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Polarization of a cell refers to the loss of symmetry that most cellsundergo in response to external or internal signals. Some of the
mechanisms for initiating and transducing polarity are shared bydifferent cell types, but, because polarization is intimately linked to
a specialized physiology, cell typespecific processes exist as well1.The mechanisms that determine where exactly in the cell periphery a
new pole will appear are best understood in yeast, migratory cells andepithelia24. In neurons, the selection of one out of several neurites
to become the cells axon rather than a dendrite has been the focus ofinterest5. Yet it is the first breakage of symmetry, by determining the
site of a second breakage at the opposite pole
6
, that defines the axison which future axonal and dendritic domains will form7,8 and alongwhich neurons will migrate9. For this essential mechanism, we have
very little information in vitro (see below) and none in vivo.A polarized organization of the cytoplasm, marked by centrosome
localization, has been recently put forward as an early and essential eventin the generation of the first neurite and thus neuronal polarizationin vitro8. In that work, it was shown that centrosome position correlateswith the site of first neurite formation8. The importance of the centro-
some for the establishment of neuronal polarity was, however, calledinto question in work in Drosophila melanogaster, where it was recently
shown that the absence of centrioles (in dsas-4 and dsas-6mutant flies)is compatible with a fairly normal development10,11. Yet centrosome
function involves more than centriole function. Accordingly, proteins
of the pericentriolar material (PCM), acting through the -tubulin ringcomplex, determine the site of microtubule nucleation and thus makethe centrosome the main center of microtubule nucleation in the cell.
By virtue of this, centrosomes determine the position, eccentric andjuxtanuclear, of membrane organelles such as the Golgi apparatus.
These facts, added to the well established correlation between mem-brane signaling and repositioning of the centrosome in migrating cells
and the role of centrosome-based functions (microtubule polymeriza-tion and membrane trafficking) for oriented cell migration3, made us
decide to analyze the importance of both centrosome and membrane-originating signals for the occurrence of neuronal polarization in vivo,
to define the hierarchy of molecular events polarizing a neuron.
RESULTS
Live imaging of neuronal polarity establishment in vivo
To follow the establishment of neuronal polarity in vivo, we choseDrosophila sensory neurons of the notum as a readout system. They
are located at the surface and readily accessible for live imaging dur-ing their birth and further development12. These neurons polarize totransmit sensory signals from the apical ciliary dendrite, wrapped by
a sheath cell, to the basally located axon. They belong to the externalmechanosensory organs of the f ly called microchaetae. Each micro-
chaeta is formed by four cells physically and functionally associated:two external support cells (the socket and the shaft cell) and two
internal cells (the neuron and the sheath cell)13,14. These cells derivefrom a single precursor cell (PI) after four asymmetric divisions that
occur during pupal development. The neuron is generated from theneuronal precursor cell (PIIIb) together with its sister cell, the sheath
cell, in the fourth division12.Three-dimensional time-lapse in vivo microscopy, in live pupae
expressing fluorescence-labeled proteins in the PI-derived cells
(neurP72>GAL4 driver), allowed us to monitor the polarization ofsensory neurons in a fly of which only part of the pupal case wasremoved. Initially we imaged during a 3-h period, beginning at the
time of PIIIb division (20 h after pupa formation (APF)). To easilyfollow the entry of the PIIIb cell into mitosis, we used the nuclear marker
histone-2AmRFP (His-RFP)15; to visualize the cell morphology, weused the GFP-tagged End-Binding protein1 (EB1) which binds the plus
1Vlaams Instituut voor Biotechnologie, Department of Molecular and Developmental Genetics, Campus Gasthuisberg, Leuven, Belgium. 2Katholieke Universiteit
Leuven Center for Human Genetics, Campus Gasthuisberg, Leuven, Belgium. 3Centro de Biologa Molecular Severo Ochoa, Universitad Autnoma de Madrid,
Madrid, Spain. 4These authors contributed equally to this work. Correspondence should be addressed to J.G.S. ([email protected]) or
C.G.D. ([email protected]).
Received 5 July; accepted 8 September; published online 13 November 2011; doi:10.1038/nn.2976
Cytokinesis remnants define first neuronalasymmetry in vivo
Giulia Pollarolo1,2,4, Joachim G Schulz1,2,4, Sebastian Munck1,2 & Carlos G Dotti13
Polarization of a neuron begins with the appearance of the first neurite, thus defining the ultimate growth axis. Unlike late
occurring polarity events (such as axonal growth), very little is known about this fundamental process. We show here that,
in Drosophila melanogasterneurons in vivo, the first membrane deformation occurred 3.6 min after precursor division. Clustering
of adhesion complex components (Bazooka (Par-3), cadherincatenin) marked this place by 2.8 min after division; the upstream
phosphatidylinositol 4,5-bisphosphate, by 0.7 min after division; and the furrow components RhoA and Aurora kinase, from the
time of cytokinesis. Local DE-cadherin inactivation prevented sprout formation, whereas perturbation of division orientation did
not alter polarization from the cytokinesis pole. This is, to our knowledge, the first molecular study of initial neuronal polarizationin vivo. The mechanisms of polarization seem to be defined at the precursor stage.
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end of growing microtubules16 (Fig. 1a and Supplementary Fig. 1).
We observed that the PIIIb cell divided along the apicalbasal axis,with a lateral-posterior inclination. Whereas the sheath cell was gen-
erated apically, the neuron was generated basally, located between thesheath cell and the glia cell. Around 7.5 min after PIIIb cytokinesis,
the first neurite became visible at the apical neuronal pole, as markedby EB1-GFP. This neurite gave rise to the dendrite that grew toward
the apical transmitting structure where it eventually integrated. Lateron, the neuron began to grow a second, basal neurite that rapidly
elongated posteriorly to become the axon.
