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1 Institutes of Neuroscience and Molecular Biology, Howard Hughes Medical Institute, University of Oregon, Eugene, Oregon 97403, USA; 2 Department of Human Genetics, Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, Utah 84112, USA
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Abstract |
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[Keywords: Aurora-A; Drosophila; neuroblast; numb; self-renewal; spindle orientation]
Received September 1, 2006; revised version accepted November 3, 2006.
). Drosophila larval brain neuroblasts have many stem cell attributes—such as the ability to remain proliferative, undifferentiated, and nontumorous for a hundred or more cell divisions while generating post-mitotic neurons and glia—and thus they have recently served as a paradigm for studies of self-renewal versus differentiation (Betschinger et al. 2006; Lee et al. 2006a, b). Neuroblasts divide asymmetrically to self-renew and to generate differentiating ganglion mother cells (GMCs), which typically generate two post-mitotic neurons or glia.
Neuroblast self-renewal requires establishment and maintenance of proper apical/basal cortical polarity. Two protein complexes are localized to the neuroblast apical cortex and partitioned into the neuroblast during asymmetric division: the Par complex and the Pins complex. The evolutionarily conserved Par complex contains Bazooka (Baz; Par-3 in mammals)/Par-6/atypical protein kinase C (aPKC) proteins, and the Pins complex contains Partner of Inscuteable (Pins)/G
i proteins; these complexes are linked by the Inscuteable (Insc) protein, which binds both Baz and Pins (Cai et al. 2003; Fuse et al. 2003; Yu et al. 2003; Siegrist and Doe 2005). Proteins localized to the basal cortex and partitioned into the GMC include Numb, Miranda (Mira), Brain tumor (Brat), and Prospero (Pros) (for review, see Betschinger and Knoblich 2004). Mislocalization of aPKC to the basal (GMC) cortex triggers ectopic neuroblast self-renewal, resulting in a dramatic expansion of the neuroblast population (Lee et al. 2006a), whereas depletion of Pros or Brat from the GMC also leads to GMC-neuroblast transformation and overproduction of neuroblasts (Bello et al. 2006; Betschinger et al. 2006; Lee et al. 2006b). Thus, establishing proper cortical polarity is essential for neuroblast self-renewal and GMC differentiation.
A second, more speculative pathway for regulating neuroblast self-renewal involves spindle orientation. In theory, if the neuroblast mitotic spindle aligns orthogonal to the apical/basal polarity axis, both neuroblast progeny would inherit apical and basal proteins, and both might assume a neuroblast identity (similar to an epithelial cell division, which partitions apical and basolateral membrane domains equally into both siblings). The linkage between spindle and apical cortex is provided, in part, by the Mud protein (Mushroom body defective; the Drosophila NuMA ortholog), which directly binds both microtubules and cortical Pins protein; mud mutants show misalignment of the mitotic spindle without altering cortical polarity (Bowman et al. 2006; Izumi et al. 2006; Siller et al. 2006). mud mutant brains have an increase in neuroblast number (Prokop and Technau 1994; Bowman et al. 2006), and it is tempting to speculate that the symmetric divisions lead to a pair of sibling neuroblasts and thus to the observed increase in neuroblast number, but this has not been confirmed by clonal analysis.
Here we describe the results of a genetic screen for additional regulators of neuroblast self-renewal. We identify three mutants showing a massive increase in larval neuroblast numbers, without any detectable disruption of optic lobe or imaginal disc epithelia, and all are alleles of the evolutionarily conserved mitotic kinase aurora-A (aurA). Mammalian AurA kinase is localized to centrosomes and is considered an oncogene (for review, see Giet et al. 2005). In Drosophila, AurA is required for centrosome maturation, cell cycle progression, Numb protein localization during sense organ precursor asymmetric cell division, and astral microtubule length in S2 cells and larval neuroblasts (Glover et al. 1995; Berdnik and Knoblich 2002; Giet et al. 2002). Here we show that loss of AurA leads to neuroblast brain tumors, which arise primarily due to defects in Numb localization and secondarily due to defects in spindle-to-cortical polarity alignment. Our data define a role for Numb in the GMC as an inhibitor of neuroblast self-renewal, and provide direct evidence that spindle orientation can regulate neuroblast self-renewal.
