A crucial first step in asymmetric cell division is to establish an axis of cell polarity along which the mitotic spindle aligns. Drosophila melanogaster neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic polarity cues, which regulate spindle orientation and cortical polarity. In this paper, we show that the Ras-like small guanosine triphosphatase Rap1 signals through the Ral guanine nucleotide exchange factor Rgl and the PDZ protein Canoe (Cno; AF-6/Afadin in vertebrates) to modulate the NB division axis and its apicobasal cortical polarity. Rap1 is slightly enriched at the apical pole of metaphase/anaphase NBs and was found in a complex with atypical protein kinase C and Par6 in vivo. Loss of function and gain of function of Rap1, Rgl, and Ral proteins disrupt the mitotic axis orientation, the localization of Cno and Mushroom body defect, and the localization of cell fate determinants. We propose that the Rap1–Rgl–Ral signaling network is a novel mechanism that cooperates with other intrinsic polarity cues to modulate asymmetric NB division.

Introduction

Asymmetric cell division is a key process in development and stem cell biology. In an asymmetric cell division, one daughter cell retains the self-renewal capacity of the mother stem cell and keeps on dividing, whereas the other daughter cell is committed to initiating a differentiation program. A crucial first step in an asymmetric cell division is to establish an axis of cell polarity along which the mitotic spindle aligns. Extrinsic and intrinsic mechanisms regulate the spindle orientation and the final asymmetry of the division. Drosophila melanogaster stem cells have been extensively studied during the last few decades, providing a deep insight into both types of mechanisms (Doe, 2008; Knoblich, 2008; Morrison and Spradling, 2008). Drosophila neural stem cells, called neuroblasts (NBs), divide asymmetrically, mainly through intrinsic polarity cues. In the embryonic central nervous system (CNS), NBs delaminate from the neuroectoderm (NE) inheriting the apicobasal polarity of the neuroepithelial cells. Intrinsic signals, mostly polarized at the apical NB cortex, tightly couple the spindle orientation along the apicobasal axis with the asymmetric location of cell fate determinants at the basal pole of the NB. In this way, these determinants are secreted to the basal and smaller daughter cell, called the ganglion mother cell (GMC). The apical and bigger daughter cell continues dividing as an NB, always budding off smaller GMCs into the embryo in the same, highly stereotyped, basal orientation (Wodarz and Huttner, 2003; Chia et al., 2008; Knoblich, 2008; Siller and Doe, 2009). Extrinsic signals emanating from the NE also participate in regulating spindle orientation and cortical polarity in the NB, though the nature of these signals remains elusive (Siegrist and Doe, 2006).

Here, we show that the Ras-like small GTPase Rap1 contributes to regulate asymmetric NB division through the Ral guanine nucleotide exchange factor Rgl, Ral, and the PDZ domain–containing protein Canoe (Cno; AF-6/Afadin in vertebrates; Miyamoto et al., 1995; Asha et al., 1999; Mirey et al., 2003). Rap1 has a key and evolutionary conserved role in regulating morphogenesis, integrin- as well as cadherin-mediated cell–cell adhesion, and junction formation. In addition, Rap1 has adhesion-independent functions that suggest a central function of Rap1 in signal transduction (Asha et al., 1999; Knox and Brown, 2002; Caron, 2003; Mirey et al., 2003; Price et al., 2004; Wang et al., 2006; Kooistra et al., 2007; O’Keefe et al., 2009). Ral proteins are Ras-like GTPases that can be activated through a Ras-dependent mechanism in mammalian cell lines (Yaffe et al., 2001) and downstream of Rap1–Rgl in Drosophila (Mirey et al., 2003). The Rap/Ras–Rgl–Ral GTPase signaling network is highly conserved between Drosophila and mammals (Moskalenko et al., 2001; Mirey et al., 2003). Intriguingly, Rap1 interacts physically with Cno/AF-6, and the Ral guanine nucleotide exchange factor Rgl has been predicted as a potential partner of Cno (Drosophila Interactions Database; Boettner et al., 2000, 2003), a novel regulator of asymmetric NB division (Speicher et al., 2008). Our results now show that loss and gain of function of Rap1, Rgl, and Ral proteins affect the NB spindle orientation, the generation of unequal-sized progeny, and the localization of apical proteins, such as Cno and the microtubule-associated protein Mushroom body defect (Mud; Numa in vertebrates; Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006). Bazooka (Baz; Par3 in vertebrates) and the atypical PKC (aPKC; Schober et al., 1999; Wodarz et al., 1999, 2000) were affected to a lesser degree. Failures in the basal localization of the cell fate determinants Numb, Prospero (Pros), and its adaptor protein Miranda (Mira; Rhyu et al., 1994; Hirata et al., 1995; Knoblich et al., 1995; Spana and Doe, 1995; Ikeshima-Kataoka et al., 1997; Shen et al., 1997; Schuldt et al., 1998) were also detected in Rap1 and Ral mutants. Moreover, coimmunoprecipitation experiments from embryo extracts showed that Rap1 is in a complex with aPKC and Par6. Additionally, Rgl synergistically cooperated with other apical proteins, such as Partner of Inscuteable (Insc; Pins), Insc, and Mud to regulate spindle orientation. Taking all data into account, we propose that the Rap1–Rgl–Ral signaling network is a novel intrinsic mechanism that cooperates with other apical proteins to regulate cortical polarity and spindle orientation in NBs.

