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NATURE NEUROSCIENCE VOLUME 14 | NUMBER 12 | DECEMBER 2011 1525

A R T I C L E S

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.
http://www.nature.com/doifinder/10.1038/nn.2976http://www.nature.com/doifinder/10.1038/nn.2976


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1526 VOLUME 14 | NUMBER 12 | DECEMBER 2011 NATURE NEUROSCIENCE

A R T I C L E S

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

+7.5 min +12.5 min

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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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A R T I C L E S

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

Wildtype

Arl3 His-RFP FasIII Merge b

dsas-6

Arl3 Cut-FasIII Asl-YFP Merge

d

+2.5 min

+12.5 min

+62.5 min

Grasp65-GFP His-RFP

dsas-6c

+17.5 min

+2.5 min

+67.5 min

0 minGrasp65-GFP His-RFP

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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A R T I C L E S

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

d

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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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A R T I C L E S

(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

n

+6 min +19 min

+29 min +49 min +77 min

n

h

0 min

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Centrioles

o

sf+49 min

nn

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nn

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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lativeintensity

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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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A R T I C L E S

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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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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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).

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