Polarization begins normally in the absence of centrioles
To test whether centrosome localization defines the position where the
first neurite forms in vivo, we visualized centrosomes by analyzing theposition of the centriolar marker Asterless (Asl)-YFP17 in sensory neu-rons of nota, fixed at the time in which only the first neurite is present.
As demonstrated before in vitro8, the first neurite and the centrosomewere localized at the same pole of the cell (Fig. 1b). Therefore, in sensory
neurons of the fly notum, as in isolated mammalian and Drosophila neu-rons7,8, centrosome position coincides with the axis of polarization.
Recently, Drosophila mutants have been described that lack centri-
oles in most cells owing to a mutation in either the dsas-4 or dsas-6gene, each required for centriole duplication10,11. Both mutants giverise to adult flies, allowing us to directly test whether centrioles are
required for the establishment of neuronal polarityin vivo. Despitethe fact that in these mutants differentiated neurons appear mor-
phologically polarized10, it is not known whether the establishmentof polarity occurs normally and whether already neuronal precur-
sor cells lack centrioles. When we expressed the centriolar markerAsl-YFP in wild-type flies, we saw two tightly associated fluorescent
spots in each cell of the wild-type nota (Supplementary Fig. 2a,b).These spots were absent in all cells ofdsas-4 and dsas-6nota (Fig.1c
and Supplementary Fig. 2c), indicating that the centrioles were lost.Owing to the comparable phenotype of the two mutants in our stud-
ies, hereafter we will refer to them more generally as dsas mutants.In agreement with the results of others10, we found that dsas maturesensory neurons lacked the primary cilium at the tip of the dendrite,
normally visible in association with the pair of centrioles in wild-typeneurons (Supplementary Fig. 2b,c). Furthermore, mitosis in dsas
PIIIb cells was slowed (compare Fig. 1a with Fig. 1d). Together, theseobservations clearly demonstrate that the centrioles are absent in the
sensory neurons ofdsas mutant pupae.Next, we investigated the establishment of neuronal polarity in dsas
mutants (Fig. 1c,d andSupplementary Fig. 3). We observed that the ori-entation of the neuronal precursor division was conserved. Also, orienta-
tion and sequence of neurite formation were not altered by the absence ofthe centrioles. The first neurite still became the apical dendrite, appeared
from the apical pole of the neuron and projected toward the stimulus-
transmitting structure. The second neurite still became the axon, as canbe inferred from its appearance from the basal pole. Moreover, the apicalextension not only appeared with the same sequence and orientation
as in wild-type neurons, but also with the same timing after the PIIIbmitosis (compare Fig. 1a,b andSupplementary Fig. 1 with Fig. 1c,d and
Supplementary Fig. 3). Thus, in fly sensory neurons, the position andtime of appearance of the first neurite are independent of the presence
of the centriole pair in the neuron itself and in at least three generationsof precursor cells that give rise to the neuron.
Intracellular clustering and morphological breakage
Studies in mammalian hippocampal neurons differentiating in culturehave shown that a bipolar organization of microtubule polymerization
and membrane transport accompanies the establishment of the bipo-
lar axis of growth and is required for neuronal polarization7,8. Hence,the normal polarization of neurons without centrioles (dsas mutants)
suggests that the proper asymmetric organization of microtubule andmembrane transport remains intact independent of the centriole. To
prove this, we analyzed the localization ofDrosophila Arf-like protein3 (Arl3) during the polarization of wild-type and dsas mutants. Arl3
was chosen because it binds both microtubules and secretory vesiclesand mediates polarized membrane transport and fusion18. Analysis ofwild-type nota, fixed at a stage in which only the first sprout is present,
showed that Arl3 immunoreactivity was confined to the apical site ofthe neuron where the first sprout is located (Fig. 2a). In dsas mutants,
this pattern of Arl3 distribution was not altered (Fig. 2b), indicatingthat the absence of centrioles does not interfere with the asymmetric
organization of intracellular membranes.To test whether an asymmetric position of membrane organelles
such as the Golgi, and as a consequence asymmetric membrane traf-ficking, helps determine the position and appearance of the first
neurite, we performed live imaging of Golgi dynamics in wild-type(Fig. 2c and Supplementary Figs. 4a and 5a) and dsas mutant (Fig. 2d
and Supplementary Figs. 4b and 5b) pupae. The distribution of
GFP-labeled Grasp65 (Golgi reassembly and stacking protein 65), aprotein implicated in the establishment and maintenance of the Golgiarchitecture19, showed that Golgi vesicles kept a uniform organization
throughout neuronal differentiation, both in wild-type and in the dsasmutant. These results confirm that intracellular membrane dynamics
are not perturbed in the absence of centrioles and indicate that Golgipolarization is not essential to define the site of initial asymmetry.
