Intra-axonal Patterning: Intrinsic Compartmentalization of the Axonal Membrane in Drosophila Neurons

Neuron

Article

Intra-axonal Patterning: IntrinsicCompartmentalization of the Axonal Membranein Drosophila NeuronsTakeo Katsuki,1,2 Deepak Ailani,1,2 Masaki Hiramoto,1,3 and Yasushi Hiromi1,2,*1Department of Developmental Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan2Department of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuoka 411-8540, Japan3Present address: ICND 216, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA*Correspondence: [email protected]

DOI 10.1016/j.neuron.2009.08.019

SUMMARY

In the developing nervous system, distribution ofmembrane molecules, particularly axon guidancereceptors, is often restricted to specific segmentsof axons. Such localization of membrane moleculescan be important for the formation and functionof neural networks; however, how this patterningwithin axons is achieved remains elusive. Here weshow that Drosophila neurons in culture establishintra-axonal patterns in a cell-autonomous manner;several membrane molecules localize to eitherproximal or distal axon segments without cell-cellcontacts. This distinct patterning of membraneproteins is not explained by a simple temporalcontrol of expression, and likely involves spatiallycontrolled vesicular targeting or retrieval. Mobilityof transmembrane molecules is restricted at theboundary of intra-axonal segments, indicating thatthe axonal membrane is compartmentalized bya barrier mechanism. We propose that this intra-axonal compartmentalization is an intrinsic propertyof Drosophila neurons that provides a basis for thestructural and functional development of the nervoussystem.

INTRODUCTION

The process by which different parts of the body acquire distinct

properties such as morphology, function, and molecular distri-

bution is generally called patterning. Likewise, patterning events

that take place within a single cell may be called intracellular

patterning. In the nervous system of vertebrates and inverte-

brates, different regions of axons are often characterized by

differential expression of membrane molecules (Bastiani et al.,

1987; Brittis et al., 2002; Callahan et al., 1995; Dodd et al., 1988;

Kidd et al., 1998; Patel et al., 1987; Rajagopalan et al., 2000a,

2000b; Simpson et al., 2000a, 2000b), suggesting that axons

are ‘‘patterned’’ into intra-axonal segments. For example, in

the ventral nerve cord of Drosophila embryos, axon guidance

188 Neuron 64, 188–199, October 29, 2009 ª2009 Elsevier Inc.

receptor Roundabout (ROBO), and other members of this family,

ROBO2 and ROBO3, accumulate on longitudinal axon tracts,

but are excluded from commissures (Kidd et al., 1998; Rajago-

palan et al., 2000a, 2000b; Simpson et al., 2000a, 2000b)

(Figures 1A and 1C). This specific distribution pattern is due to

the localization of ROBO proteins to distal axon segments of

individual commissural neurons (Kidd et al., 1998; Simpson

et al., 2000a) (Figure 1D). The distribution of another guidance

receptor, Derailed (DRL), which, by contrast, is enriched on

the anterior commissures, provides a second example of this

regulated distribution (Bonkowsky et al., 1999; Callahan et al.,

1995) (Figures 1B–1D). In the spinal cord of mouse embryos,

commissural axons express elevated levels of Robo1 and

Robo2 proteins on the axon segments that have crossed the

floor plate, while an isoform of Rig-1/Robo3 is mostly present

on the precrossing axon segment (Chen et al., 2008; Sabatier

et al., 2004). Thus, the spatial regulation of axon guidance

receptors within an axon appears to be conserved across

species.

Despite the generality of the intra-axonal localization of

membrane molecules, little is known about how such elabo-

rate patterns emerge. These patterns may result from the influ-

ence of extrinsic cues, an intrinsic ability of cells, or both. It is

also elusive how polarized distribution of these membrane

proteins along axons can be established and maintained

without diffusing into a uniform distribution. Here, using

a low-density primary cell culture prepared from Drosophila

embryos, we show that neurons possess an ability to generate

intra-axonal patterns of membrane molecules cell-autono-

mously. ROBO3 and ROBO2 are localized to distal axon

segments, while DRL is localized to proximal axon segments

in a complementary manner. These localization patterns are

not explained by a simple temporal control of receptor expres-

sion, and likely involve spatially controlled vesicular targeting

or retrieval pathways. The temperature-sensitive dynamin

mutant reveals that DRL requires Dynamin-dependent endocy-

tosis for its localization to proximal axon segments, whereas

ROBO3 localization is relatively insensitive to blocking Dyna-

min function. We also show that the exchange of membrane

proteins between the distal and proximal axon segments is

restricted at a medial point of the axon, which may maintain

the compartmentalized distribution of membrane proteins

along axons.

