Current Biology 18, 831–837, June 3, 2008 ª2008 Elsevier Ltd All rights reserved
DOI 10.1016/j.cub.2008.04.072
Report
The PDZ Protein Canoe Regulates
the Asymmetric Division of Drosophila
Neuroblasts and Muscle Progenitors
Stephan Speicher,1 Anja Fischer,2 Juergen Knoblich,2
and Ana Carmena1,*
1Instituto de Neurociencias de Alicante
Consejo Superior de Investigaciones Cientı́ficas/University
Miguel Hernandez
Sant Joan d’Alacant
03550 Alicante
Spain
2IMBA-Institute of Molecular Biotechnology
1030-Vienna
Austria
Summary
Asymmetric cell division is a conserved mechanism to generate cellular diversity during animal development and a key
process in cancer and stem cell biology [1, 2]. Despite the increasing number of proteins characterized, the complex network of proteins interactions established during asymmetric
cell division is still poorly understood. This suggests that
additional components must be contributing to orchestrate
all the events underlying this tightly modulated process.
The PDZ protein Canoe (Cno) and its mammalian counterparts AF-6 and Afadin are critical to regulate intracellular signaling and to organize cell junctions throughout development [3–13]. Here, we show that Cno functions as a new
effector of the apical proteins Inscuteable (Insc)-Partner of
Inscuteable (Pins)-Gai during the asymmetric division of
Drosophila neuroblasts (NBs) [2]. Cno localizes apically
in metaphase NBs and coimmnunoprecipitates with Pins
in vivo. Furthermore, Cno functionally interacts with the
apical proteins Insc, Gai, and Mushroom body defect (Mud)
to generate correct neuronal lineages. Failures in muscle
and heart lineages are also detected in cno mutant embryos.
Our results strongly support a new function for Cno regulating key processes during asymmetric NB division: the localization of cell-fate determinants, the orientation of the mitotic spindle, and the generation of unequal-sized daughter
cells.
Results and Discussion
Cno Colocalizes with the Apical Protein Bazooka/Par-3
in Metaphase NBs
NBs delaminate from the neuroectoderm inheriting the apicobasal polarity of the neuroectodermal cells, in which the
PDZ proteins Bazooka (Baz)/Par-3 and DmPar-6 and the
kinase DaPKC localize apicolaterally [14–17]. After delamination, NBs maintain the apical localization of Baz/DmPar-6/
DaPKC. The cytoplasmic PDZ protein Cno localizes at the adherens junctions of some epithelial cells [9], and we wondered
whether Cno was also present in the neuroectoderm and in
the delaminated NBs. Double immunofluorescences with
antibodies against Cno and Baz showed that these proteins
*Correspondence: [email protected]
colocalize both apicolaterally at the adherens junctions of neuroepithelial cells and apically in the delaminated metaphase
NBs (mNBs) (Figures 1A–1A000 ). At later phases of the NB division, Cno was no longer detected.
Cno Is Required for the Basal Distribution of Cell-Fate
Determinants and for the Correct Orientation of the
Mitotic Spindle
Apical proteins, such as Baz/Par-3, are critically involved in
regulating cell-fate determinants localization and spindle orientation at metaphase [2, 15, 17, 18]. Given that Cno was detected in an apical crescent in mNBs, we next wondered
whether Cno was also required for modulating those events.
In control embryos, the cell-fate determinant Numb [19] was
basally located in 95.4% of mNBs (n = 65) (Figure 1B). In
cno2 zygotic mutants, Numb was uniform or undetectable (Figure 1C) or was present in nonbasal crescents (Figure 1D) in
47.9% of the mNBs analyzed (n = 137). cno2 has been defined
as the strongest allele of cno, although the particular lesion
associated is unknown [20]. However, cno2 is probably a null
allele because cno2 over the Df(3R)6-7 (covering the cno
gene) showed a similar percent of Numb localization failures
(44.9%, n = 78). Additionally, cno3, another strong allele of
cno considered as a null (D. Yamamoto, personal communication [9]) displayed defects in Numb localization in comparable
cases (41.8%, n = 67). The basal distribution of the scaffolding
protein Miranda (Mira) [21–23] was also altered in 16.9% of
mNBs (n = 83) of cno2 mutants (Figures 1G and 1G0 ). Indeed,
the localization of two Mira cargo proteins, the cell-fate determinants Prospero (Pros) [24–26] and Brain Tumor (Brat) [27–29],
was affected in mNBs (70%, n = 80 and 95.2%, n = 126,
respectively) (Figures 1F, 1F00 , 1G, 1G00 , and 1I–1J0 ). The variable penetrance of the cno2 mutant phenotype observed for
the different proteins analyzed may reflect, at least in part,
the different sensitivity of the antibodies used.