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Results |
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To identify genes that regulate neuroblast self-renewal, we screened a collection of pupal lethal mutants (L. Wang, J. Evans, H. Andrews, R. Beckstead, C.S. Thummel, and A. Bashirullah, in prep.) for changes in central brain neuroblast number. Three mutants [l(3)LL-8839, l(3)LL-14641, l(3)LL-17961] had a massive increase in larval neuroblasts, developing >1000 central brain neuroblasts at stages where wild-type larvae have only 95–100 neuroblasts; in addition, the mutant neuroblasts were maintained into pupal stages, whereas wild-type pupae have few neuroblasts (Fig. 1A–C). The mutants had normal optic lobe and imaginal disc epithelial morphology (Fig. 1B'; data not shown), unlike previously identified tumor suppressor mutants (for review, see Hariharan and Bilder 2006). The mutations were deficiency and complementation mapped to the aurA locus, and the aurA87ac-3 mutant (Glover et al. 1995) showed a similar supernumerary brain neuroblast phenotype (data not shown). Sequencing of the mutant alleles revealed that l(3)LL-14641 has a single base change resulting in a V 
E change at position 302, within the kinase activation loop (Cheetham et al. 2002); l(3)LL-17961 has a single base change resulting in a D N change at position 334 that is predicted to destabilize -helical packing; and l(3)LL-8839 has a single base change resulting in a K stop change at position 377 that deletes the C terminus of the protein (Fig. 1D); thus these are new aurA alleles. The AurA kinase is detected in the cytoplasm and on centrosomes in neuroblasts and sense organ precursors (Berdnik and Knoblich 2002), as is an AurA:GFP fusion protein expressed in neuroblasts (Supplementary Fig. 1; data not shown), consistent with a role in neuroblast asymmetric cell division.
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Single aurora-A mutant neuroblasts generate clones containing multiple neuroblasts
To determine the origin of the extra neuroblasts in the mutant brains, we generated positively marked clones in single neuroblasts within aurA mutant brains, at a low frequency of one to two per brain lobe to keep each clone well separated. In wild type, genetically marked 
gal+ clones induced in a single neuroblast always mark one large neuroblast and a population of smaller GMC/neuronal progeny (Fig. 1K). In contrast, single neuroblast clones induced in aurA mutants typically contained multiple neuroblasts as well as a pool of smaller GMC/neuronal progeny (Fig. 1L). Thus, aurA mutant neuroblasts undergo occasional divisions in which both siblings assume the neuroblast fate, leading to an expansion of the neuroblast population (Fig. 1M). Importantly, neuroblast-specific expression of AurA:GFP (wor-gal4 UAS-aurA:GFP) is able to significantly rescue the aurA supernumerary neuroblast phenotype, showing that the phenotype is due to loss of AurA within neuroblast lineages (Supplementary Fig. 2).We conclude that the evolutionarily conserved AurA kinase is required to inhibit neuroblast self-renewal and promote GMC/neuronal differentiation.
Aurora-A regulates aPKC and Numb cortical polarity
In wild-type neuroblasts, Baz/Par6/aPKC and Insc/Pins/Gi proteins are localized to the apical cortex of metaphase neuroblasts (Fig. 2A–C; data not shown). In aurA mutants, we find that aPKC can be uniform cortical or show ectopic cortical patches; all other apical proteins assayed show normal localization (Fig. 2D–G; data not shown). Ectopic aPKC is observed in a small fraction of neuroblasts at early larval stages (48–72 h after larval hatching [ALH]) and becomes much more common at later stages when the increase in neuroblast number is the most dramatic (96–120 h ALH). aPKC; aurA double mutants completely suppress ectopic neuroblast formation (Supplementary Fig. 2), showing that aPKC is required for the aurA supernumerary neuroblast phenotype. This rescue is specific (e.g., not simply due to aPKC mutants arresting the neuroblast cell cycle or inducing neuroblast cell death) because aPKC mutants can only weakly suppress the brat mutant supernumerary neuroblast phenotype (Lee et al. 2006b). We conclude that aurA mutants disrupt aPKC but not other tested apical polarity proteins; that aPKC delocalization coincides with the increase in neuroblast numbers; and that aPKC is required for the ectopic neuroblasts observed in aurA mutants.