Adhesion proteins are symmetry breakage hallmarks
In epithelial cells, the clustering of the adhesion proteins called cad-herins demarcates the future apical domain4. Adhesion molecules
cluster apically in the neural progenitors of the mammalian cortex 20.However, it is not known whether adhesion molecules are inherited by
postmitotic neurons and whether or not they define the polarity axis.To investigate this, we examined the distribution of the E-cadherin
(E-cad) complex. This complex is an apical marker of polarized epithe-lial cells and it is involved in the structural and intracellular rearrange-
ments of different cell types21. Indeed, -catenin (-cat), a part of the
E-cad complex22 and an essential constituent of the signal transduc-tion from cadherins to actin23, was enriched at the tip of the dendrite
in polarized sensory neurons (Fig. 3a). This observation encouragedus to analyze -catGFP distribution and dynamics in time-lapse
mode before and during polarization (Supplementary Fig. 6and Supplementary Video 1). We detected -cat at the apical pole
of the dividing PIIIb cell and of the emerging sheath cell, whereasthe newborn neuron did not show any local -cat enrichment. Yet
around 2.5 min after the completion of cytokinesis, GFP-positive
puncta started to accumulate at the apical-anterior pole, where thefirst sprout would later form. The -cat puncta grew larger, becamemore compact and, during neurite elongation, moved as a U-shaped
structure in the apical-anterior direction. Given the previous visu-alization of-cat at the tip of the dendrite in f ixed nota, we conclude
that the migrating GFP-positive U-shaped structure represents the tipof the elongating dendrite. These results suggest -cat as a very early
landmark of neuronal polarity.To set -cat assembly in relation to the first membrane deforma-
tion of the neuron in as narrow a time frame as possible, we imaged-catGFP together with Partner of Numb fused to RFP (PON-RFP),
(Fig. 3bd). PON is a cell fate, membrane-associated protein, preferen-tially segregating into the neuron during PIIIb division24. It therefore
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a
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Figure 1 In vivodifferentiation of wild-type and dsasmutant sensory neurons. (a) Representative confocal stills taken from movies of living wild-type pupae
expressing EB1-GFP (green) and nuclear His-RFP (red) in sensory organs of the Drosophilanotum. Timing relative to the end of cytokinesis. Shown are PIIIb
cell division (15 min to 2.5 min), apical sheath cell and basal neuron generation (0 min), first neurite formation at the apical neuronal pole (+7.5 min),
first neurite elongation (+12.5 to +95 min) and second neurite formation and elongation at the basal pole (+105 to +115 min). White arrows, dividing PIIIb
cell. Dashed lines, circumference of the neuron. Open arrows, sheath (s) or glial (g) cells. White arrowheads, first sprout. Open arrowheads, second sprout.
(b,c) Confocal images of nota fixed 21 h APF, from wild-type (b) or dsas-4 (c) mutant pupae ubiquitously expressing Asl-YFP to mark centrioles. Dashed
lines, circumference of the neuron. White arrowheads, neuronal centrioles at the base of the first neurite in wild-type pupa. (Asl-YFP spots belonging to the
surrounding apical cells have been deleted; for complete image, see Supplementary Fig. 1a.) Anti-HRP (red) marks the neuron; anti-Cut (blue) marks all
sensory organ cells. (d) Representative confocal stills taken from movies of living dsas-6pupae showing orientation of the neuronal precursor division (25
to 0 min), orientation and sequence of neurite formation (first apical neurite, +10 min; second basal neurite, +147.5 min) in the absence of the centrioles.
White arrows, dividing PIIIb cell. Dashed lines, circumference of the neuron. Open arrows, sheath (s) or glial (g) cells. White arrowheads, first sprout. Open
arrowhead, second sprout. Top panels, surface view: anterior is left, lateral is up. Bottom panels, lateral view: anterior is left and apical is up. Scale bars, 5 m.
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marks the neuronal membrane immediately after mitosis. In addition,
to determine the exact end of cytokinesis and to relate that to -catpolarization and sprout formation even more accurately, we used a
shortened cycle time (43 s) in our image analysis (versus 2.5 min usedfor the previous recording). With these settings, 172 s after cytokine-
sis, as judged by the formation of a continuous neuronal PON-RFPring, we already observed the formation of membrane ruffles at the
contact site of sheath cell and neuron and the appearance of-catGFP
puncta where protrusions coming from the two cells touched eachother. Remarkably, 215 s after cytokinesis, we could already visualize
the appearance of a sprout at the apical pole of the neuron, followedby a progressive enrichment of -catGFP signal around this
sprout (Fig. 3bd and Supplementary Video 2). The timing of theobserved events was very consistent between different experiments.
a
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Arl3 His-RFP FasIII Merge b
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Arl3 Cut-FasIII Asl-YFP Merge
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Wild type
0 min
Figure 2 dsasmutant neurons retain proper intracellular asymmetry during polarization. (a,b) Confocal images of sensory organs fixed 21 h APF, from
wild-type (a) or dsas-6(b) mutant pupae. Dashed lines, neuronal circumference. White arrowheads, anti-Arl3 (indicated in red) marking neuronal
intracellular vesicles polarized in the first neurite. (a) His-RFP (indicated in blue) marks all sensory organ nuclei. Anti-Fasciclin III (anti-FasIII;
indicated in green) marks neuronal membranes. (b) Anti-Cut (blue) marks all sensory organ nuclei. Anti-FasIII (blue) marks neuronal membranes.
Asl-YFP (green), centriolar marker absent in all mutant cells. Top panels, surface view. Bottom panels, lateral view: apical is up. ( c,d) Confocal stills
of living wild-type (c) and dsas-6mutant (d) pupal sensory organs expressing Grasp65-GFP (green) to show Golgi dispersal and His-RFP (red) to mark
sensory organ cell nuclei. Golgi vesicles are dispersed throughout the cytoplasm at the end of the PIIIb mitosis (0 min), undergo fast reassembly into
larger structures with a symmetric distribution (+2.5 min) and keep a uniform organization throughout neuronal differentiation (+17.5 to 67.5 min ( c),
+12.5 to 62.5 min (d)). Dashed lines, neuronal circumference. Top panels, surface view: anterior is left, lateral is up. Bottom panels, lateral view:
anterior is left, apical is up. Scale bars, 5 m.