Intriguingly, the orientation of the mitotic spindle in mNBs of
cno2 mutants was randomized in 18.3% of the cases (n = 71)
(Figures 1C and 1D). In control embryos, the spindle is tightly
aligned with the center of Numb crescents in mNBs (Figure 1B).
In cno2 mutants, the spindle was uncoupled with the Numb
crescent in 7.7% of the mNBs that showed these crescents
(either basal or at other incorrect localizations, n = 39) (Figure 1D). The maternal contribution of cno might reduce the
penetrance of these phenotypes [30].
The overexpression of Cno also caused Numb localization
failures (45.8%) and aberrant spindle orientations (39%) in
mNBs (n = 59). Hence, our results showed that Cno regulates
essential processes during asymmetric NB division: the basal
localization of cell-fate determinants and the proper orientation of the mitotic spindle.
Cno Participates in the Generation of Unequal-Sized NB
Daughter Cells
Another characteristic feature of asymmetric NB division is the
different cell size of the progeny [31, 32]. Hence, we next analyzed whether Cno was also regulating this process. Control
telophase NBs (tNBs) showed unequal-sized daughter cells
in 100% of the cases analyzed (n = 19; Figures 1K and 1K0 ).
Current Biology Vol 18 No 11
832
Figure 1. Cno Localizes Apically in Metaphase
NBs Regulating Cell-Fate Determinants Localization, Spindle Orientation, and the Generation of
Unequal-Sized Daughter Cells
(A–A000 ) Confocal micrograph shows Cno (red) colocalization (yellow) with Baz (green) at the adherens junctions in the neuroectoderm (NE) and at
the apical pole of mNB (arrow). Neurotactin (Nrt,
blue) labels cell membranes. The DNA (PH3)
is shown both in red (A and A0 ) and in green
(A and A00 ).
(B–D) PH3 is shown in yellow, and Numb is shown
in green. The discontinuous line depicts the orientation of the mitotic spindle. (B) shows that in
control embryos, Numb is basally located in
mNBs (arrows), and the spindle is orientated
along the apico-basal axis of cell polarity. (C)
and (D) show that in cno2 mutant embryos,
Numb is uniformly distributed at the cortex (C)
or irregularly accumulated (arrow in [D]). The mitotic spindle is misorientated at mNBs in cno2
mutants.
(E–G00 ) Pros is shown in green, and Mira is shown
in red. PH3 is shown in both green and red (yellow
in the merge). (E), (E0 ), and (E00 ) show that in control embryos, Pros and Mira colocalize at the
basal pole of mNBs (arrow). (F)–(G00 ) show that
in cno2 mutants, Pros was either undetectable
(arrow in [F] and [F00 ]) or mislocalized (arrows in
[G] and [G00 ]). Mira localization was also altered
in cno2 mutant embryos (arrows in [G] and [G0 ]).
(H–J0 ) Brat is shown in green, PH3 is shown in red,
and Nrt is shown in blue. In control embryos (H
and H0 ), Brat localizes basally in mNBs (arrow).
In cno2 mutants (I–J0 ), Brat was either undetectable (arrow in [I] and [I0 ]) or mislocalized (arrow
in [J] and [J0 ]).
(K–M0 ) a-tubulin and Nrt are shown in blue, Baz is
shown in red, and PH3 is shown in yellow.
(K0 , L0 , and M0 ) Only the ‘‘blue channel’’ showing
a-tubulin and Nrt is shown in black and white.
Red arrows point to the equator of the mitotic
spindle. (K) and (K0 ) show that in control embryos,
tNBs show unequal cell-sized daughter cells. (L)
and (L0 ) show that in cno2 mutants, a percentage
of tNBs shows equal-sized progeny. In (M) and
(M0 ), inscP49; cno2 double mutants show equalsized progeny with more penetrant phenotype
than cno2 single mutants (see also text).
Canoe Functions during Asymmetric Cell Division
833
Figure 2. Cno Functionally Interacts with Apical
Proteins for the Correct Generation of Neuronal
Lineages
(A–D) High magnifications of several segments of
the ventral nerve cord at stage 16 showing Eve
expression in the RP2 neuron (arrows) in control
embryos (A and C) and in cno2 mutant embryos
(B and D). (A) and (B) show that in control embryos, only one RP2 neuron is detected per hemisegment (arrows in [A]). In cno2 mutant embryos,
two RP2s are detected in some hemisegments
(double-arrow in [B]; see also Table 1). (C) and
(D) show confocal micrographies containing expression of Eve (red) and Zfh1 (green). In control
embryos shown in (C), Zfh1 colocalizes (yellow)
with Eve in the RP2 neuron (arrows). In cno2
mutants shown in (D), neither Eve nor Zfh1 are
detected in the position of the RP2 neuron
in some hemisegments (arrowheads; see also
Table 1).