Results and discussion

Rap1 is present in the embryonic neuroepithelium and in NBs

In an attempt to further characterize the protein network that along with Cno modulates asymmetric NB division (Speicher et al., 2008), we wanted to analyze in detail the expression and function of the Cno-interacting partner Rap1 (Boettner et al., 2000). GFP-Rap1, a fusion protein that is expressed under the control of the endogenous Rap1 promoter (Knox and Brown, 2002), was detected throughout the NE, evenly distributed in the cytoplasm, and in the delaminated metaphase NBs (mNBs), with a slight enrichment at the apical pole along with the Baz apical crescent (Fig. 1, A and A′). Looking in more detail throughout the NB mitotic cycle, GFP-Rap1 was found uniformly distributed at prophase and started to accumulate apically at metaphase (Fig. 1, B–C′). During anaphase, this apical enrichment was still detected, and by telophase, GFP-Rap1 began to delocalize (Fig. 1, D–E′). Hence, Rap1 was a potential candidate for regulating the process of asymmetric NB division.

Rap1 is required for the mitotic axis orientation and apical proteins localization in mNBs

To determine a possible function of Rap1 in the cell division axis orientation, we analyzed the effect of expressing wild-type (WT), constitutively active (V12), and dominant-negative (N17) forms of Rap1 (hereafter referred to as Rap1 mutants). A maternal Gal-4 line (V32) was used to drive expression of these transgenes. The spindle orientation was altered when the Rap1 signal was impaired, in clear contrast with control embryos, in which most NBs showed a normal spindle orientation along the apicobasal axis of cell polarity (Fig. 1, F–I). To further support these results, we decided to look at additional Rap1 mutant conditions: Rap1P5709 germline clones, a complete loss of maternal and zygotic product, Rap1P5709-only zygotic loss, and R1 (Roughened1), a gain-of-function mutation in the Rap1 locus (Hariharan et al., 1991). Clear defects in the spindle orientation were detected in all these mutants (Fig. 1 J). Apical cortical polarity was also affected in Rap1 mutants. The Par complex proteins Baz and aPKC were mislocalized in 10.1% of mNBs (n = 178) and 1.4% (n = 143) in UAS-Rap1WT embryos, respectively, in 11.2% (n = 161) and 11.3% (n = 115) in UAS-Rap1V12 embryos, and in 28.8% (n = 145) and 19.8% (n = 101) in UAS-Rap1N17 embryos (Fig. 1, G–I). No defects in Baz (n = 65 mNBs) or aPKC (n = 66) were observed in control embryos. Much more penetrant phenotypes were observed for the apical proteins Cno and Mud in those mutant backgrounds (Fig. 2, A–I). The localization of the Gαi subunit and Pins, other apical proteins key for regulating spindle alignment (Parmentier et al., 2000; Schaefer et al., 2000; Yu et al., 2000), were, however, not affected (Fig. 2, J–M; and not depicted). Hence, Rap1 is required for the correct establishment of cortical polarity and spindle orientation through a pathway that includes Cno and Mud but is Gαi and Pins independent.

Rap1 mutants show cell fate determinant mislocalization and equal-sized daughter cells

Given the defective localization of apical proteins we observed in Rap1 mutants, we predicted that cell fate determinants would be misplaced in mNBs. In fact, whereas in control embryos Numb was found in basal crescents in most mNBs, clear defects in Numb localization were detected in Rap1 mutants, including Rap1P5709 germline clones and R1 mutant conditions (Fig. 3, A–Q). Another cell fate determinant, the transcription factor Pros and its adaptor protein Mira also showed altered location in a significant number of the mNBs analyzed compared with control embryos (Fig. 3, R–Y). These defects were partially or completely rescued at telophase (compensatory mechanism known as telophase rescue; Fig. 3 Y). Therefore, Rap1 is required for the correct establishment of apical polarity in the NB and for the proper location of asymmetrically segregating factors. Mud, whose distribution was altered in Rap1 mutants, is not required for apicobasal cortical polarity (Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006; Cabernard and Doe, 2009). Hence, the failure in cell fate determinant location in Rap1 mutants was caused, at least in part, by the mislocalization in these mutants of Cno and aPKC/Baz, all key factors for the proper formation of determinant basal crescents in mNBs (Speicher et al., 2008; Wirtz-Peitz et al., 2008). Intriguingly, another distinctive feature of asymmetric NB division, the generation of unequal-sized daughter cells, was also altered in Rap1 mutants (Fig. S1, A–D). In addition, the asymmetric division within the well-characterized RP2 neuron lineage (Broadus et al., 1995) was affected in these mutants (Fig. S1, E–H). Hence, Rap1 regulates multiple aspects of the asymmetric NB division.