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The -catGFP always appeared between theneuron and the sheath cell in the image 172 s
after cytokinesis, and the first neuronal sproutalways became visible in the subsequent image
(215 s after cytokinesis).As mentioned before, -cat is a component
of the E-cad complex, which is conservedthroughout evolution and forms adherens
junctions at the apical portion of cell-cell
junctions in epithelial cells. We thereforeinvestigated whether, during neuronal polari-
zation, other members of the E-cad complexshow the same localization as -cat. We
looked at the dynamics of the transmembraneprotein Drosophila E-cad (DE-cad) and the
membrane-associated protein Armadillo
(Arm), the -catenin homolog in flies. Liveimaging analysis of the PIIIb division andneuron differentiation revealed that both DE-cad (SupplementaryFig. 7) and Arm (Supplementary Fig. 8) shared the same distributionpattern and dynamics as -cat.
During the initiation of epithelial polarity, Par-3 (Bazooka (Baz)in flies) induces the proper apical repositioning of the DE-cadherin
clusters4. The apical membrane of mammalian cortical neural progeni-tors contains Par-3 (together with cadherin molecules), but the relation-
ship between Par-3 polarity in neural progenitors and dendriteaxonpolarity in postmitotic neurons is undefined20. We thus wondered
whether assembly of adherens junction components was accompaniedby Baz during the establishment of neuronal polarity. We analyzed
-cat and Baz dynamics together by expressing -catGFP with
Baz-mCherry in the sensory organs of the notum (SupplementaryFig. 9). By time-lapse recording of PIIIb division and neuronal
differentiation, we observed that Baz clusters arose simultaneouslywith -cat and were juxtaposed to -cat clusters during the entire
process. In conclusion, molecules of classical adherens junctions,and the apical cue Baz, mark, precede and accompany polarization of
Drosophila sensory neurons in vivo.In recent studies, cadherin molecules and centrosome localization
were found to be linked during neuronal migration25. We also found-cat and centrioles at the tip of the first neurite in polarized neurons
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Figure 3 -cat assembly precedes and marks the
area of symmetry breakage. (a) Confocal images
of wild-type nota, expressing -catGFP (green)
and nuclear His-RFP (red) in the sensory organs,
fixed 25h APF. White arrowheads, neuronal -cat
at the tip of the dendrite (white arrows). Anti-HRP
(blue) marks the neuron. Top panels, surface
view. Bottom panels, lateral view: apical is up.
(b) Confocal stills, taken every 43 s, of living
wild-type pupa expressing -catGFP (left),
PON-RFP (right, neuronal membrane) and
nuclear His-RFP (right) in sensory organs. White
arrowheads, -catGFP puncta becoming visible
at the apical, anterior neuronal pole (+172 s)
and later concentrated around the growing sprout
(+301 to +559 s). White arrows, PON-RFP
positive first sprout appears at the same pole
(+215 s). Open arrowheads, neuronal membrane.
Surface views: anterior is left; lateral at bottom.
Dashed rectangle indicates area shown in d.
(c) Line plots from each still in b, quantifying
intensity along the sheath cellneuron axis. Black
line, -catGFP intensity. Gray line, His-RFP
intensity, revealing the position of neuronal and
sheath cell nuclei; and PON-RFP intensity, showingthe membrane enrichment in between neuron and
sheath cell. Black arrowheads, -catGFP peaks.
For details of quantification, see Online Methods.
(d) Same stills as in b, with RFP in red and the GFP
signal displayed in a color code ranging from black
through green to white with increase in intensity;
yellow arrowheads, points of high GFP intensity
corresponding to the white arrowheads in b.
Scale bars, 5 m.
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(Supplementary Fig. 2b and Fig. 3a). We therefore investigated the
spatiotemporal relationship between the assembly of adherens junc-tion components and centrosome position at the apical neuronal pole
(Fig. 4). Immediately after the PIIIb division was completed, neuro-nal centrioles were located opposite the plane of cleavage; that is, at
the posterior-basal pole of the neuron. Approximately 30 min later,the centriolar pair moved, clockwise or counterclockwise, toward the
anterior-apical pole (Fig. 4a,b). Time-lapse recording of-cat dynam-
ics in the dsas mutants confirmed normal clustering of adherens junc-tion molecules even in the absence of the centrioles (Supplementary
Fig. 10). Together with our previous observations, these data clearly
show that centrosome repositioning to the apical pole is subsequentto the clustering of adherens junction components and first sprout
formation. In addition, centriole rotation toward the site of adhe-sion complex formation suggests that the newly established punctum
adherens at the apical neuronal pole is already functional, thus pro-moting reorganization of the intracellular machinery.
PtdIns(4,5)P2 enrichment precedes E-cadherin recruitment
Direct binding of phosphatidylinositol-4,5-bisphosphate (PtdIns
(4,5)P2) to Baz recruits Baz to the membrane in Drosophila epi-
thelial cells and neuroblasts26. We thus wondered whether, duringneuronal polarization, asymmetric localization of PtdIns(4,5)P2precedes the formation of the DE-cad complex. By visualizing
PtdIns(4,5)P2 dynamics with pleckstrin homology domainphos-pholipase C1GFP (PH-PLC-GFP)27 (Fig. 5ac), we were already
able to detect, 43 s after cytokinesis and thusearlier than DE-cad complex recruitment,
high enrichment of PtdIns(4,5)P2, at thepole where the first neuronal sprout would
later form. After the onset of sprout forma-tion, PtdIns(4,5)P2 continued to accumulate
around the sprout itself, at a location similarto that of Baz and the DE-cad complex (Fig. 3
a b0 min
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Figure 4 Centriole rotation at the apical pole follows
the assembly of adherens junction components.