In cno2 mutants, equal-size divisions were observed in 21.3%
of tNBs (n = 75; Figures 1L and 1L0 ). Two redundant pathways,
Baz/DaPKC/Insc and Pins-Gai, regulate cell size and mitoticspindle asymmetry at the NB apical pole [32]. Only when
both pathways are compromised is the different size of the
daughter cells affected [32, 33]. Our data suggested that Cno
functions downstream of Gai. Thus, Cno might belong to the
Pins-Gai pathway. Indeed, when both insc and cno were eliminated, 85.2% of tNBs showed equal-sized daughter cells (n =
40), a much more penetrant phenotype than those displayed
by each single mutant [32] (Figures 1M and 1M0 ; see above).
Moreover, DGai, cno2 double mutants showed a much lower
percentage of equal-sized divisions (30.4%, n = 69) than the
inscP49; cno2 double mutants. Hence, these results strongly
suggest that Cno participates within the Pins-Gai pathway to
regulate NB progeny size.
Cno Functionally Interacts with Apical Proteins
for the Correct Generation of Neuronal Lineages
Given the defects observed in cno2 mutant embryos during
NBs division, we next wondered whether neuronal lineages
were altered in cno2 mutants. The lineage of the ganglion
mother cell (GMC) 4-2a has been extensively studied [34].
This GMC expresses the transcription factor Even-Skipped
(Eve) and divides asymmetrically to give rise to two different
neurons called RP2 and RP2 sibling. Both maintain the expression of Eve initially; however, at later stages of embryogenesis,
only the RP2 neuron keeps expressing Eve [34] (Figures 2A and
2C). In control embryos, 0.9% of the segments analyzed (n =
423) showed defects in the number of RP2s. In cno2 mutants,
two or no RP2s were detected per hemisegment in 5.7% of the
segments analyzed (n = 245). Such a result that suggested failures in the GMC 4-2a asymmetric division (Figures 2B and 2D
and Table 1). This phenotype was also observed in cnomis1 hypomorph mutants (4.6%, n = 304 segments) as well as in mutants for genes that are critical during asymmetric cell division.
For example, homozygotes for DaPKCk06403, inscP49, DGai,
and mud4 (zygotic null mutant embryos) showed defects in
the GMC 4-2a lineage in 6.4% (n = 327), 13.8% (n = 280),
2.5% (n = 367), and 8.3% (n = 326) of the segments analyzed,
respectively (Table 1). Hence, we next investigated whether
Cno was interacting with these proteins to properly generate
the GMC 4-2a neuronal lineage. Double heterozygotes
DaPKCk06403/+; cno2/+ showed defective RP2 number in
0.8% of segments (n = 382). This result is consistent with
a lack of functional interactions between DaPKC and Cno. However, double heterozygotes inscP49/+; cno2/+ and DGai, +/+,
cno2 showed an altered RP2 lineage in 14.4% (n = 403) and
7.6% (n = 397) of the segments analyzed, respectively (Table
1). In addition to the analysis of double heterozygotes, we
found that the cnomis1 phenotype (see above) was significantly
enhanced in a mud4 zygotic null mutant background (Table 1).
Altogether, these results indicated that Cno functionally interacts with the apical proteins Insc, Gai, and Mud during the
Table 1. Percentage of Segments of the Indicated Genotype that Display Failures in the Number of RP2 Neurons, including Losses and Duplications
Total Failures
Genotype
RP2 Loss
RP2 Duplication
Number of Segments
Number of Embryos
Number
Percentage
yw
cno2
cnomis1
DaPKCk06403
inscP49
DGai
mud4
DaPKCk06403/+; cno2/+
inscP49/+; cno2/+
DGai, +/+, cno2
mud4/Y; cnomis1/cnomis1
cno2/+
inscP49/+
DGai/+
4
11
13
18
91
9
27
3
31
2
67
2
3
3
0
3
1
3
296
0
0
0
27
28
0
1
2
0
423
245
304
327
280
367
326
382
403
397
302
282
302
356
45
35
40
40
40
38
40
39
50
46
40
32
37
40
4
14
14
21
387
9
27
3
58
30
67
3
5
3
0.9
5.7
4.6
6.4
13.8
2.5
8.3
0.8
14.4
7.6
22.2
1.0
1.6
0.8
Current Biology Vol 18 No 11
834
Figure 3. Epistatic and Physical Interactions between Cno and Apical Proteins
(A–C0 ) Cno and PH3 are shown in green, and Nrt is shown in blue. As shown in (A) and (A0 ), in control embryos, Cno localizes at the apical pole of mNBs
(arrow). In (B)–(C0 ), Cno is undetectable or mislocalized at mNBs in both insc lof (arrowhead in [B] and [B0 ]) and insc gof mutant embryos (arrow in [C]
and [C0 ]). See also text.