Neuron

Intra-axonal Patterning in Drosophila Neurons

RESULTS

Intra-axonal Localization of Axon Guidance ReceptorsCan Be Generated Cell AutonomouslyWe sought to examine whether Drosophila neurons possess any

intrinsic ability to generate intra-axonal distribution patterns of

membrane molecules, and if so, by what mechanism. To explore

the cell-autonomous properties of Drosophila neurons, we used

a low-density primary cell culture system, in which neurons

extend their axons in the absence of cell-cell contacts. We tested

whether or not neurons placed in culture exhibit a localized distri-

bution of ROBO receptors. Immunostaining revealed that ROBO

A

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H

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J

K

L

B C D Figure 1. Intra-axonal Localization of Axon Guid-

ance Receptors in Cultured Drosophila Neurons

(A–D) Distribution of ROBO3 (A) and DRL (B) axon guid-

ance receptors in the ventral nerve cord of stage 16

Drosophila embryos. The anti-HRP antibody stains all

neuronal surfaces (C). (D) Trajectory of a commissural

neuron illustrating the localization of ROBO3 (magenta)

and DRL (green) within a single axon.

(E–I) Cultured neurons in isolation labeled for ROBO (E),

ROBO2 (F), ROBO3 (G), DRL (H), and BP102 (I) (magenta).

GAP-GFP (green) labels the entire plasma membrane, and

was used as a membrane marker for the quantitative anal-

ysis of localization (see Experimental Procedures).

(J) Overlapping distribution patterns of ROBO3 (magenta)

versus ROBO2 (green).

(K and L) Complementary distribution patterns of ROBO3

(magenta) versus DRL (green) (K) or BP102 (green) (L).

Throughout figures, arrowheads pointing downward and

upward indicate the boundaries of distal and proximal

localization, respectively. Right-most panels, fluores-

cence intensity profiles along axons. Scale bars, 10 mm.

is distributed uniformly along axons, suggesting

that ROBO requires extrinsic signals for its

distal localization pattern (Figure 1E, Table 1).

By contrast, ROBO2 and ROBO3 were localized

to distal axon segments in many neurons, with

a discrete boundary at a medial point in the

axonal process (Figures 1F and 1G, Table 1).

Likewise, DRL protein and the antigen of

a monoclonal antibody BP102 (Seeger et al.,

1993) were also localized cell-autonomously to

the proximal region of axons (Figures 1H and

1I, Table 1). Thus, we conclude that Drosophila

neurons possess an ability to create intra-

axonal localization patterns via a cell-intrinsic

mechanism (or mechanisms).

Intra-axonal Localization of GuidanceReceptors Shares a Common BoundaryTo unveil the mechanism for this intrinsic intra-

axonal patterning, we first asked how many

distinct localization patterns can be formed in

a single axon. We reasoned that if each localized

molecule is based on a distinct localization

mechanism, there could be as many distinct

localization patterns and boundaries as the number of different

molecules. Alternatively, if multiple molecules employ the same

mechanism or related mechanisms, their localization patterns

could share a common boundary. To test this, we visualized the

distribution of multiple receptors simultaneously using double im-

munostaining.WhenROBO2andROBO3weredetectedbydouble

staining, their distal localization pattern overlapped and shared

a common boundary (Figure 1J). Moreover, the distal pattern of

ROBO3 and the proximal pattern of DRL (or BP102) were comple-

mentaryandtheir boundaries largelycoincided (Figures 1K and1L).

These results suggest that intra-axonal patterning may involve

a mechanism that generates or maintains multiple molecules.