(a) Confocal stills of wild-type living pupa
ubiquitously expressing Asl-YFP (green) showing
centriolar dynamics during neuronal polarization.
Cycle time, 1 min. His-RFP (red) marks sensory
organ nuclei. n, neuronal nucleus. White arrows:
centrioles opposite to the plane of cleavagethat
is, at the basal, posterior neuronal poleat the end
of PIIIb cytokinesis (0 min), moving to the apical,
anterior neuronal pole (+6 to +29 min). Surface
views: anterior is left and lateral is up. Scale bar,
5 m. (b) Schematic representation of centriole
rotation toward the anterior-apical neuronal pole.
PON-RFP, His-RFPPH-PLC-GFP
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Figure 5 PtdIns(4,5)P2 enrichment at the place
of first sprout formation precedes and marks
the assembly of DE-cad complex. (a) Confocal
stills of living wild-type pupa expressing
PH-PLC-GFP (left) to mark PtdIns(4,5)P2,
His-RFP (right) to mark sensory organ cell
nuclei, and PON-RFP (right) to mark the
neuronal membrane after completion of
cytokinesis. White arrowheads, PH-PLC-GFP
starting to accumulate at the site of cytokinesis
at +43 s and remaining concentrated in the
growing sprout. White arrows, neuronal sprout
becoming visible at +215 s. Open arrowheads,
neuronal membrane. Surface views: anterior is
left; lateral is up. Dashed rectangle indicates
area shown in c. (b) Line plots from each stillin a, quantifying intensity along the sheath
cellneuron axis. Black line, PH-PLC-GFP
intensity. Gray line, His-RFP intensity, revealing
the position of neuronal and sheath nuclei; and
PON-RFP intensity, showing the membrane
enrichment between neuron and sheath cell.
Black arrowheads, PH-PLC-GFP peaks. For
details of quantification, see Online Methods.
(c) Same stills as in a, with RFP in red and the
GFP signal displayed in a color code ranging
from black through green to white with increase
in intensity; yellow arrowheads, PH-PLC-GFP
clusters corresponding to the white arrowhead
in a. Scale bar, 5 m.
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and Supplementary Figs. 79). These data show that PtdIns(4,5)P2
becomes enriched at the site of future sprout formation in less thanone minute after cytokinesis and, shortly thereafter, adherens junction
components accumulate at the same neuronal pole; they are all stil lpresent when the sprout starts to form.
To demonstrate that the E-cadherin complex is not only a markerbut also of functional importance for sprout formation, we tried to
disrupt this complex by overexpression in precursor cells of a domi-nant negative DE-cadherin, dCR4h-GFP, that lacks a part of the
extracellular domain28. As expected, this caused an inhibition of cell
division (Supplementary Fig. 11), precluding analysis of sprout for-mation in the neuron. To avoid the effect of DE-cad loss on neuronalbirth, we moved to an alternative strategy. We used GFP knocked
into the endogenous DE-cad (shg) locus (DE-cad::GFP) to create aDE-cadGFP fusion protein expressed at endogenous levels29 as a target
for chromophore-assisted laser inactivation (CALI)30. InactivatingDE-cad in the region of future sprout formation immediately after the
neuron was born caused a failure to sprout in three of five homozygousflies but in none of the heterozygous flies that still express 50% non-
targeted DE-cad (Fig. 6).
Mitotic cleavage site defines the symmetry breakage site
Of note, we observed that the place of the first neuronal membranedeformation corresponded to the site of cleavage during neuronal
precursor mitosis. In addition, PtdIns(4,5)P2 is known to be required
at the cleavage furrow during cytokinesis of several cell types3133.Thus, we hypothesized that the cues that polarize the neuronalmembrane before sprout formation are inherited from the cell divi-
sion of the precursor cell. To test this, we analyzed the localizationof two markers of cytokinesis, from the end of PIIIb division on.
We first examined Rho1 (the fly homolog of mammalian RhoA)dynamics because Rho1 is a main component of the midzone and
is required for furrow ingression and constriction during mitosis34.In addition, it regulates actin remodeling spatially, at the position
of adherens junctions in epithelial cells3537. Time-lapse analysis ofRho1-GFP confirmed Rho1 accumulation
at the midzone/midbody in late mitosis.Rho1 remained at the same location in the
a bDE-cad::GFP/+ DE-cad::GFP/DE-cad::GFP
BeforeCALI
6minafterCALI
sh
sh
nsh
n
n
sh
n
Figure 6 CALI inactivation of DE-cad blocks initial bud formation.
(a,b) Confocal stills of living heterozygous (a) and homozygous (b)
DE-cad::GFP(ref. 29) pupae, expressing His-RFP to mark sensory organ
cell nuclei and PON-RFP to mark the neuronal membrane. Pseudocolor
black-blue-green-white indicates increasing RFP intensity. Yellow squares,
region to which CALI inactivation of DE-cad was applied: neuronal sprout
formation was impaired in the homozygous DE-cad::GFPpupa (b, white
arrowhead), whereas it was not affected in the heterozygote (a, white arrow).