Given that Rap1 is required for a proper cell–cell adhesion in epithelial tissues and that this might influence the analysis of the underlying NBs in Rap1 mutants, we looked in detail to epithelial cell polarity in Rap1V12 and Rap1N17 mutant embryos (Fig. S2). Different polarity proteins, such as Drosophila E-cadherin (DE-cad), aPKC, and Scribble (Scrib), were analyzed. In Rap1V12 mutants, aPKC was reduced, but DE-cad and Scrib were not affected. Similar results were found in Rap1N17, though in this case, clear defects in the morphology/integrity of the epithelia were observed. Remarkably, however, no correlation was found between epithelial morphology and NB polarity/orientation defects in these mutants (for an example see Fig. S2, C, C′, F, and F′). From this analysis, we conclude that Rap1 is required for both processes, epithelial integrity and NB polarity, and that these functions are independent. Additionally, to discard that the defects observed in Rap1 mutant NBs were caused by earlier defects in NB specification, we analyzed in detail the process of NB delamination at stage 9 in these mutants. In Rap1V12, NBs were organized in the windowlike arrangement typical of this stage in WT embryos (Fig. S3, A and D). About 31% of Rap1N17 mutant embryos showed NB disorganization, probably caused by the early role of Rap1 in morphogenetic events, such as gastrulation (Fig. S3, G and H; see also Fig. 5 in Asha et al. [1999]). In a percentage of Rap1V12 and Rap1N17 mutant embryos, the number of NBs seemed also to be affected, but importantly, NB delamination was properly achieved in both cases (Fig. S3, compare E, F, and I–K with B and C). Thus, NB alterations in polarity and spindle orientation are not merely a consequence of an impaired NB specification process.

Rgl and Ral mutants display abnormal NB spindle orientation and apicobasal cortical polarity

Given the phenotypes we found in Rap1 mutants in the CNS NBs, we wondered whether the Rap1 effectors Rgl and Ral also had a function in this system. We found that ΔRgl mutant embryos (Mirey et al., 2003) displayed mitotic spindle misorientation (Fig. 4, B and E), the same phenotype that showed embryos expressing activated (RalCA) and dominant-negative forms (RalDN) of Ral (Fig. 4, C, D, F, and G). Moreover, double mutants Rap1N17; ΔRgl showed a spindle phenotype more similar to that shown by ΔRgl single mutants (Fig. 4), suggesting that Rgl is acting in the same pathway of Rap1, downstream of it (Fig. 1 I). Additionally, in both ΔRgl and Ral mutants, the apicobasal cortical polarity was altered. Baz was misplaced in 9.3% of the ΔRgl NBs analyzed (n = 75; 0% in control embryos, n = 65) and in 16.4% (n = 79) and 15.4% (n = 65) of NBs in RalCA or RalDN embryos (Fig. 4, B–G). The localization of Cno and Mud also showed (similar to that observed in Rap1 mutants) more penetrant phenotypes. Although in control embryos, 4.2 and 12.5% of mNBs displayed failures in Cno and Mud localization, respectively, in RalCA mutants, the percentages were 71% of NBs (n = 62) for Cno localization and 51.2% for Mud (n = 80; Fig. 4, H–I′ and K–L′). In RalDN mutant embryos, the failures observed corresponded to 76.8% (n = 56) for Cno and 64.3% (n = 56) for Mud (Fig. 4, J, J′, M, and M′). Finally, the basal localization of Numb was also affected in ΔRgl, in 48% of mNBs (n = 75; Fig. 4, B and E), and in Ral mutants. Specifically, in RalCA mutants, Numb failures were 27.8% (n = 79) and 29.2% (n = 65) in RalDN mutant embryos (Fig. 4, C, D, F, and G). Thus, Rgl–Ral functions in the embryonic NBs of the CNS to regulate cortical polarity and spindle orientation downstream of Rap1.