Neuron 64, 188–199, October 29, 2009 ª2009 Elsevier Inc. 189

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Intra-axonal Patterning in Drosophila Neurons

Localization Patterns of Receptors Are Not Determinedby Their Temporal Expression ProfilesA simple mechanism for intra-axonal localization of guidance

receptors would be to control distribution by the temporal

expression profile of the receptors at the growth cone during

axonal elongation (Dodd et al., 1988; Keleman et al., 2002). For

example, the complementary distribution pattern of DRL and

ROBO3 could be generated if the expression of DRL and

ROBO3 switch at a certain time point in axonal elongation;

DRL may be expressed during the earlier stages of axonal elon-

gation, whereas ROBO3 may be expressed only after the

expression of DRL has been turned off. This hypothesis implies

that the spatial pattern along the proximal-distal axis of the

axon is generated from information on the time axis.

To test this possibility, we manipulated the timing of receptor

expression using GAL4/UAS-based systems. When EGFP-

tagged receptors (hereafter referred to as ROBO-EGFP, etc.)

were expressed throughout axonal elongation under the control

of elav-GAL4, EGFP-tagged receptors showed localization

patterns similar to those of endogenous proteins; ROBO-EGFP

was uniformly distributed, ROBO2-EGFP and ROBO3-EGFP

were localized to the distal axon segment, and DRL-EGFP was

localized to the proximal axon segment (Figures 2A–2D, Table 2).

Even when ROBO3-mRFP and DRL-EGFP were simultaneously

expressed in the same cell, they showed complementary locali-

zation patterns with similar or identical boundary positions (Fig-

ures 2E–2G). Complementary localization of tagged ROBO3 and

DRL molecules was also verified in vivo, by coexpression in

a subset of identified neurons under the control of a GAL4 driver

(Figure S1 available online). These results indicate that localiza-

tion is not determined by the temporal profile of receptor expres-

sion at the transcriptional level.

Because the above result does not rule out the possibility that

localization is determined by temporal control at the posttran-

scriptional level, we next used an expression system that can

switch on the expression of transgenic receptors by a brief

heat pulse (see Experimental Procedures for details). This

system enables us to delay expression, including at the posttran-

Table 1. Localization Patterns of Receptors in Cultured

Drosophila Neurons

Localization Patterns (%)

Molecule

Neurons

Scored N.D. Uniform Distal Proximal

Position of

Boundary (mm)

ROBO 131 41.2 46.6 2.3 9.9 41.1 ± 17.3

ROBO2 156 82.7 5.8 10.9 0.6 25.5 ± 17.9

ROBO3 119 63.0 10.9 26.1 0 24.7 ± 11.4

DRL 109 79.8 12.8 0 7.3 31.4 ± 14.8

BP102 163 8.0 26.4 0 65.6 24.6 ± 14.6

Drosophila embryonic neurons were immunostained after 24 hr in culture,

and fluorescence signals along axons were examined for their distribu-

tion patterns. N.D., not detectable. Position of boundary is indicated as

the distance from soma (average ± SD). N.B.: When visually assessed,

about 30% of neurons showed BP102 staining localized in a middle

region of the axon; however, the majority (�80%) of them were classified

into ‘‘Uniform’’ pattern by our quantification algorithm. See Experimental

Procedures for details of quantification.

190 Neuron 64, 188–199, October 29, 2009 ª2009 Elsevier Inc.

scriptional level, to any time point in axonal elongation. Expres-

sion was induced in neurons that had extended axons for 1

day, and then the distribution of receptors was re-examined after

24 hr of receptor expression (Figure 2H). If timing of expression is

the determinant of the localization patterns, these receptors

should only be detected in the distal axon segments that elon-

gated after the heat pulse. In contrast to this expectation,

DRL-EGFP was localized to proximal axon segments, showing

that a proximal localization pattern can be generated irrespective

of the timing of expression (Figures 2I–2K). The localization

pattern of distally localized ROBO3-EGFP also did not match

its expression profile; its boundary was located more proximal

to the cell body than the site where the tip of the axon was

located at the time of receptor induction (Figures 2L–2N).

