI division [3,4]. It is possible that, in the SOP lineage, Fmi/Fz functions like Bazooka to regulate an Inscuteablelike activity, which in turn couples spindle orientation and protein localization. Despite the difference between the planar polarity pathway and the Bazooka/Inscuteable pathway for the orthogonal apical–basal polarity, both pathways appear to regulate the localization of Numb through Pon. Further studies will help clarify how the two pathways impinge on Pon to control Numb localization. Supplementary material Supplementary material including additional methodological detail and a figure showing that Fz also functions with Dfz2 in the Wingless pathway to pattern the sensory bristles is available at http://currentbiology.com/supmat/supmatin.htm. Acknowledgements We thank Su Guo for critically reading the manuscript; members of the Jan lab for discussions; Susan Younger for advice on fly genetics; and Jay Brenman, Feng-Biao Gao, Richard Carthew, Michel Gho and Francois Schweisguth for fly stocks. This work was supported by a NIMH grant to the Silvo Conte Center for Neuroscience Research at UCSF and a National Research Service Award from NIH to B.L. Y-N.J. and L.J. are Investigators of the Howard Hughes Medical Institute. References 1. Rhyu MS, Jan LY, Jan YN: Asymmetric distribution of Numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 1994, 76:477-491. 2. Uemura T, Shepherd S, Ackerman L, Jan LY, Jan YN: numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. Cell 1989, 58:349-360. 3. Kraut R, Chia W, Jan LY, Jan Y N, Knoblich JA: Role of inscuteable in orienting asymmetric cell division in Drosophila. Nature 1996, 383:50-55. 4. Gho M, Schweisguth F: Frizzled signaling controls orientation of asymmetric sense organ precursor cell divisions in Drosophila. Nature 1998, 393:178-181. 5. Usui T, Shima Y, Shimada Y, Hirano S, Burgess RW, Schwarz TL, et al.: Flamingo, a seven-pass transmembrane cadherin, regulates epithelial planar polarity under the control of Frizzled. Cell 1999, 98:585-595. 6. Gao F-B, Brenman JE, Jan LY, Jan YN: Genes regulating dendritic outgrowth, branching and routing in Drosophila. Genes Dev 1999, in press. 7. Vinson CR, Adler PN: Directional non-cell autonomy and the transmission of polarity information by the frizzled gene of Drosophila. Nature 1987, 329:549-551. 8. Krasnow RE, Adler PN: A single Frizzled protein has a dual function in tissue polarity. Development 1994, 120:1883-1893. 9. Phillips RG, Whittle JRS: wingless expression mediates determination of peripheral nervous system elements in late stages of Drosophila wing disc development. Development 1993, 118:427-438. 10. Bhanot P, Brink M, Samos CH, Hsieh JC, Wang Y, Macke JP, et al.: A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 1996, 382:225-230. 11. Zhang J, Carthew RW: Interaction between Wingless and DFz2 during Drosophila wing development. Development 1998, 125:3075-3085. 12. Lu B, Rothenberg M, Jan LY, Jan YN: Partner of Numb colocalizes with Numb during mitosis and directs Numb asymmetric localization in Drosophila neural and muscle progenitors. Cell 1998, 95:225-235. 13. Axelrod JD, Miller JR, Shulman JM, Moon RT, Perrimon N: Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev 1998, 12:2610-2622. 14. Kuchinke U, Grawe F, Knust E: Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka. Curr Biol 1998, 8:1357-1365. S1 Supplementary material Flamingo controls the planar polarity of sensory bristles and asymmetric division of sensory organ precursors in Drosophila Bingwei Lu, Tadao Usui, Tadashi Uemura, Lily Jan and Yuh-Nung Jan Current Biology 25 October 1999, 9:1247–1250 Supplementary materials and methods Figure S1 Drosophila genetics The FRT–FLP recombination system was used to generate fmiE59 mutant clones on the notum. Second or third instar larvae of the genotype yw, hsFLP/+; FRTG13fmiE59/FRTG13fmi+ y+ or yw, hsFLP/+; FRTG13fmi+/FRTG13fmi+ y+ (control) were heat-shocked at 38°C for 1 h to induce mitotic recombination. For overexpression studies, transgenic UAS lines were crossed to appropriate Gal4 driver lines at 25°C. Adult flies coming out of the mitotic recombination cross and overexpression crosses were dissected and the nota were mounted and photographed. Immunostaining For immunostaining of SOPs, 15–16 h old pupae were dissected and the nota were processed for immunostaining as described [S1]. Primary antibodies used were guinea pig anti-Numb (1:1000), mouse anti-Fmi (1:10), rabbit anti-Asense (1:5000), and mouse anti-Tubulin (1:1000). Z-series of confocal images were obtained on a BioRad MRC600 confocal microscope and merged images were processed using Adobe Photoshop programs. Supplementary reference S1. Rhyu MS, Jan LY, Jan YN: Asymmetric distribution of Numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 1994, 76:477-491. (a) (b) (c) Current Biology The overexpression phenotypes of Dfz2 and dominant-negative forms of fz (fzN) and Dfz2 (Dfz2N) suggest that Fz and Dfz2 function together in the Wingless pathway to pattern the sensory organs. The bristles of (a) apterous–GAL4 > UAS–Dfz2, (b) scabrous–Gal4 > UAS–fzN, and (c) scabrous–Gal4 > UAS–Dfz2N are shown. The scabrous gene is expressed in the SOPs and a subset of epithelial cells surrounding the SOPs. (a) After overexpression of Dfz2, ectopic scutellar bristles (arrows) similar to those in apterous–Gal4 > UAS–fz flies was observed. Conversely, overexpression of (b) fzN or (c) Dfz2N resulted in a loss of certain macrochaetes (arrowheads). Note that overexpression of Dfz2, fzN or DfzN has no effect on bristle polarity.