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; Betschinger et al. 2006; Lee et al. 2006b), so we tested whether aurA mutants have defects in basal protein localization. Wild-type neuroblasts localize Numb, Miranda, Pros, and Brat to the basal cortex at metaphase (Fig. 2H–K). In aurA mutants, we observed normal Mira/Pros/Brat protein basal crescents at metaphase (Fig. 2I–K), but Numb was either relatively normal (Fig. 2L) or delocalized around the cortex (Fig. 2M). This is similar to the Numb localization defects in aurA mutant mitotic sense organ precursors (Berdnik and Knoblich 2002; see below).
Numb inhibits neuroblast self-renewal
The role of Numb during larval neuroblast asymmetric division has never been investigated, so we assayed the phenotype of numb mutant clones generated in single larval neuroblasts. Wild-type clones always show one neuroblast and its family of neuronal progeny (Fig. 3A), whereas numb mutant clones always contain multiple neuroblasts plus neuronal progeny (Fig. 3B). Thus, the numb clones are similar to the aurA mutant brains in their ability to expand the neuroblast population while still producing differentiated Pros+ Elav+ neurons. Interestingly, the numb and AurA mutant phenotypes are distinct from mira, pros, or brat mutant single neuroblast clones, which contain few or no differentiated neurons (Supplementary Fig. 3). We conclude that AurA is required to restrict Numb to the basal cortex and that Numb inhibits neuroblast selfrenewal.
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175 neuroblasts per lobe) at the same larval stage (Fig. 3C–E); this is similar to the number of neuroblasts seen in mutants that affect spindle orientation but not cortical polarity such as cnn (Fig. 3C) or mud (Prokop and Technau 1994; Bowman et al. 2006). Thus, Numb overexpression appears to suppress the cortical polarity component of the aurA supernumerary neuroblast phenotype. We conclude that Numb acts downstream from AurA to inhibit neuroblast self-renewal.
Aurora-A regulates alignment of the mitotic spindle with the cortical polarity axis
We next tested whether spindle orientation defects could contribute to the production of ectopic neuroblasts in aurA mutants. In wild-type metaphase neuroblasts, the mitotic spindle is aligned within 15° of the center of the apical/basal cortical polarity axis (Siller et al. 2005, 2006; Bowman et al. 2006). In aurA metaphase neuroblasts, the mitotic spindle is essentially randomized relative to the apical/basal cortical polarity axis (Fig. 4B,C). Because some aurA neuroblasts have multiple centrosomes (wild type: 2.0, n = 25; aurA: 3.8, n = 45) and multipolar spindles, we only scored neuroblasts where a clear bipolar spindle could be observed. In addition, aurA mutant neuroblasts have defects in telophase spindle orientation, with 15% of the neuroblasts dividing symmetrically (Fig. 4D,F; see below)—a phenotype that is never observed in wild-type neuroblasts (Fig. 4E). Spindle orientation defects are not due to loss of cortical Mud protein (which is essential for proper spindle orientation) (Bowman et al. 2006; Izumi et al. 2006; Siller et al. 2006), because Mud remains cortical in aurA mutant neuroblasts (Fig. 4G,H). Interestingly, overexpression of Numb does not rescue the aurA spindle orientation defect (Fig. 4D), consistent with AurA regulating cell polarity and spindle orientation phenotypes via two distinct pathways (see Discussion). We conclude that AurA is essential for proper alignment of the mitotic spindle to the neuroblast cortical polarity axis.
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). In addition, we find that the spindle anchoring protein Mud is centrosomal and enriched at the anterior and posterior cortex (Fig. 4K). In aurA mutant SOPs, cortical polarity is established normally based on proper anterior Pins crescent formation, but Numb is delocalized (confirming previous findings; Berdnik and Knoblich 2002). Importantly, we observe nearly random spindle orientation relative to the SOP cortical polarity axis (Fig. 4L–N), similar to the neuroblast spindle orientation phenotype. The most frequent spindle orientation is along the proximodistal axis, perhaps reflecting a secondary spindle orientation cue from the planar cell polarity system. We conclude that AurA is required in both neuroblasts and SOPs to align the mitotic spindle with the cortical polarity axis.