(D–F0 ) Cno and PH3 are shown in red, Mud is shown in green, and Nrt is shown in blue. In (E)–(F0 ), Cno localization was altered in both Gb13F maternal and
zygotic lof mutant embryos (arrowhead in [E] and [E0 ]) and in Gai gof mutants (arrows in [F] and [F0 ]).
(G–I0 ) Mud is shown in green, PH3 is shown in red, and Nrt is shown in blue. As shown in (G) and (G0 ), in control embryos, Mud is detected in an apical crescent
(arrow) and at the two centrosomal regions in mNBs. In (H)–(I0 ), Mud localization failed in both cno lof (arrowhead in [H] and [H0 ]) and cno gof mutant embryos
(arrow in [I] and [I0 ]) at mNBs. See also text.
(J) Cno coimmunoprecipitates with Pins in embryo extracts. Immunoprecipitation from embryo extracts was performed with rabbit a-Pins and rabbit a-GFP
antibodies. After immunoblotting, the immunoprecipitate was probed with a-Pins and a-Cno antibodies.
(K) Working model of protein interactions. The diagram shows the proposed network of interactions between apical proteins during asymmetric NB division.
DaPKC/Baz/Insc pathway is shown in orange, and the Pins/Gai/Cno pathway appears in blue (see also text). Lines between proteins indicate physical
interactions. Arrows between proteins show epistatic relationships. The discontinuous line between Pins and Cno indicates that their interaction may be
indirect.
(L) At prophase, the DmPar6/DapKC/Baz/Insc pathway is localized at the apical pole of the neuroblast. At metaphase, the microtubule (MT)-Khc-73-Dlg
pathway is active [39], tightly aligning the mitotic spindle (MTs) with the Pins/Gai/Cno crescent. The microtubule-binding protein Mud also contributes
to this process.
asymmetric cell divisions that generate specific neuronal lineages in the CNS.
Cno Acts Downstream of the Apical Proteins Insc-Pins-Gai
and Upstream of the NuMA-Related Protein Mud
Since Cno functionally interacted with Insc, Gai, and Mud
(Table 1, see above), we next analyzed the epistatic relationships between them. To investigate whether Cno was acting
upstream of the apical proteins, we examined the localization
of Baz, Insc, and Gai in cno2 mutant embryos. The distribution
of all these proteins was normal (Baz: 96.3%, n = 27; Insc:
100%, n = 24; and Gai: 96.5%, n = 29) in cno2 mutants. This result suggested that Cno acts either downstream or in parallel
to Baz, Insc, and Gai. To clarify this point, we analyzed the distribution of Cno in loss- and gain-of-function (lof and gof) mutants for several apical proteins. In inscP49 lof mutants, Cno
was untraceable or showed a wrong orientation in 78.8% of
the mNBs analyzed (n = 33) (Figures 3B and 3B0 ). Insc overexpression also caused failures in Cno localization (76.2% n =
21); Cno was either undetectable (13/21) or present in not-apical crescents (3/21) (Figures 3C and 3C0 ). Likewise, in Gb13F
maternal and zygotic null mutant embryos, in which Gai is
lost [35], Cno was mislocalized or undetectable in 93.5% of
the mNBs (n = 46) (Figures 3E and 3E0 ). Moreover, the
overexpression of Gai caused a striking mislocalization of
Cno in 100% of the mNBs analyzed (n = 25, Figures 3F and
3F0 ). The NuMA-related protein Mud binds the apical protein
Pins and functions downstream of Pins-Gai to regulate spindle orientation [36–38]. In mud mutant NBs, the spindle fails
to tightly align with the basal crescent [36–38], and this failure
is also shown by cno2 mutant NBs (Figure 1D, see also
above). Additionally, Cno and Mud interacted genetically (Table 1). Hence, we asked whether Cno functions along with
Mud to regulate spindle orientation. In control embryos,
Mud localized at the apical cortex of mNBs (97.2%, n = 36)
and at the two centrosomal regions (100%, n = 36) [36–38]
(Figures 3G and 3G0 ). In cno2 lof mutants, Mud failed to accumulate apically in 48.9% of mNBs (n = 47), and 14.9% of NBs
showed Mud localization in one or none of the two centrosomes (Figures 3H and 3H0 ). cno gof also caused failures in
Mud localization (38.2%, n = 34; Figures 3I and 3I0 ). Altogether, our results strongly support a function of Cno downstream of Insc and Pins-Gai and upstream of Mud during
asymmetric NB division.