These results demonstrate that the temporal profile of

receptor expression at the growth cone is not essential for

receptor localization. We thus hypothesize that this localization

is achieved by compartmentalization of an axon; a single axonal

process is divided into two compartments, proximal and distal,

each of which can accommodate specific membrane molecules,

and distribution of membrane molecules to each compartment is

determined via compartment-specific trafficking pathways.

Dynamin-Dependent Endocytosis Is Requiredfor the Localization of DRLCompartment-specific distribution of membrane proteins can be

achieved via compartment-specific targeting, retrieval, or both.

To test whether retrieval from the plasma membrane via endocy-

tosis is involved in the formation of intra-axonal localization

patterns, we examined the distribution of guidance receptors

in a temperature-sensitive Dynamin mutant called shibirets1

(shits1). The shibire (shi) gene product Drosophila Dynamin

(dDyn) is known to regulate endocytosis in a variety of fly tissues,

and shits1 animals show defects in endocytosis at temperatures

29�C or higher (Guha et al., 2003; Kosaka and Ikeda, 1983a;

Kosaka and Ikeda, 1983b). Because complete loss of Dynamin

function prevents axonal growth, axons were first allowed to

grow at the permissive temperature (22�C) for 24 hr, and then

the temperature was shifted to restrictive conditions (29�C,

31�C, or 33�C), at which point the expression of ROBO3-EGFP

or DRL-EGFP was initiated by the hormone-inducible Gene-

Switch system (Osterwalder et al., 2001) (see Experimental

Procedures). This enabled us to examine the distribution of

receptors that are synthesized under reduced Dynamin function.

At the permissive temperature, DRL was localized to proximal

segments in around 80% of neurons in both wild-type and shits1

backgrounds. At nonpermissive temperatures (29�C and 31�C)

the percentage of neurons with proximal localization decreased

by 60% in shits1 backgrounds (Figures 3A, 3B, and 3F, and Table

S1). At 33�C, both control and experimental conditions showed

significant decrease in the number of neurons with proximal

pattern compared to the permissive temperature, which may

be due to the intrinsic sensitivity of Dynamin proteins to high

temperature (Guha et al., 2003) (Figure 3F). The change in the

distribution of DRL-EGFP at restrictive temperatures is likely to

be on the plasma membrane, since immunostaining for DRL

under nonpermeabilized conditions (surface labeling) also

resulted in uniform distribution (data not shown). When Dynamin

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Intra-axonal Patterning in Drosophila Neurons

function was restored by down-shifting the temperature to

a permissive condition (a 5 hr chase), the proximal localization

pattern increased by about 30% (data not shown). These results

suggest that the proximal localization of DRL on the axonal

membrane requires Dynamin-dependent endocytosis.

In contrast to DRL, the distal localization of ROBO3 was only

mildly affected by the shits1 mutation: the number of neurons

A

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NML

B

C

D

Figure 2. Localization Patterns of Receptors

Are Not Determined by Their Temporal Expres-

sion Profiles

(A–E) Receptors localize even if they are constitutively

expressed under the control of elav-GAL4. (A–E)

Neurons after 24 hr in culture. (A) ROBO-EGFP, (B)

ROBO2-EGFP, (C) ROBO3-EGFP, and (D) DRL-

EGFP (green) were continuously expressed from the

onset of neuronal differentiation using elav-GAL4.

Membrane-targeted myr-mRFP (magenta) was coex-

pressed to facilitate visualization of morphology and

quantification of localization. Right-most panels, fluo-

rescence intensity profiles along axons. (E–G), DRL-

EGFP and ROBO3-mRFP show complementary local-

ization patterns, and the positions of their boundaries

correlate with each other. (E) DRL-EGFP (green),

ROBO3-mRFP (magenta), and myr-EBFP2 (not

shown) were simultaneously expressed under the

control of elav-GAL4. Membrane-targeted myr-

EBFP2 was used to facilitate quantification of localiza-

tion. Right-most panel, fluorescence intensity profiles

along the axon. (F) Higher magnification of the

boundary region indicated with a rectangle in (E). (G)