aurora-A neuroblasts divide symmetrically to expand the neuroblast population
It has been proposed that physically symmetric cell divisions can lead to expansion of the larval neuroblast population during mutant or wild-type insect development (Nordlander and Edwards 1970; Farris et al. 1999; Bowman et al. 2006; Siller et al. 2006), but this attractive hypothesis has never been directly tested. Here we use time-lapse imaging to assay the frequency and cell fate consequences of neuroblast symmetric divisions in aurA14641 mutants. We imaged histone:GFP (HisAvD:GFP) in explanted larval brains; these whole-brain cultures are healthy enough to allow us to follow >10 wild-type neuroblast cell divisions (see Materials and Methods). We assayed division symmetry by measuring both sibling nuclear size (with histone:GFP) and sibling cell size (using background fluorescence signal). We found that wild-type neuroblasts always divide asymmetrically with regard to sibling cell and nuclear size (100%, n = 11) (Fig. 5A,D; Supplementary Movie 1). In contrast, aurA mutant neuroblasts show both asymmetric cell divisions (Fig. 5B,D; Supplementary Movie 2) and symmetric cell divisions (17%, n = 42) (Fig. 5C,D; Supplementary Movies 3, 4). Thus, aurA14641 neuroblasts show a significant number of symmetric divisions; this could contribute to the supernumerary neuroblast phenotype if both progeny retained a neuroblast identity. In support of this model, we observed a neuroblast lineage tree containing two symmetric divisions followed by an asymmetric cell division (Fig. 5E). This shows that a symmetric neuroblast division can produce additional neuroblasts with the potential for asymmetric cell division—a hallmark of neuroblast identity—and suggests that symmetric divisions can expand the neuroblast population.
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; Du and Hannon 2004). Time-lapse analysis of aurA mutant neuroblasts revealed delays in prometaphase and metaphase (Fig. 5F) as well as increased overall cell cycle length (data not shown). In addition, we observe an increased mitotic index (wild type: 7.6% prometaphase–metaphase, n = 690; aurA: 14.2% prometaphase–metaphase; n = 2753) in fixed preparations. It is formally possible that the cell cycle delays are the cause of the neuroblast cell polarity and supernumerary neuroblast phenotype (Fichelson and Gho 2004). However, we have previously shown that metaphase-arrested neuroblasts have normal cortical polarity, and that Lis1 and Glued mutants have cell cycle delays without a matching neuroblast cortical polarity or supernumerary neuroblast phenotype (Spana and Doe 1995; Broadus and Doe 1997; Siller et al. 2005). We conclude that aurA mutant neuroblasts show cell cycle delays, but that these delays do not prevent or cause the observed increase in brain neuroblasts. |
Discussion |
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). Consistent with this hypothesis, aPKC aurA double mutants show strong suppression of the aurA supernumerary neuroblast phenotype, consistent with aPKC functioning downstream from AurA. While it is possible that loss of aPKC suppresses the phenotype in a nonspecific way (e.g., by arresting neuroblast cell proliferation or inducing neuroblast apoptosis), we fail to observe similarly strong suppression of the brat supernumerary neuroblast phenotype in aPKC brat double mutants (Lee et al. 2006b). This shows that aPKC functions more specifically in the AurA pathway than in the Brat pathway.
The only other detectable cortical polarity defect seen in aurA mutant neuroblasts is a delocalization of Numb from the basal cortex. A similar Numb defect is seen during asymmetric cell division of pupal SOPs in aurA mutants (Fig. 4K; Berdnik and Knoblich 2002), perhaps reflecting a specific and direct regulation of Numb by AurA, although Numb is not phosphorylated by AurA in vitro (Berdnik and Knoblich 2002). The importance of the Numb delocalization phenotype is revealed by the ability of Numb overexpression in neuroblasts to rescue most of the aurA mutant phenotype (all except the component due to spindle orientation defects; see below). Thus, Numb acts downstream from AurA to inhibit neuroblast self-renewal. Numb joins Mira/Pros/Brat as proteins that are partitioned into the GMC during neuroblast asymmetric cell division, where they function to inhibit neuroblast self-renewal (Fig. 6; Bello et al. 2006; Betschinger et al. 2006; Lee et al. 2006b).