Cno Forms a Complex In Vivo with Pins
Given the functional relationships we found between Cno and
apical proteins during asymmetric NB division, we next
Canoe Functions during Asymmetric Cell Division
835
Figure 4. Cno Is Also Required for Dorsal Muscle
and Heart Lineage Formation
(A) Diagram depicting the muscle and heart lineages of the dorsal progenitors P2 and P15.
(B–E) High magnification of the most dorsal part
of the mesoderm in one hemisegment at stage
12 (B) or stage 14 (C–E). Eve is shown in red,
Runt is shown in blue, and Svp (Svp-lacZ) and
the general cytoplasmic muscle marker 3E2 are
shown in green. As shown in (B) and (C), in control
embryos, Svp colocalizes with Eve at stage 12 in
the founder of two Svp cardial cells (FSvpCs), either the two Svp pericardial cells or the two Svp
cardioblasts detected at later stages per hemisegment (see [C]); Svp also colocalizes with
Runt in the founder of the dorsal muscle DO2
(FDO2) at this stage (B). At stage 14 (C), Svp is detected in two pericardial cells (SvpPCs) and two
cardioblasts (SvpCBs) per hemisegment and in
the dorsal muscle DO2. As shown in (D) and (E),
in cno2 mutant embryos, altered P2 and P15 muscle and heart lineages are observed at stage 14.
Altered P15 lineage is detected in some hemisegments that show DA1 muscle duplications and
reduction of SvpCs (D) or DA1 muscle loss (arrowhead in [E]) and gain of SvpCs. Altered P2 lineage is also observed in some hemisegments
showing loss of muscle DO2 (arrowhead) and
gain of EPCs (D).
(F–I0 ) Numb localization in the dorsal progenitor
P2 is basal in control embryos (arrow in [F]–[G0 ]),
but it is all around the cortex in cno2 mutants (arrowheads in [H]–[I0 ]).
wondered whether Cno was physically interacting with some
of these proteins. Coimmunoprecipitation experiments from
Drosophila embryo extracts showed that Cno is forming
a complex with Pins (Figure 3J). Cno did not physically interact
with DmPar6, Baz, DaPKC, or other apical proteins tested such
as Insc, Gai, and Mud (not shown).
Pins also forms a complex in mNBs with the tumor-suppressor protein Discs Large (Dlg) and the kinesin Khc-73, an astral
microtubule-binding protein [39] (Figures 3K and 3L). First,
at prophase, the DmPar6/Insc pathway is required to polarize
Pins/Gai at the apical pole of the NB. Then, at metaphase,
the Pins/Gai/Dlg/Khc-73 complex forms,
and it is key for tightly coupling cortical
polarity with spindle orientation (Figure 3L) [39]. Hence, we wondered
whether Cno was also forming part of
this complex. Our experiments showed
that neither Dlg nor Khc-73 coimmunoprecipitate Cno in embryo extracts (not
shown). This result indicated that Cno
is not forming part of the Dlg/Khc-73
complex.
Altogether, we propose a working network of protein interactions as depicted
in Figure 3K. Our analysis of epistatic relationships between apical proteins and
Cno showed that Cno is acting downstream of Insc-Pins-Gai and upstream
of Mud. Indeed, our genetic analysis
suggests that, at least for the control
of daughter cells size asymmetry, Cno
functions within the Pins-Gai pathway
(Figures 3K and 3L, in blue), in parallel to the DaPKC-BazInsc pathway (Figures 3K and 3L, in orange). Accordingly, we
found that Cno forms a complex with Pins in vivo (Figure 3J).
Cno did not coimmunoprecipitate with Gai, though. One possibility is that Cno and Gai are mutually exclusive in the complex that each of them forms with Pins. Additionally, we cannot
discard transient or labile interactions between Cno and Gai
that we are unable to detect. Another Pins interacting partner,
the microtubule-binding protein Mud contributes to coordinate spindle orientation with cortical polarity [36–38]. Given
the functional relationships that we found between Cno and
Current Biology Vol 18 No 11
836
Mud (Table 1 and Figures 3G–3I0 ), Cno could act in a complex
with Pins to modulate Mud localization and, consequently,
spindle orientation.
Cno Also Regulates the Asymmetric Division of Muscle
and Heart Progenitors
Finally, we wondered whether the function of Cno during
asymmetric cell division was conserved in different tissues.