Open arrows, sheath (sh) or neuronal (n) cells. Surface views: anterior is
left; lateral at bottom. Scale bars, 5 m.
a
PON-RFP, His-RFPRho1-GFP
86 s
43 s
0 s
+86 s
+215 s
+688 s
b
0
40
80
120
160
0 2 4 6 8 10m
0
40
80
120
160
0 2 4 6 8 10m
0
40
80
120
160
0 2 4 6 8 10m
0
40
80
120
160
0 2 4 6 8 10m
0
40
80
120
160
0 2 4 6 8 10m
0
40
80
120
160
0 2 4 6 8 10m
c
0 s
+86 s
+215 s
+688 s
86 s
43 s
Relativeintensity
Relativeintensity
Relativeintensity
Relativein
tensity
Relativeintensity
Relativeintensity
Figure 7 Mitosis-inherited cortical furrow
molecules are retained in the neuron and mark
the area of symmetry breakage. (a) Confocal
stills of living pupae expressing Rho1-GFP (left),
His-RFP (right) to mark sensory organ cell nuclei
and PON-RFP (right) to mark the neuronal
membrane after completion of cytokinesis.
White arrowheads, Rho1-GFP concentration
during anaphase at the cleavage site (86 to
43 s), which becomes the antero-apical pole
of the neuron after cytokinesis (0 to +86 s)
and colocalizes with the emerging sprout
(white arrows, +215 s). Rho1 remains highly
enriched at the same neuronal pole during
sprout maturation (+688 s). Open arrowheads,
neuronal membrane. Dashed rectangle indicatesarea shown in c. (b) Line plots from each still
in a, quantifying the intensity along the sheath
cellneuron axis of Rho1-GFP (black line).
His-RFP intensity, revealing the position of
the neuronal and sheath nuclei, and PON-RFP
intensity, showing the membrane enrichment
between neuron and sheath cell (gray line).
Black arrowheads, Rho1-GFP peaks. For details
of quantification, see Online Methods. (c) Same
stills as in a, with RFP in red and the GFP signal
displayed in a color code ranging from black
through green to white with increase in intensity;
yellow arrowheads, GFP clusters corresponding
to the white arrowheads in a. Scale bar, 5 m.
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1532 VOLUME 14 | NUMBER 12 | DECEMBER 2011 NATURE NEUROSCIENCE
A R T I C L E S
newborn neuronthat is, at the pole from which the first sprout will
formand stayed there, highly enriched, during sprout maturation(Fig. 7 and Supplementary Video 3). Second, we analyzed dynam-
ics of the pericentriolar protein Aurora-A during the establishmentof neuronal polarity. Aurora-A38 accumulates at the midbody at the
end of cytokinesis39 and is required for neurite growth40 and properaxonal specification41. Live imaging of GFPAurora-A dynamics
confirmed an accumulation of this protein at the midbody duringthe end of cytokinesis and revealed that Aurora-A enrichment main-
tained the same position in the newborn neurons, until a few minutes
after sprout formation (Supplementary Fig. 12 and Supplementary
Video 4). The same pattern of Aurora-A dynamics at the midbody
and at the pole of neuronal sprout formation was detected in the dsasmutant pupae (Supplementary Fig. 13). These results show that, in
immediately postmitotic neurons, polarized accumulation of corticalfurrow markers precedes the enrichment of PtdIns(4,5)P2 and sub-
sequent assembly of-cat and Baz clusters at the pole of symmetryrupture, before the first detectable membrane deformation.
To distinguish whether the site of first sprout formation is deter-mined by the axis of PIIIb division or by the orientation of the neuron
in the surrounding epithelial tissue, we analyzed sprout formation and
DE-cad clustering in a dishevelledmutant (dsh1) where, owing to arandom orientation of the PI division in the epithelial plane, sensoryneurons occupy a random position in this plane (that is, relative to
the anteriorposterior and lateralmedial axis)42. The site of DE-cadaccumulation always occurred on the axis of PIIIb division, between
the sheath cell and the neuron, even if the orientation of the PIIIbmitotic axis with respect to the surrounding tissue was altered (n = 25)
(Supplementary Fig. 14). Moreover, in dsh1 mutants, DE-cadGFP,after clustering at the cleavage plane, showed the same dynamics and
remodeling as in the wild type, indicating that, in dsh1 mutants as well,DE-cadGFP marks the first sprout formation and later localizes at
the tip of the growing dendrite (data not shown). Thus, our results,by showing that dsh1 mutation induces misorientation of the neu-
ronalsheath cell axis without altering neuronal polarization at thecleavage plane, confirm that the site of DE-cad accumulation and first
sprout formation is determined intrinsically by cytokinesis remnants
and not extrinsically by the environment.All in all, our results show that mitosis-inherited furrow molecules
are the earliest landmarks of neuronal polarityin vivo (see model andtime course, Supplementary Fig. 15). We conclude that the spatial land-
mark of neuronal polarity is already defined by mitotic inheritance.