The Rap1–Rgl–Ral signaling network functions in a complex with aPKC and Par6 cooperating with other apical proteins to regulate asymmetric NB division

Here, we have shown that Rap1 functions in asymmetric NB division in the embryonic CNS to modulate NB apicobasal cortical polarity and mitotic axis orientation. Rap1 would act through the Rgl–Ral–Cno signaling network to regulate Mud localization and, hence, spindle alignment. The localization of the apical protein Pins, which is attached to the cortex through the heterotrimeric Gαi subunit (Fig. 5 F; Schaefer et al., 2000; Nipper et al., 2007), was not dependent on Rap1 signaling and neither was the location on the Gαi subunit. Pins, through Discs Large and Khc-73, is key for spindle location (Siegrist and Doe, 2005; Johnston et al., 2009). Hence, both pathways, Rap1–Rgl–Ral and Gαi–Pins could cooperate to properly orientate the mitotic axis. To test this possibility, we analyzed the spindle orientation in double mutants ΔRgl, pinsΔ50. In this genetic background, 46.4% of the mNBs (n = 69) showed a correct spindle alignment (0–15° window) compared with the 64.0% (n = 75; P = 0.0439) and the 66.1% (n = 56; P = 0.0314) of mNB defects found in ΔRgl or pinsΔ50 single mutants, respectively (Fig. 5 A). In addition, inscP49; ΔRgl and mud4; ΔRgl double mutants also showed a strong cooperation in regulating spindle orientation (Fig. 5, B and C). In inscP49; ΔRgl double mutants, 36.1% of the mNBs (n = 72) displayed a WT spindle orientation (0–15° window) versus the 64.0% (n = 75; P = 0.0009) and the 57.9% (n = 57; P = 0.0204) of mNB failures observed in the single mutants ΔRgl and inscP49, respectively. The interaction between Rgl and mud was statistically significant when comparing the expressivity, not the penetrance, of the phenotype. In other words, mud4; ΔRgl double mutants showed many more cases of spindle alignment defects in the 75–90° window (11.8%, n = 59) compared with the single mutants ΔRgl (2.7%, n = 75; P = 0.0429) and mud4 (1.6%, n = 63; P = 0286). This last interaction between mud and Rgl suggests that the Rap1–Rgl–Ral pathway is modulating something else, independent of Mud, which is important for spindle alignment. It has previously been reported that there are two independent and redundant apical pathways in NBs. One of these pathways is formed by Baz, aPKC, Par6, and Insc, and the other pathway is formed by Gαi–Pins (Cai et al., 2003). Even though there are some interdependence between both pathways at prophase for their apical location (Yu et al., 2000), they become much more independent from metaphase onwards. For example, Pins localizes asymmetrically in 81% of the insc mutant mNBs analyzed (Cai et al., 2003). This would explain why the Baz and aPKC failures found in Rap1 mutants, only at metaphase, are not affecting Pins location. Hence, the Rap1–Rgl–Ral pathway synergistically cooperates with other apical polarity cues to correctly position the mitotic spindle.

The Rap1 signaling network also contributes to the establishment of the cortical polarity and, consequently, to the proper segregation of determinants at the basal NB pole. This effect seems to be mainly mediated through its interacting partner Cno, which has been shown to be required for this process (Speicher et al., 2008). Trying to understand how Rap1 is initially polarized at the apical NB pole, we performed in vivo coimmunoprecipitation assays with GFP-Rap1 and different apical proteins. Although Pins, Gαi, or Mud did not show any positive result, we found that both aPKC and Par6 were able to coimmunoprecipitate with Rap1 (Fig. 5 D). Thus, these Par complex proteins can help to locate Rap1 at the apical NB pole. The localization of these proteins, Baz and aPKC, also key for cell fate determinant localization, were altered to a lesser degree in Rap1 mutant mNBs. This effect of Rap1–Rgl–Ral on Baz and aPKC may respond to the establishment in normal conditions of a positive feedback loop of the Rap1 pathway on the Par protein complex (Par6-Baz/Par3–aPKC) to facilitate their stabilization at metaphase (Fig. 5 E). Intriguingly, Rap1B has been shown to act upstream of this complex and of Cdc42 to regulate neuronal polarity in mammalian cell cultures (Schwamborn and Püschel, 2004). The low penetrant phenotypes observed for Baz and aPKC location in Rap1 mutants suggests though that Rap1 signal is not the major driving force initially positioning the Par proteins. This might be driven, at least in part, by extrinsic signals coming from the NE (Siegrist and Doe, 2006). The nature of those extrinsic cues remains, however, elusive.

Materials and methods

Drosophila strains and genetics

The following mutant stocks and fly lines were used: GFP-Rap1 (Knox and Brown, 2002), UAS-Rap1WT, UAS-Rap1V12, and UAS-Rap1N17 (a gift from R. Reuter, Universität Tübingen, Tübingen, Germany), Rap1P5709 (a gift from N. Brown, University of Cambridge, Cambridge, England, UK), R1 (Bloomington Stock Center), pinsΔ50 (Schaefer et al., 2000), inscP49 (Bloomington Stock Center), mud4 (Bloomington Stock Center), UAS-RalCA, UAS-RalDN, ΔRgl (a gift from J. Camonis, Institut Curie, Institut National de la Santé et de la Recherche Médicale, Paris, France; Mirey et al., 2003), and maternal-GAL4 V32 (a gift from J.A. Knoblich, Institute of Molecular Biotechnology, Vienna, Austria). All the crosses of GAL4-UAS were performed at 29°C. yw was used as the reference control WT strain. Balancer chromosomes containing different lacZ or GFP transgenes were used for identifying homozygous mutant embryos.