Positions of the boundary of DRL-EGFP and

ROBO3-mRFP in each neuron are plotted as the

distance from soma. In about 70% of neurons the

boundaries of DRL-EGFP and ROBO3-mRFP coin-

cided within the range of 10 mm (gray region). Neurons

whose boundaries differ more than 10 mm were found

to have bright side branches juxtaposing the main

axon, a condition that our current algorithm cannot

handle well. (H–N) Expression of DRL-EGFP (I–K)

and ROBO3-EGFP (L–N) was induced after 24 hr in

culture by using a heat-shock-triggered induction

system (H). (I and L) Just before induction. (J and M)

Twenty-four hours after induction. (K and N) Intensity

profiles of EGFP fluorescence along the axon in (J)

and (M). Arrows indicate the position of the growth

cone at the onset of induction. Asterisks indicate the

growth cone 24 hr after induction. Scale bars, 10 mm,

except for (F), for which scale bar = 2 mm.

that show distal localization of ROBO3

decreased by 7%, 10%, and 20% at 29�C,

31�C, and 33�C, respectively (Figures 3C,

3D, and 3F, and Table S1 available online).

At restrictive temperatures some neurons

categorized as ‘‘uniform distribution’’

showed bright punctate signals of ROBO3-

EGFP along the entire length of axons

(Figure 3E). These punctate signals seen by

EGFP fluorescence were not labeled by

surface labeling using anti-ROBO3 antibody,

suggesting that they are likely to be vesicles

or aggregates that are inaccessible from the surface (Figure 3E).

About half of the neurons that were classified as uniform distribu-

tion by the quantitation of EGFP fluorescence showed distal

localization under the surface labeling condition, and thus the

severe reduction of Dynamin function at 33�C causes only

10% reduction of the distal ROBO3 pattern generated on the

cell surface. These results suggest that the formation of distal

Neuron 64, 188–199, October 29, 2009 ª2009 Elsevier Inc. 191

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Intra-axonal Patterning in Drosophila Neurons

localization of ROBO3 is largely independent of Dynamin

function.

We next addressed the possibility of selective targeting of DRL

to the membrane. The dependence of DRL on shi function for

endocytosis enabled us to design an experiment to distinguish

between directed and uniform targeting. We reasoned that if

DRL is preferentially targeted to the proximal segment, proteins

newly appearing on the cell surface should exhibit proximal

localization even when endocytosis is blocked. To test this, the

distribution of DRL-EGFP in shits1 mutant was examined at

2 hr, 4 hr, and 6 hr after the temperature was shifted to a restric-

tive condition (29�C). To assay only newly synthesized proteins

appearing on the cell surface, the expression of DRL-EGFP

was initiated by the GeneSwitch system simultaneously with

shift up to the restrictive temperature, and DRL-EGFP on the

axonal surface was detected by surface labeling. At 2 hr from

DRL-EGFP induction, a large fraction (64%) of neurons exhibited

proximal localization pattern, even in shits1 mutant (Figure 3G).

This suggests that newly synthesized molecules appear prefer-

entially on the proximal axonal membrane. The severe effect of

compromised Dynamin function on DRL localization manifested

only after longer chase periods; at 4 hr from DRL-EGFP induc-

tion, shits1 cells showed a significant reduction in the number

of proximal localizations, with further reduction at 6 hr (Fig-

ure 3G). This likely reflects a specific targeting property of

DRL, because similar experiments using ROBO3 revealed that

ROBO3-EGFP was distally localized at all time points (Fig-

ure 3G). These results suggest that there is a preferential target-

ing of DRL to the proximal axon compartment, although this

does not exclude the possibility that DRL is also selectively

retrieved from the distal axon compartment.