As the NBs of the CNS, the Drosophila somatic muscle and
heart progenitors divide asymmetrically to give rise to two different founder cells [40, 41]. Cno is present in the somatic mesoderm and is required for muscle and heart progenitor specification [4]. Hence, we investigated whether Cno was also
functioning during the asymmetric division of muscle and
heart progenitors. For this analysis, we focused on two dorsal
progenitors called P2 and P15 that express the transcription
factor Eve and whose lineages have been characterized in detail [40, 42, 43] (Figure 4A). In this study, we found that the transcription factor Seven-up (Svp), a characteristic marker of
a subset of cardial cells [44], was expressed in a dorsal founder
cell of unknown identity until now, which we have named
founder of Svp cardial cells (FSvpCs) (Figures 4A and 4B).
With all these markers, specific for individual derivatives, we
analyzed whether dorsal muscle and cardial lineages were altered in cno2 mutants. We found that at late stages (stage 14),
3.1% of hemisegments (n = 96) showed simultaneously either
loss of EPCs and gain of DO2 muscle or gain of EPCs and loss
of the DO2 muscle (P2 lineage) (Figure 4D). In control embryos,
we did not observe this phenotype in any of the hemisegments
analyzed (n = 216). Indeed, Numb localization, which was basal
in 100% of the metaphase P2s analyzed (n = 17) in control embryos, was altered in 92.8% of metaphase P2s in cno2 mutants
(n = 14) (Figures 4 F–4I0 ). Hemisegments showing duplication
of DA1 muscle and loss of SvpCs or DA1 muscle loss and
gain of SvpCs (P15 lineage) were also detected in cno2 mutants (Figures 4D and 4E). Hence, Cno was required for the
asymmetric division of progenitor cells both in the CNS and
in the mesoderm.
Conclusions
Numerous proteins have been shown to be required during
asymmetric cell division. The lack of fully penetrant phenotypes found in single mutants for genes participating in this
process, the existence of redundant pathways, and the increasing number of proteins involved make apparent how extremely well regulated the process of asymmetric cell division
must be. Hence, the discovery of new modulators, as we have
shown here for the PDZ protein Cno, is key for our complete
understanding of this intricate process. Especially challenging
in the next future will be unraveling the complete network of
connections between all the players required for an accurate
asymmetric cell division.
Supplemental Data
Experimental Procedures are available at http://www.current-biology.com/
cgi/content/full/18/11/831/DC1/.
Acknowledgments
We thank C. Doe, Y.N. Jan, T.C. Kauffman, F. Matsuzaki, F. Moya, H. Nash,
C. Petritsch, T. Raabe, R. Wharton, A. Wodarz, D. Yamamoto, the Bloomington Drosophila Stock Center, and the Hybridoma Bank for kindly providing
fly strains and antibodies. We also thank the Network of European Neuroscience Institutes (ENINET) for partly financing this work and anonymous
reviewers for helpful and pointed comments. This work was supported by
Grants from the Austrian Academy of Sciences, the Austrian Research
Fund (FWF), and the WWTF and the EU FP7 networks ONCASYM and
EuroSyStem to J.A.K. and by grants from the Marie Curie Program (MIRGCT-2005-016536), the Spanish Ministry of Education and Science
(BFU2006-09130), and the Spanish Ministry of Education and Science Grant
CONSOLIDER-INGENIO 2010 CSD2007-00023 to A.C. A.F. is supported by
the EU Marie Curie grant IMBA PhD Programme.
Received: December 20, 2007
Revised: April 26, 2008
Accepted: April 28, 2008
Published online: May 22, 2008
References
1. Knoblich, J.A. (2008). Mechanisms of asymmetric stem cell division. Cell
132, 583–597.
2. Wodarz, A., and Huttner, W.B. (2003). Asymmetric cell division during
neurogenesis in Drosophila and vertebrates. Mech. Dev. 120,
1297–1309.
3. Boettner, B., Harjes, P., Ishimaru, S., Heke, M., Fan, H.Q., Qin, Y., Van
Aelst, L., and Gaul, U. (2003). The AF-6 homolog canoe acts as a Rap1
effector during dorsal closure of the Drosophila embryo. Genetics
165, 159–169.
4. Carmena, A., Speicher, S., and Baylies, M. (2006). The PDZ protein
Canoe/AF-6 Links Ras-MAPK, Notch and Wingless/Wnt signaling
pathways by directly interacting with Ras, Notch and Dishevelled.
PLoS ONE 1, e66.
5. Gaengel, K., and Mlodzik, M. (2003). Egfr signaling regulates ommatidial
rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling
and the Ras effector Canoe/AF6. Development 130, 5413–5423.
6. Ikeda, W., Nakanishi, H., Miyoshi, J., Mandai, K., Ishizaki, H., Tanaka, M.,
Togawa, A., Takahashi, K., Nishioka, H., Yoshida, H., et al. (1999).
Afadin: A key molecule essential for structural organization of cell-cell
junctions of polarized epithelia during embryogenesis. J. Cell Biol.
146, 1117–1132.