Generation of germline clones

w; pr pwn P {ry+t7.2=hsFLP}38/Cyo; Rap1P5709 P{FRT (whs)}2A/TM2 females were crossed with w; P{ovoD1-18}3L P{FRT(whs)}2A/TM3, Sb males. Mitotic recombination was induced in 24–48-h larvae for 2 h at 37°C. Virgins from this cross were mated with Rap1P5709/TM6lacZ males, and the embryos (without maternal and zygotic Rap1 products) were used for the phenotypic analysis.

Immunofluorescence

Embryo fixation and antibody staining were performed by standard protocols (4% formaldehyde for 20 min) with the exceptions mentioned at the end of this paragraph. The following primary antibodies were used: sheep anti-GFP at 1:400 (Osenses); rabbit anti–β-galactosidase at 1:1,000–1:10,000 (Cappel); mouse anti–β-galactosidase at 1:8,000 (Promega); rabbit anti–PKC-ζ at 1:1,000 (C-20; Santa Cruz Biotechnology, Inc.); rabbit anti-Cno at 1:400 (Speicher et al., 2008); guinea pig anti-Numb at 1:250 (a gift from Y.-N. Jan, University of California, San Francisco, San Francisco, CA; Rhyu et al., 1994); rabbit anti-PH3 at 1:400 (Millipore); rabbit anticentrosomine (Cnn) at 1:400 (a gift from T.C. Kaufman, Indiana University, Bloomington, IN); mouse anti-Neurotactin at 1:200 (Speicher et al., 1998); rabbit anti-Baz at 1:200 (a gift from A. Wodarz, Georg-August-Universität Göttingen, Göttingen, Germany; Wodarz et al., 1999); rabbit anti-Mira at 1:2,000 (a gift from F. Matsuzaki, RIKEN Center for Developmental Biology, Kobe, Japan; Ikeshima-Kataoka et al., 1997); mouse anti-Pros at 1:100 (Developmental Studies Hybridoma Bank); mouse anti-Mud at 1:100 (a gift from F. Matsuzaki; Izumi et al., 2006); rabbit anti-Scrib at 1:4,000 (a gift from C. Doe, University of Oregon, Eugene, OR); rabbit anti–Even-skipped at 1:3,000 (Frasch et al., 1987); rat anti–DE-cad at 1:20 (Developmental Studies Hybridoma Bank); rabbit anti-Pins at 1:200 and rabbit anti-Gαi at 1:1,000 (both gifts from J.A. Knoblich); mouse anti–α-tubulin at 1:1,000 (Sigma-Aldrich); rat anti–Lethal of Scute (L’sc) at 1:2,000 (Martín-Bermudo et al., 1991); and mouse anti-Wingless at 1:50 (Developmental Studies Hybridoma Bank). Secondary antibodies coupled to biotin (Vector laboratories) plus streptavidin 488 (Invitrogen), Alexa Fluor 488, Alexa Fluor 546, or Alexa Fluor 633 (Invitrogen) were used. For immunostaining with the anti-Cno antibody, embryos were fixed by using the heat–methanol method (Tepass, 1996). For α-tubulin staining, embryos were fixed with 37% formaldehyde for 1 min. L’sc signal was enhanced by use of reagents (Tyramide Signal Amplification; DuPont).

Spindle orientation analysis

Taking as a reference the overlying epithelia, angles formed between the axis delineated by the NB spindle and the apicobasal polarity axis of epithelial cells were measured using Photoshop (Adobe).

Microscope image acquisition

Fluorescent images were recorded by using an upright microscope (DM-SL with Spectral Confocal acquisition software; Leica). All images were taken with an HCX Plan Apochromat 63×/1.32-0.6 NA oil confocal scanning objective. Figs. 1 (A–E′) and 5 (A–C) were acquired with an additional electronic zoom (z = 4). Fig. S1 (E–H) was recorded by using a microscope (Axio Imager.A1; EC Plan Neofluar 63×/1.25 NA oil objective; Carl Zeiss) and a camera (AxioCam HRc; Carl Zeiss). Images were assembled by using Photoshop CS3.