Mobility of Membrane Molecules Is Restrictedat the Compartment Boundary by a Diffusion BarrierMechanismWhile compartment-specific retrieval and targeting pathways

likely contribute to the establishment and/or maintenance of

the compartmentalized distribution of receptors, an additional

effective mechanism for pattern maintenance over time is the

mobility restriction of proteins on the fluid plasma membrane,

either within the compartment or at the compartment bound-

Table 2. Localization Patterns of EGFP-Tagged Receptors in

Cultured Drosophila Neurons

Localization Patterns (%)

Molecule

Neurons

Scored Uniform Distal Proximal

Position of

Boundary (mm)

ROBO-EGFP 108 67.6 4.6 27.8 25.8 ± 11.2

ROBO2-EGFP 98 2.0 98.0 0 23.8 ± 9.8

ROBO3-EGFP 120 2.5 97.5 0 22.4 ± 8.7

DRL-EGFP 99 16.2 0 83.8 31.8 ± 14.8

Drosophila embryonic neurons 24–36 hr in culture expressing EGFP-

tagged receptors and myr-mRFP under the control of a panneuronal

driver elav-GAL4 were imaged live, and fluorescence signals along axons

were examined for their distribution patterns. Position of boundary is indi-

cated as the distance from soma (average ± SD). See Experimental

Procedures for details of quantification.

192 Neuron 64, 188–199, October 29, 2009 ª2009 Elsevier Inc.

aries. Compartmentalized membrane proteins may become

immobile by being anchored to the cytoskeleton (Garrido et al.,

2003; Pan et al., 2006), or their mobility could be restricted at

certain locations on the membrane by a barrier mechanism

(Kobayashi et al., 1992; Nakada et al., 2003; Takizawa et al.,

2000; Winckler et al., 1999). To test whether or not localized

molecules are mobile, we measured the mobility of ROBO3-

EGFP and DRL-EGFP, which are localized to distal and proximal

axon segments, respectively, using fluorescence recovery after

photobleaching (FRAP) (Lippincott-Schwartz et al., 1999).

When ROBO3-EGFP fluorescence was bleached in a part of

the distal axon segment, the fluorescence recovered to the

bleached area from both ends (Figures 4A–4D). Similar results

were obtained with DRL-EGFP in proximal axon segments

(Figures 4E–4H). These results suggest that these molecules

are laterally mobile within an axonal compartment.

We next asked whether or not a barrier exists that could retain

membrane molecules within one axon compartment. To this

end, we examined whether CD8-GFP, a model transmembrane

protein that labels the entire plasma membrane of Drosophila

neurons (Lee and Luo, 1999), can move from one axon segment

to another. CD8-GFP and ROBO3-mRFP were coexpressed in

neurons in culture, and photobleaching of CD8-GFP was per-

formed in regions neighboring the boundary of ROBO3-mRFP

(Figure 4I–4K). The recovery at the side abutting the boundary

was significantly slower than that at the side away from the

boundary, indicating that the movement of CD8-GFP across

the compartment boundary is restricted (Figure 4K–4M). Similar

mobility restriction was observed with ROBO-EGFP and a GPI-

linked GFP (GFPgpi, Greco et al., 2001), but not with GAP-

GFP, which is targeted to the inner membrane (Figure 4N–4P).

These data suggest that the exchange of transmembrane

proteins between axon segments is generally limited at the

boundary region.

To gain further insight into the nature of this constraint, we per-

formed spot-size (�1 mm2) FRAP experiments on CD8-GFP

along the axon (Figures 5A and 5B). In contrast to the quick

recovery at locations away from the boundary of ROBO3-

mRFP, the region in proximity of the boundary exhibited dimin-

ished levels of recovery, with both a larger immobile fraction

and a longer half-recovery time (Figures 5C and 5D). Such

regions of reduced mobility were confined to within 10 mm of

axon length around the boundary (Figures 5E and 5F). These

data suggest that a diffusion barrier, which is thought to hinder

the mobility of molecules through the anchoring and friction-

like effects of membrane proteins (Nakada et al., 2003), exists

at the boundary region of intra-axonal compartments. This

barrier may contribute to the maintenance of intra-axonal

patterns of guidance receptors by limiting the exchange of

membrane proteins between distinct axon segments, and may

also serve as a positional cue for the specification of intra-axonal

compartments.

