Download Flamingo controls the planar polarity of sensory

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
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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)
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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,
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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
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et al.: Flamingo, a seven-pass transmembrane cadherin, regulates
epithelial planar polarity under the control of Frizzled. Cell 1999,
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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
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8. Krasnow RE, Adler PN: A single Frizzled protein has a dual
function in tissue polarity. Development 1994, 120:1883-1893.
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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
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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.