Coimmunoprecipitations

For immunoprecipitations, 0–7-h embryos were homogenized in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 1 mM EDTA, 1% Triton X-100, 1 mM NaF, 100 μM Na3VO4, 2 mM PMSF, and protease inhibitors [Complete; Roche]). Embryo extracts were centrifuged at 4°C for 15 min at 14,000 rpm (18,700 g) and then for 5 min in the same conditions. Precleared extracts were incubated with rabbit polyclonal antibody to GFP Sepharose beads (Abcam) for 2 h at 4°C. The beads then were washed three times with lysis buffer without inhibitors, resuspended in protein set buffer (Fluka), and heated at 95°C for 5 min. Precipitates were resolved by SDS-PAGE and immunoblotted with mouse anti-GFP (Takara Bio, Inc.) at 1:2,000, rabbit anti–PKC-ζ at 1:500, or rabbit anti-Par6 (a gift from J.A. Knoblich) at 1:2,000. Each experiment was repeated at least three times.

Online supplemental material

Fig. S1 shows that Rap1 mutants show equal-sized daughter cells and defects in the RP2 lineage. Fig. S2 shows that epithelial polarity defects in Rap1 mutants are not correlated with NB polarity and spindle orientation failures. Fig. S3 shows that NB delamination is not affected in Rap1 mutant embryos.

Acknowledgments

We thank Rolf Reuter, Jacques Camonis, Nick Brown, Andreas Wodarz, Fumio Matsuzaki, Juergen Knoblich, Yuh-Nung Jan, Thomas Kaufman, Chris Doe, the Bloomington Drosophila Stock Center at the University of Indiana, and the Developmental Studies Hybridoma Bank at the University of Iowa for kindly providing fly strains and antibodies.

This work was supported by grants to our laboratory from the Spanish government (BFU2006-09130, BFU2009-08833, and CONSOLIDER-INGENIO 2010 CSD2007-00023).