7. Kuriyama, M., Harada, N., Kuroda, S., Yamamoto, T., Nakafuku, M.,
Iwamatsu, A., Yamamoto, D., Prasad, R., Croce, C., Canaani, E., and
Kaibuchi, K. (1996). Identification of AF-6 and canoe as putative targets
for Ras. J. Biol. Chem. 271, 607–610.
8. Matsuo, T., Takahashi, K., Kondo, S., Kaibuchi, K., and Yamamoto, D.
(1997). Regulation of cone cell formation by Canoe and Ras in the developing Drosophila eye. Development 124, 2671–2680.
9. Matsuo, T., Takahashi, K., Suzuki, E., and Yamamoto, D. (1999). The Canoe protein is necessary in adherens junctions for development of ommatidial architecture in the Drosophila compound eye. Cell Tissue Res.
298, 397–404.
10. Miyamoto, H., Nihonmatsu, I., Kondo, S., Ueda, R., Togashi, S., Hirata, K.,
Ikegami, Y., and Yamamoto, D. (1995). canoe encodes a novel protein
containing a GLGF/DHR motif and functions with Notch and scabrous
in common developmental pathways in Drosophila. Genes Dev. 9,
612–625.
11. Prasad, R., Gu, Y., Alder, H., Nakamura, T., Canaani, O., Saito, H., Huebner, K., Gale, R.P., Nowell, P.C., Kuriyama, K., et al. (1993). Cloning of the
ALL-1 fusion partner, the AF-6 gene, involved in acute myeloid leukemias with the t(6;11) chromosome translocation. Cancer Res. 53,
5624–5628.
12. Takahashi, K., Matsuo, T., Katsube, T., Ueda, R., and Yamamoto, D.
(1998). Direct binding between two PDZ domain proteins Canoe and
ZO-1 and their roles in regulation of the jun N-terminal kinase pathway
in Drosophila morphogenesis. Mech. Dev. 78, 97–111.
13. Zhadanov, A.B., Provance, D.W., Jr., Speer, C.A., Coffin, J.D., Goss, D.,
Blixt, J.A., Reichert, C.M., and Mercer, J.A. (1999). Absence of the tight
junctional protein AF-6 disrupts epithelial cell-cell junctions and cell
polarity during mouse development. Curr. Biol. 9, 880–888.
14. Petronczki, M., and Knoblich, J.A. (2001). DmPAR-6 directs epithelial
polarity and asymmetric cell division of neuroblasts in Drosophila.
Nat. Cell Biol. 3, 43–49.
15. Schober, M., Schaefer, M., and Knoblich, J.A. (1999). Bazooka recruits
Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts. Nature 402, 548–551.
Canoe Functions during Asymmetric Cell Division
837
16. Wodarz, A., Ramrath, A., Grimm, A., and Knust, E. (2000). Drosophila
atypical protein kinase C associates with Bazooka and controls polarity
of epithelia and neuroblasts. J. Cell Biol. 150, 1361–1374.
17. Wodarz, A., Ramrath, A., Kuchinke, U., and Knust, E. (1999). Bazooka
provides an apical cue for Inscuteable localization in Drosophila neuroblasts. Nature 402, 544–547.
18. Kuchinke, U., Grawe, F., and Knust, E. (1998). Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka.
Curr. Biol. 8, 1357–1365.
19. Rhyu, M.S., Jan, L.Y., and Jan, Y.N. (1994). Asymmetric distribution of
numb protein during division of the sensory organ precursor cell confers
distinct fates to daughter cells. Cell 76, 477–491.
20. Jürgens, G., Wieschaus, E., Nüsslein-Volhard, C., and Kluding, H. (1984).
Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. Rouxs Arch. Dev. Biol. 193, 283–295.
21. Ikeshima-Kataoka, H., Skeath, J.B., Nabeshima, Y., Doe, C.Q., and
Matsuzaki, F. (1997). Miranda directs Prospero to a daughter cell during
Drosophila asymmetric divisions. Nature 390, 625–629.
22. Schuldt, A.J., Adams, J.H., Davidson, C.M., Micklem, D.R., Haseloff, J.,
St Johnston, D., and Brand, A.H. (1998). Miranda mediates asymmetric
protein and RNA localization in the developing nervous system. Genes
Dev. 12, 1847–1857.
23. Shen, C.P., Jan, L.Y., and Jan, Y.N. (1997). Miranda is required for the
asymmetric localization of Prospero during mitosis in Drosophila. Cell
90, 449–458.
24. Hirata, J., Nakagoshi, H., Nabeshima, Y., and Matsuzaki, F. (1995).
Asymmetric segregation of the homeodomain protein Prospero during
Drosophila development. Nature 377, 627–630.