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How does Numb inhibit neuroblast self-renewal in the GMC? Numb is a well-characterized inhibitor of Notch signaling that is segregated into the GMC (Frise et al. 1996; Guo et al. 1996; Zhong et al. 1996; O’Connor-Giles and Skeath 2003; Hutterer and Knoblich 2005), and Notch signaling is active in larval neuroblasts but not in GMCs (Almeida and Bray 2005). Thus the most obvious model is that Numb blocks Notch receptor signaling in the GMC. However, Notch mutant clones generated in larval neuroblasts do not affect neuroblast survival or clone size (Almeida and Bray 2005). Similarly, we have seen no change in neuroblast number in two different Notch-ts mutants (although we observed the expected small wing imaginal disc phenotype) (C.-Y. Lee, unpubl.). In addition, we did not observe supernumerary neuroblasts in larval neuroblast clones overexpressing the constitutively active Notch intracellular domain (C.-Y. Lee, unpubl.), although the same Notch intracellular domain generates the expected sibling neuron phenotype when expressed in the embryonic CNS (J. Boone and K. Robinson, unpubl.). Thus, Notch is an excellent candidate for promoting neuroblast self-renewal, but additional experiments will be needed to test this model more rigorously. In this context, it is interesting to note that Notch promotes stem cell self-renewal in mammals (Androutsellis-Theotokis et al. 2006; Fan et al. 2006).
We show that aurA mutant neuroblasts have essentially random orientation of the mitotic spindle relative to the apical/basal cortical polarity axis, resulting in a some neuroblasts dividing symmetrically (in size and cortical polarity markers). This phenotype may arise due to lack of astral microtubule interactions with the neuroblast cortex, as aurA mutant neuroblasts have reduced astral microtubule length (Giet et al. 2002). Alternatively, AurA may affect spindle orientation by phosphorylating proteins required for spindle orientation, such as Cnn, Pins, or Mud. For example, Mud has a consensus AurA/Ipl1 phosphorylation site within its microtubule-binding domain, and it will be interesting to determine if this site needs to be phosphorylated for Mud to bind microtubules. Spindle orientation defects only generate part of the supernumerary neuroblast phenotype in aurA mutant brains, however, because overexpression of Numb can rescue most of the phenotype without rescuing spindle alignment, and cnn or mud mutants have nearly random spindle alignment but only a modest increase in neuroblast number (Fig. 3C; Bowman et al. 2006). Thus, we propose that spindle orientation defects and cortical polarity defects combine to generate the dramatic supernumerary neuroblast phenotype seen in aurA mutants (Fig. 6).
Mammalian aurA has been termed an oncogene due to its overexpression in several cancers, its ability to promote proliferation in certain cell lines, and the fact that reduced levels lead to multiple centrosomes, mitotic delay, and apoptosis (for review, see Giet et al. 2005). However, an in vivo aurA mutant phenotype has not yet been reported. In contrast, we find that aurA loss-of-function mutations result in a neuroblast "brain tumor" phenotype, including prolonged neuroblast proliferation during pupal stages when wild-type neuroblasts have stopped proliferating. aurA mutants do not, however, have the imaginal disc epithelial overgrowth seen in other Drosophila tumor suppressor mutants (for review, see Hariharan and Bilder 2006), and aurA mutant neuroblasts have a delay in cell cycle progression. We propose that the aurA supernumerary neuroblast phenotype is not due to loss of growth control or a faster cell cycle time, but rather due to a cell fate transformation from a differentiating cell type (GMC) to a proliferating cell type (neuroblast).
We conclude that AurA restrains neuroblast numbers using two pathways: first by promoting Numb localization into the GMC, and second by promoting alignment of the mitotic spindle with the cortical polarity axis. Absence of the first pathway leads to increased neuroblasts at the expense of GMCs, whereas absence of the second pathway leads to increased neuroblasts due to symmetric cell division. It will be interesting to determine whether mammalian AurA uses one or both pathways to regulate stem cell asymmetric division and self-renewal.
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Materials and methods |
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All mutant chromosomes were balanced over CyO, actin::GFP or TM3 actin::GFP, Ser, e. We used Oregon R as wild type, and the following mutant chromosomes:
) ) ; Lee et al. 2006a) ) ) ) ) ) ) ) ) )
All clone experiments (MARCM, actin-Gal4, or actinnuc.lacZ) were done using standard methods (Lee and Luo 2001; Lee et al. 2006b) by giving the larval progeny a 1-h 37°C heat shock at 24 h ALH, development at 25°C, and clone analysis at 96 h ALH.