References

References
Asha
H.
,
de Ruiter
N.D.
,
Wang
M.G.
,
Hariharan
I.K.
.
1999
.
The Rap1 GTPase functions as a regulator of morphogenesis in vivo
.
EMBO J.
18
:
605
615
.
Boettner
B.
,
Govek
E.E.
,
Cross
J.
,
Van Aelst
L.
.
2000
.
The junctional multidomain protein AF-6 is a binding partner of the Rap1A GTPase and associates with the actin cytoskeletal regulator profilin
.
Proc. Natl. Acad. Sci. USA.
97
:
9064
9069
.
Boettner
B.
,
Harjes
P.
,
Ishimaru
S.
,
Heke
M.
,
Fan
H.Q.
,
Qin
Y.
,
Van Aelst
L.
,
Gaul
U.
.
2003
.
The AF-6 homolog canoe acts as a Rap1 effector during dorsal closure of the Drosophila embryo
.
Genetics.
165
:
159
169
.
Bowman
S.K.
,
Neumüller
R.A.
,
Novatchkova
M.
,
Du
Q.
,
Knoblich
J.A.
.
2006
.
The Drosophila NuMA Homolog Mud regulates spindle orientation in asymmetric cell division
.
Dev. Cell.
10
:
731
742
.
Broadus
J.
,
Skeath
J.B.
,
Spana
E.P.
,
Bossing
T.
,
Technau
G.
,
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
.
Cabernard
C.
,
Doe
C.Q.
.
2009
.
Apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation in Drosophila
.
Dev. Cell.
17
:
134
141
.
Cai
Y.
,
Yu
F.
,
Lin
S.
,
Chia
W.
,
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
.
Caron
E.
2003
.
Cellular functions of the Rap1 GTP-binding protein: a pattern emerges
.
J. Cell Sci.
116
:
435
440
.
Chia
W.
,
Somers
W.G.
,
Wang
H.
.
2008
.
Drosophila neuroblast asymmetric divisions: cell cycle regulators, asymmetric protein localization, and tumorigenesis
.
J. Cell Biol.
180
:
267
272
.
Doe
C.Q.
2008
.
Neural stem cells: balancing self-renewal with differentiation
.
Development.
135
:
1575
1587
.
Frasch
M.
,
Hoey
T.
,
Rushlow
C.
,
Doyle
H.
,
Levine
M.
.
1987
.
Characterization and localization of the even-skipped protein of Drosophila
.
EMBO J.
6
:
749
759
.
Hariharan
I.K.
,
Carthew
R.W.
,
Rubin
G.M.
.
1991
.
The Drosophila roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination
.
Cell.
67
:
717
722
.
Hirata
J.
,
Nakagoshi
H.
,
Nabeshima
Y.
,
Matsuzaki
F.
.
1995
.
Asymmetric segregation of the homeodomain protein Prospero during Drosophila development
.
Nature.
377
:
627
630
.
Ikeshima-Kataoka
H.
,
Skeath
J.B.
,
Nabeshima
Y.
,
Doe
C.Q.
,
Matsuzaki
F.
.
1997
.
Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions
.
Nature.
390
:
625
629
.
Izumi
Y.
,
Ohta
N.
,
Hisata
K.
,
Raabe
T.
,
Matsuzaki
F.
.
2006
.
Drosophila Pins-binding protein Mud regulates spindle-polarity coupling and centrosome organization
.
Nat. Cell Biol.
8
:
586
593
.
Johnston
C.A.
,
Hirono
K.
,
Prehoda
K.E.
,
Doe
C.Q.
.
2009
.
Identification of an Aurora-A/PinsLINKER/Dlg spindle orientation pathway using induced cell polarity in S2 cells
.
Cell.
138
:
1150
1163
.
Knoblich
J.A.
2008
.
Mechanisms of asymmetric stem cell division
.
Cell.
132
:
583
597
.
Knoblich
J.A.
,
Jan
L.Y.
,
Jan
Y.N.
.
1995
.
Asymmetric segregation of Numb and Prospero during cell division
.
Nature.
377
:
624
627
.
Knox
A.L.
,
Brown
N.H.
.
2002
.
Rap1 GTPase regulation of adherens junction positioning and cell adhesion
.
Science.
295
:
1285
1288
.
Kooistra
M.R.
,
Dubé
N.
,
Bos
J.L.
.
2007
.
Rap1: a key regulator in cell-cell junction formation
.
J. Cell Sci.
120
:
17
22
.
Martín-Bermudo
M.D.
,
Martínez
C.
,
Rodríguez
A.
,
Jiménez
F.
.
1991
.
Distribution and function of the lethal of scute gene product during early neurogenesis in Drosophila
.
Development.
113
:
445
454
.
Mirey
G.
,
Balakireva
M.
,
L’Hoste
S.
,
Rossé
C.
,
Voegeling
S.
,
Camonis
J.
.
2003
.
A Ral guanine exchange factor-Ral pathway is conserved in Drosophila melanogaster and sheds new light on the connectivity of the Ral, Ras, and Rap pathways
.
Mol. Cell. Biol.
23
:
1112
1124
.
Miyamoto
H.
,
Nihonmatsu
I.
,
Kondo
S.
,
Ueda
R.
,
Togashi
S.
,
Hirata
K.
,
Ikegami
Y.
,
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
.
Morrison
S.J.
,
Spradling
A.C.
.
2008
.
Stem cells and niches: mechanisms that promote stem cell maintenance throughout life
.
Cell.
132
:
598
611
.
Moskalenko
S.
,
Henry
D.O.
,
Rosse
C.
,
Mirey
G.
,
Camonis
J.H.
,
White
M.A.
.
2001
.
The exocyst is a Ral effector complex
.
Nat. Cell Biol.
4
:
66
72
.
Nipper
R.W.
,
Siller
K.H.
,
Smith
N.R.
,
Doe
C.Q.
,
Prehoda
K.E.
.
2007
.
Galphai generates multiple Pins activation states to link cortical polarity and spindle orientation in Drosophila neuroblasts
.
Proc. Natl. Acad. Sci. USA.
104
:
14306
14311
.
O’Keefe
D.D.
,
Gonzalez-Niño
E.
,
Burnett
M.
,
Dylla
L.
,
Lambeth
S.M.
,
Licon
E.
,
Amesoli
C.
,
Edgar
B.A.
,
Curtiss
J.
.
2009
.
Rap1 maintains adhesion between cells to affect Egfr signaling and planar cell polarity in Drosophila
.