DISCUSSION
The current view on the establishment of neuronal polarity is based
mainly on in vitro studies focusing on the accumulation or activa-tion of proteins in a single neurite of already multipolar cells5. The
data presented here shed light on the mechanisms underlying the
true establishment of neuronal polarity; that is, the generation ofthe first neurite. Previous workin vitro concluded that elongation ofthe first neurite requires the early polarization of membrane organelles
under the master organization of the centrosome7,8. Our workin vivoshowed that centrioles were required neither for the establishment of
the polarity axis nor for the occurrence of proper intracellular organi-zation. We showed, in fact, that absence of centrioles did not affect
intracellular membrane and cytoskeletal organization and dynamics,normally controlled by centrosomes (centrioles and pericentriolar
material). However, the presence of Aurora-A dynamics at the mitoticspindle poles in cells lacking centrioles suggests that pericentriolar
material might organize polar cytoplasm in the absence of centrioles.This is worth highlighting, as it might explain the ability of cells to
divide and differentiate in the absence of centrioles10. Irrespective of
the conserved membrane and cytoskeletal organization in the dsasmutants, our results indicate that a polarized intracellular organi-
zation does not determine the site of appearance of morphologicalpolarity. Instead, the first membrane deformation is marked by corti-
cal specialization, linked to the inheritance of cortical furrow markersat the apical neuronal pole. The molecules we found to be clustered
before first neurite formation have been shown to act in the polariza-tion of non-neuronal cells3,4,41,4347. The loss of sprout formation afteracute inactivation of DE-cad provides functional evidence that the
cadherin complex is indeed required. The site of cortical specializa-tion in the neuron and thus of the first sprout formation is defined by
the cleavage plane even when the orientation of neuronal precursorcell division is altered in respect to the animal body axis. Altogether,
these observations support our model that cadherin-mediated signal-ing, restricted to the apical pole of the neuron corresponding to the
site of the last mitotic cleavage, is functionally important in directingneuronal polarization.
Of note, the apical pole of the sheath cell is opposite the plane ofcleavage, indicating that apical domain and cleavage site do not neces-
sarily coincide in polarized cells. The presence of the apical domain
at the site of cleavage could confer specific properties on the apicaldomain, such as the ability to induce neurite formation.
On the basis of our data and knowledge of cell polarization in other
systems, we propose a model of neuronal polarization (Supplementary
Fig. 15). In this model, the neuron is born with asymmetrically
distributed remnants of the last mitotic cleavage, such as Rho1 orAurora-A. Rho1 induces an increase in PtdIns(4,5)P2 (ref. 48) at the
former cleavage pole. PtdIns(4,5)P2 recruits Baz, E-cad complexesare stabilized, and a punctum adherens forms4,26,44,49. Rho1-induced
myosin II activation generates a cortical tension that is transducedto the plasma membrane through actomyosin tethering to adherens
junctions components, thus promoting the remodeling of the punc-tum adherens and the consequent alteration of cell shapethat is,
the sprout formation
35
. The resultant cortical flow then promotesmicrotubule reorganization and reorientation of intracellular asym-metry to reinforce the polarity axis and, ultimately, allow polarized
neurite growth43.In our work, we thus unravel the hierarchy of events during neuro-
nal polarization in vivo and identify a cortical specialization, acquiredthrough mitotic inheritance, as the earliest landmark of symmetry
breakage. Our work provides the first experimental evidence for alink between cytokinesis and polarization in postmitotic cells other
than budding yeast, as recently suggested1. Both processes, cytokine-sis and sprout formation, require spatially restricted membrane and
cytoskeleton remodeling. Therefore, the core machinery assembledfor cytokinesis may be reused to initiate symmetry breakage at the
same site, before its diffusion or degradation. In fact, we demonstrate
a tight spatial and temporal (3 min elapsing) association betweencytokinesis and first sprout formation. We thus hypothesize that thecytokinesis machinery is adopted by neurons to polarize. This recy-
cling might save energy because it avoids new protein synthesis andassembly in early-born neurons, and/or it might ensure fidelity and
coordination among neighboring cells during polarity induction. Bylinking the establishment of neuronal polarity to the last mitotic event
generating the neuron and to the formation of dynamic cell adhesion,our findings in the sensory neurons of the Drosophila notum can be
easily envisaged as setting the ground for further research. It will bechallenging to explore the mitotic inheritance of neuronal polarity
and the involvement of cell adhesion in the establishment of mam-malian neuronal polarization, in different neuronal subtypes.
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NATURE NEUROSCIENCE VOLUME 14 | NUMBER 12 | DECEMBER 2011 1533
A R T I C L E S
METHODS
Methods and any associated references are available in the onlineversion of the paper at http://www.nature.com/natureneuroscience/.
Note: Supplementary information is available on the Nature Neuroscience website.
ACKNOWLEDGMENTSWe would like to thank C. Gonzalez, F. Calderon de Anda, A. Grtner andF. Feiguin for discussions; M. Morgan for technical help and discussions; T. Lecuit
and R. Levayer for sharing their adapted CALI protocol; and Y. Bellaiche (CurieInstitute), H. Bellen (Baylor College of Medicine), C. Gonzalez (Institut de RecercaBiomedica, Barcelona), T.J.C. Harris (University of Toronto), S. Hayashi (RIKENCenter for Developmental Biology), Y. Hong (University of Pittsburgh), J. Knoblich(Institute of Molecular Biotechnology, Wien), H. Oda (JT Biohistory Research Hall,Takatsuki), M. Rolls (Pennsylvania State University), the Bloomington DrosophilaStock Center and the Developmental Studies Hybridoma Bank for providingus with stocks and reagents. G.P. was supported by a Boehringer IngelheimFoundation scholarship. This work was supported by Katholieke UniversiteitLeuven and Fonds Wetenschappelijk Onderzook-Vlaanderen.
AUTHOR CONTRIBUTIONSG.P.: experimental design, data collection and assembly, data interpretation,manuscript writing. J.G.S.: experimental design, data assembly and interpretation,manuscript writing. S.M.: technical and imaging assistance, data analysis. C.G.D.:leading and coordinating the project, manuscript writing and editing. J.G.S. and
C.G.D.: supervision of the project.
COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.