Antibodies and imaging
Larvae brains were dissected in Schneider’s medium (Sigma), fixed in 100 mM Pipes (pH 6.9), 1 mM EGTA, 0.3% Triton X-100, and 1 mM MgSO4 containing 4% formaldehyde for 23 min and blocked for 1 h in 1x PBS containing 1% BSA and 0.3% Triton X-100 (PBS-BT) supplemented with 0.01 M glycine and 0.1% goat normal serum. After blocking, specimens were washed in PBS-BT for 1 h and incubated with primary antibodies in PBS-BT overnight at 4°C. Brains were stained as described previously (Albertson and Doe 2003; Lee et al. 2006a). Pupal nota were dissected and stained as described previously (Manning and Doe 1999). We used guinea pig anti-Mira (1:400), rat anti-Mira (1:500), guinea pig anti-Numb (1:1000; J. Skeath), rat anti-Brat (1:100), mouse anti-Pros MR1A bioreactor supernatant (1:100), rabbit anti-aPKC
(1:1000; Santa Cruz Biotechnology), rabbit anti-Gi (1:1000), mouse anti-Insc (1:1000; Bill Chia), rat anti-Pins (1:500), rat anti-Par6 (1:50), guinea pig anti-Baz (1:400), rabbit anti-Scribble (1:2500), mouse anti-Discs large (1:100; Developmental Study Hybridoma Bank [DSHB]), rabbit anti-Mud (1:1000), guinea pig anti-Dpn (1:1), mouse anti-Elav (1:50; DSHB), rat anti-Elav (1:10; DSHB), mouse anti-BrdU (1:50, Sigma), rabbit anti-gal (1:1000), mouse anti--tubulin (1:2000; Sigma), Rat anti--tubulin (1:100; Serotec), mouse anti-
-tubulin (1:2000; Sigma), rabbit anti-AurA (1:200; J. Knoblich), rabbit anti-phosphohistoneH3 (1:1000; Upstate Biotechnology), rabbit anti-Cnn (1:1000; T. Kaufman), rabbit anti-GFP (1:1000; Torrey Pine), mouse anti-CD8 (1:100; Caltag), mouse anti-Hindsight "concentrated" (1:100; DHSB), and secondary antibodies from Molecular Probes. Antibodies without named sources were made in the laboratory; details are available on request.
BrdU pulse/chase experiments
Larvae were fed with BrdU (1 mg/mL; Roche) in media for 4 h, and then one pool was processed for BrdU staining (pulse experiments) and a second pool was grown without BrdU for 24 h before fixation and BrdU staining (pulse/chase experiments). Larval brains were dissected, processed, and antibody-stained as described above with the exception that larval brains were treated in 2N HCl for 30 min prior to primary anti-BrdU antibody staining.
Neuroblast counting and brain orientation
A larval brain lobe consists of the medially localized central brain and the laterally localized optic lobe. Neuroblasts can be unambiguously identified by expression of Wor, Dpn, and Mira and the absence of the neuronal/glial differentiation markers Elav and Repo (Albertson and Doe 2003; Lee et al. 2006a). Central brain neuroblasts (the focus of this study) can be distinguished from optic lobe neuroblasts due to their medial–superficial location in the brain, larger size, and dispersed pattern (optic lobe neuroblasts laterally positioned in the brain and spaced very closely to each other, forming a ribbon that flanks and encircles the highly stereotypical epithelial-shaped optic lobe cells) (Lee et al. 2006a). All images of neuroblasts shown were collected from central brain,; all brains were mounted with dorsal surface up and ventral surface down, and the midline is to the left in all panels.
Time-lapse imaging of larval neuroblasts
aurA14641 was recombined with His2AvD:GFP (Clarkson and Saint 1999) and crossed to aurA17961. Late second and/or early third instar aurA mutant larvae were picked based on the lack of the dominant marker Tubby and dissected as previously described (Siller et al. 2005, 2006). Larvae were dissected and mounted in D-22 media (US Biological) with 1% bovine growth serum (BGS; HyClone) and imaged on a Bio-Rad Radiance 2000 confocal or Leica SP2 microscope using a 60x 1.4NA oil-immersion objective. Time-lapse sequences were processed using ImageJ and Imaris 5.0.1, 64-bit (Bitplane).
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Footnotes |
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4 Present address: Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.
E-MAIL cdoe{at}uoneuro.uoregon.edu; FAX (541) 346-4736.
Supplemental material is available at http://www.genesdev.org.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1489406
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11 for each genotype.