Dev. Biol.
333
:
143
160
.
Parmentier
M.L.
,
Woods
D.
,
Greig
S.
,
Phan
P.G.
,
Radovic
A.
,
Bryant
P.
,
O’Kane
C.J.
.
2000
.
Rapsynoid/partner of inscuteable controls asymmetric division of larval neuroblasts in Drosophila
.
J. Neurosci.
20
:
RC84
.
Price
L.S.
,
Hajdo-Milasinovic
A.
,
Zhao
J.
,
Zwartkruis
F.J.
,
Collard
J.G.
,
Bos
J.L.
.
2004
.
Rap1 regulates E-cadherin-mediated cell-cell adhesion
.
J. Biol. Chem.
279
:
35127
35132
.
Rhyu
M.S.
,
Jan
L.Y.
,
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
.
Schaefer
M.
,
Shevchenko
A.
,
Shevchenko
A.
,
Knoblich
J.A.
.
2000
.
A protein complex containing Inscuteable and the Galpha-binding protein Pins orients asymmetric cell divisions in Drosophila
.
Curr. Biol.
10
:
353
362
.
Schober
M.
,
Schaefer
M.
,
Knoblich
J.A.
.
1999
.
Bazooka recruits Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts
.
Nature.
402
:
548
551
.
Schuldt
A.J.
,
Adams
J.H.
,
Davidson
C.M.
,
Micklem
D.R.
,
Haseloff
J.
,
St Johnston
D.
,
Brand
A.H.
.
1998
.
Miranda mediates asymmetric protein and RNA localization in the developing nervous system
.
Genes Dev.
12
:
1847
1857
.
Schwamborn
J.C.
,
Püschel
A.W.
.
2004
.
The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity
.
Nat. Neurosci.
7
:
923
929
.
Shen
C.P.
,
Jan
L.Y.
,
Jan
Y.N.
.
1997
.
Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila
.
Cell.
90
:
449
458
.
Siegrist
S.E.
,
Doe
C.Q.
.
2005
.
Microtubule-induced Pins/Galphai cortical polarity in Drosophila neuroblasts
.
Cell.
123
:
1323
1335
.
Siegrist
S.E.
,
Doe
C.Q.
.
2006
.
Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts
.
Development.
133
:
529
536
.
Siller
K.H.
,
Doe
C.Q.
.
2009
.
Spindle orientation during asymmetric cell division
.
Nat. Cell Biol.
11
:
365
374
.
Siller
K.H.
,
Cabernard
C.
,
Doe
C.Q.
.
2006
.
The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts
.
Nat. Cell Biol.
8
:
594
600
.
Spana
E.P.
,
Doe
C.Q.
.
1995
.
The prospero transcription factor is asymmetrically localized to the cell cortex during neuroblast mitosis in Drosophila
.
Development.
121
:
3187
3195
.
Speicher
S.
,
García-Alonso
L.
,
Carmena
A.
,
Martín-Bermudo
M.D.
,
de la Escalera
S.
,
Jiménez
F.
.
1998
.
Neurotactin functions in concert with other identified CAMs in growth cone guidance in Drosophila
.
Neuron.
20
:
221
233
.
Speicher
S.
,
Fischer
A.
,
Knoblich
J.
,
Carmena
A.
.
2008
.
The PDZ protein Canoe regulates the asymmetric division of Drosophila neuroblasts and muscle progenitors
.
Curr. Biol.
18
:
831
837
.
Tepass
U.
1996
.
Crumbs, a component of the apical membrane, is required for zonula adherens formation in primary epithelia of Drosophila
.
Dev. Biol.
177
:
217
225
.
Wang
H.
,
Singh
S.R.
,
Zheng
Z.
,
Oh
S.W.
,
Chen
X.
,
Edwards
K.
,
Hou
S.X.
.
2006
.
Rap-GEF signaling controls stem cell anchoring to their niche through regulating DE-cadherin-mediated cell adhesion in the Drosophila testis
.
Dev. Cell.
10
:
117
126
.
Wirtz-Peitz
F.
,
Nishimura
T.
,
Knoblich
J.A.
.
2008
.
Linking cell cycle to asymmetric division: Aurora-A phosphorylates the Par complex to regulate Numb localization
.
Cell.
135
:
161
173
.
Wodarz
A.
,
Huttner
W.B.
.
2003
.
Asymmetric cell division during neurogenesis in Drosophila and vertebrates
.
Mech. Dev.
120
:
1297
1309
.
Wodarz
A.
,
Ramrath
A.
,
Kuchinke
U.
,
Knust
E.
.
1999
.
Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts
.
Nature.
402
:
544
547
.
Wodarz
A.
,
Ramrath
A.
,
Grimm
A.
,
Knust
E.
.
2000
.
Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts
.
J. Cell Biol.
150
:
1361
1374
.
Yaffe
M.B.
,
Leparc
G.G.
,
Lai
J.
,
Obata
T.
,
Volinia
S.
,
Cantley
L.C.
.
2001
.
A motif-based profile scanning approach for genome-wide prediction of signaling pathways
.
Nat. Biotechnol.
19
:
348
353
.
Yu
F.
,
Morin
X.
,
Cai
Y.
,
Yang
X.
,
Chia
W.
.
2000
.
Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization
.
Cell.
100
:
399
409
.

    Abbreviations used in this paper:
     
  • aPKC

    atypical PKC

  •  
  • Baz

    Bazooka

  •  
  • Cnn

    centrosomine

  •  
  • Cno

    Canoe

  •  
  • CNS

    central nervous system

  •  
  • DE-cad

    Drosophila E-cadherin

  •  
  • GMC

    ganglion mother cell

  •  
  • Insc

    Inscuteable

  •  
  • L’sc

    Lethal of Scute

  •  
  • Mira

    Miranda

  •  
  • mNB

    metaphase NB

  •  
  • Mud

    Mushroom body defect

  •  
  • NB

    neuroblast

  •  
  • NE

    neuroectoderm

  •  
  • Pins

    Partner of Insc

  •  
  • Pros

    Prospero

  •  
  • Scrib

    Scribbled

  •  
  • WT

    wild type

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