25. Knoblich, J.A., Jan, L.Y., and Jan, Y.N. (1995). Asymmetric segregation
of Numb and Prospero during cell division. Nature 377, 624–627.
26. Spana, E.P., and Doe, C.Q. (1995). The prospero transcription factor is
asymmetrically localized to the cell cortex during neuroblast mitosis in
Drosophila. Development 121, 3187–3195.
27. Lee, C.Y., Wilkinson, B.D., Siegrist, S.E., Wharton, R.P., and Doe, C.Q.
(2006). Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal. Dev. Cell 10, 441–449.
28. Sonoda, J., and Wharton, R.P. (2001). Drosophila Brain Tumor is a translational repressor. Genes Dev. 15, 762–773.
29. Bello, B., Reichert, H., and Hirth, F. (2006). The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central
brain of Drosophila. Development 133, 2639–2648.
30. Bellotto, M., Bopp, D., Senti, K.A., Burke, R., Deak, P., Maroy, P.,
Dickson, B., Basler, K., and Hafen, E. (2002). Maternal-effect loci
involved in Drosophila oogenesis and embryogenesis: P elementinduced mutations on the third chromosome. Int. J. Dev. Biol. 46,
149–157.
31. Albertson, R., and Doe, C.Q. (2003). Dlg, Scrib and Lgl regulate neuroblast cell size and mitotic spindle asymmetry. Nat. Cell Biol. 5, 166–170.
32. Cai, Y., Yu, F., Lin, S., Chia, W., and Yang, X. (2003). Apical complex
genes control mitotic spindle geometry and relative size of daughter
cells in Drosophila neuroblast and pI asymmetric divisions. Cell 112,
51–62.
33. Parmentier, M.L., Woods, D., Greig, S., Phan, P.G., Radovic, A., Bryant,
P., and O’Kane, C.J. (2000). Rapsynoid/partner of inscuteable controls
asymmetric division of larval neuroblasts in Drosophila. J. Neurosci.
20, RC84.
34. Broadus, J., Skeath, J.B., Spana, E.P., Bossing, T., Technau, G., and
Doe, C.Q. (1995). New neuroblast markers and the origin of the aCC/
pCC neurons in the Drosophila central nervous system. Mech. Dev.
53, 393–402.
35. Yu, F., Cai, Y., Kaushik, R., Yang, X., and Chia, W. (2003). Distinct roles of
Galphai and Gbeta13F subunits of the heterotrimeric G protein complex
in the mediation of Drosophila neuroblast asymmetric divisions. J. Cell
Biol. 162, 623–633.
36. Bowman, S.K., Neumuller, R.A., Novatchkova, M., Du, Q., and Knoblich, J.A.
(2006). The Drosophila NuMA Homolog Mud regulates spindle orientation in
asymmetric cell division. Dev. Cell 10, 731–742.
37. Izumi, Y., Ohta, N., Hisata, K., Raabe, T., and Matsuzaki, F. (2006). Drosophila Pins-binding protein Mud regulates spindle-polarity coupling
and centrosome organization. Nat. Cell Biol. 8, 586–593.
38. Siller, K.H., Cabernard, C., and Doe, C.Q. (2006). The NuMA-related Mud
protein binds Pins and regulates spindle orientation in Drosophila neuroblasts. Nat. Cell Biol. 8, 594–600.
39. Siegrist, S.E., and Doe, C.Q. (2005). Microtubule-induced Pins/Galphai
cortical polarity in Drosophila neuroblasts. Cell 123, 1323–1335.
40. Carmena, A., Murugasu-Oei, B., Menon, D., Jimenez, F., and Chia, W.
(1998). Inscuteable and numb mediate asymmetric muscle progenitor
cell divisions during Drosophila myogenesis. Genes Dev. 12, 304–315.
41. Ruiz Gomez, M., and Bate, M. (1997). Segregation of myogenic lineages
in Drosophila requires numb. Development 124, 4857–4866.
42. Carmena, A., Buff, E., Halfon, M.S., Gisselbrecht, S., Jimenez, F.,
Baylies, M.K., and Michelson, A.M. (2002). Reciprocal regulatory interactions between the Notch and Ras signaling pathways in the Drosophila embryonic mesoderm. Dev. Biol. 244, 226–242.
43. Carmena, A., Gisselbrecht, S., Harrison, J., Jimenez, F., and Michelson,
A.M. (1998). Combinatorial signaling codes for the progressive determination of cell fates in the Drosophila embryonic mesoderm. Genes Dev.
12, 3910–3922.
44. Ward, E.J., and Skeath, J.B. (2000). Characterization of a novel subset of
cardiac cells and their progenitors in the Drosophila embryo. Development 127, 4959–4969.