Published online at http://www.nature.com/natureneuroscience/.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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NATURE NEUROSCIENCE doi:10.1038/nn.2976
ONLINE METHODSDrosophila stocks. All stocks were maintained at 25 C and were raised on
cornmeal-yeast-agar medium. Bloomington Stock Center provided the following
stocks: P{lacW}dsas-4s2214, PBac{PB}dsas-6c02901, P{UAS>Grasp65-GFP},
P{UAS>-cat-GFP}, P{arm-GFP}, P{UAS>Rho1-GFP} andw1 dsh1. P{Gal4}neurP72
was kindly provided by Y. Bellaiche; P{UAS>His2A-RFP}, P{UAS>pon-RFP},
P{UAS>GFP-aur} (encoding GFPAurora-A) by J. Knoblich; P{UAS>Eb1-GFP}
by M. Rolls; P{ubi>asl-YFP} (ubiquitously expressing Asl-YFP) by C. Gonzalez;
P{ubi>shg-GFP} (ubiquitously expressing DE-cadGFP) and P{UAS>shgdCR4h
}by H. Oda; P{UAS>baz-mCherry} by T.J.C. Harris; P{UAS>PLC-PH-GFP} by
H. Bellen; DE-cad::GFPby Y. Hong.
Fluorescence live imaging. Pupae were prepared 20 h APF for time-lapse analy-
sis as reported previously12. Live imaging was performed at 25 C. Images were
acquired on a Radiance Bio-Rad 2100 upright confocal microscope with a 60,
1.4 numerical aperture oil objective lens. Images were acquired every 2.5 min
(Figs. 1a,c, 2c,d and 7a,b), every minute (Fig. 4a) or every 43 s (Figs. 3b, 5a
and 6a). Settings for the imaging were as follows: for 2.5-min cycle time, 0.9-m
steps, 20-m total stack size; for 1-min cycle time, 2.05-m steps, 17.50-m total
stack size; for 43-s cycle time, 1.7-m steps, 11.20-m total stack size. Pinhole
settings were adapted to ensure coverage of all stack information. For each time-
lapse, the pupa was placed under the microscope with the anterior side of the pupa
on the left on the computer screen. For each experiment regarding the different
marker proteins, in wild-type or dsas mutants, at least five independent groupswere checked. For the dsh experiments, 25 groups were analyzed for DE-cad
polarization and 8 groups located in the same notum position were analyzed for
orientation of PIIIb division.
Immunohistochemistry. Nota from pupae 2125 h APF were dissected and
processed as previously described50. Primary antibodies used were mouse anti-
Cut (2B10; Developmental Studies Hybridoma Bank), rabbit anti-HRP (ICN
Biomedicals), rabbit anti-HRP conjugated with Cy5 (Jackson Laboratories),
rabbi anti-Arl3 (a gift from S. Hayashi), mouse anti-FasIII (7G10; Developmental
Studies Hybridoma Bank). All Alexa-coupled secondary antibodies (488, 568 and
647) were from Molecular Probes.
Chromophore-assisted laser inactivation. CALI experiments in DE-cad::
GFP/+ or DE-cad::GFP/DE-cad::GFPpupae bearing GFP fused to the C termi-
nus of endogenous DE-cad29 were performed as previously described30, with
modifications. Around 20 h APF, right after completion of the precursor cell
mitosis and closure of the PON-RFP ring marking the neuronal membrane, CALI
was applied to a region of interest 2.25 m 2.25 m in 20 cycles, at the site of
potential sprout formation, for a total time of 36 s using a 160-mW krypton-
argon laser at 477 and 488 nm with 100% power. The pixel size was 90 nm, pixel
dwell time 2.6 s. We used a 60, 1.4 numerical aperture oil objective lens. For
membrane visualization and spatial orientation, PON-RFP and His-RFP were
expressed under neurP72>GAL4. Absence or presence of sprout formation was
assessed 2, 4 and 6 min after CALI.
Image processing. Images were processed using ImageJ 1.40g (US National
Institutes of Health) and Adobe Photoshop CS2 9.0.2 software. Figures were
assembled with Adobe Illustrator CS2. For each time-lapse, representative
stills are shown at the time indicated, referring to the time of completion of
PIIIb division.
To better reveal differences in intensity of the observed GFP-tagged polarity
markers, an extended color range was chosen. GFP intensities in the image were
coded in a range from black through green to white, displaying the highest val-
ues in white. Images were smoothed once for noise reduction and contrast was
adjusted. The false-color display therefore gives a qualitative representation of dif-
ferences in GFP intensity. To better visualize the dynamics of the mCherry-tagged
Baz, the same approach was taken, and the fluorescent signal was displayed in a
color code ranging with increasing intensity from black through red to white.
To analyze the intensity distribution quantitatively, line profiles along theneuronalsheath cell axis (that is, the axis of PIIIb division), from the surface
and lateral views depicted in the figures, were used. The graphs represent the
intensity distribution across that axis. For the generation of the line profiles, the
average of eight pixels perpendicular to the line itself was used to sample more
pixels. To further reduce noise, the line was smoothed with a running average
of four values.
To clearly visualize the dispersion of Golgi membranes, surface plots of the
intensity distribution of Grasp65-GFP were generated. Relative GFP intensity is
revealed by the peaks height and color, ranging with increasing intensity from
blue through black to orange.
Time-lapse movies were assembled using Volocity 5.0 and Imaris 6.2 software.
50. Gho, M., Lecourtois, M., Geraud, G., Posakony, J.W. & Schweisguth, F. Subcellular
localization of Suppressor of Hairless in Drosophilasense organ cells during Notch
signalling. Development122, 16731682 (1996).