Presynaptic Proteins Accumulate to the Distal AxonCompartmentThe formation of the functional neuronal circuit relies on correct

axonal wiring and the subsequent establishment of synaptic

connections. In the Drosophila ventral nerve cord, synaptic

Neuron

Intra-axonal Patterning in Drosophila Neurons

A

B

C

D

E

F G

Figure 3. Dynamin-Dependent Endocytosis

Is Required for the Localization of DRL

(A–D) Distribution of DRL-EGFP (A and B, green)

and ROBO3-EGFP (C and D, green) expressed in

a control (A and C) or in shits1 background (B and

D) at a nonpermissive temperature (29�C). Mem-

brane-targeted myr-mRFP (magenta) was coex-

pressed to facilitate visualization of morphology

and quantification of localization. Right-most

panels, fluorescence intensity profiles along the

axons.

(E) Distribution of ROBO3-EGFP (green) and

surface distribution of ROBO3-EGFP (cyan) in

shits1 mutant at 33�C. Arrows indicate bright punc-

tate signals of ROBO3-EGFP that are not labeled

under the nonpermeabilized condition.

(F) Localization patterns of DRL-EGFP and

ROBO3-EGFP in shits1 experiments. The numbers

22, 29, 31, and 33 indicate the incubation temper-

ature (�C) after the induction of EGFP-tagged

reporter genes. The number of neurons examined

for each condition is indicated below the bars.

***p < 1.0e�10, **p < 1.0e�7, *p < 1.0e�3, no

asterisk: p > 0.02; G-test.

(G) Time course of the percentage of neurons that

show proper localization patterns of newly synthe-

sized protein in shits1 mutant background at a

restrictive temperature (29�C). DRL-EGFP and

ROBO3-EGFP on the plasma membrane were de-

tected by surface labeling. In a shits1 mutant back-

ground, the percentage of neurons that show

proximal localization of DRL-EGFP decreases

with time, whereas controls do not show signifi-

cant differences between different time points.

Due to the weak expression of myr-mRFP at

2 hr, images were visually analyzed (see Experi-

mental Procedures). The number of neurons

examined for each condition is indicated below

the bars. ***p = 6.8e�7, **p = 0.0045, *p = 0.041,

no asterisk: p > 0.1; G-test. The statistical signifi-

cance between control and shits1 of DRL at each

time point is p = 6.3e�4, p = 3.2e�10, and

p = 3.7e�15 (G-test), for 2 hr, 4 hr, and 6 hr,

respectively.

Scale bars, 10 mm.

proteins such as synaptotagmin and synapsin are localized to

the distal axon segment of individual neurons, and synapse

formation takes place mainly on the longitudinal axon tracts (Fur-

rer et al., 2007; Littleton et al., 1993; Lohr et al., 2002; Sanchez-

Soriano et al., 2005). This raises a possibility that intra-axonal

compartmentalization could also be employed to provide spatial

information for synaptic differentiation. To test this idea, we

examined the distribution of EGFP-tagged synaptic vesicle

proteins in cultured neurons. A postsynaptic protein Discs-Large

(Dlg-YFP) exhibited a uniform distribution along axons (Figure 6A,

Table S2). In contrast, presynaptic proteins synaptotagmin (syt-

eGFP) and n-synaptobrevin (n-syb-eGFP) were localized to the

distal axon segment, and the boundary of syt-eGFP or n-syb-

eGFP localization largely coincided with that of ROBO3-mRFP

(Figures 6B and 6C, Table S2). This suggests that the distal

axon compartment can accommodate not only axon guidance

receptors but also presynaptic proteins. Each compartment

may serve as a platform for various intracellular events, for

example by tethering synaptic vesicles to the target membrane

via Rab and its effectors (Zerial and McBride, 2001), causing

the retention of n-syb-eGFP and syt-eGFP in the distal segment.

We propose that the intra-axonal compartment is a fundamental

structure of Drosophila neurons that could contribute to both the

structural and functional development of the nervous system.

DISCUSSION

Development of the nervous system depends on the expression

of various axon guidance molecules; however, relatively little is

known about how their expression pattern is controlled in space

and time (Dickson, 2002). It is believed that extrinsic factors such

as signals from midline cells or floor plate cells play instructive

roles in regulating the expression of the guidance receptors on

growing axons, because the localization patterns on axons

highly correlate with the position of these cells (Dickson and

Gilestro, 2006). In mice, it has been reported that responsiveness

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Intra-axonal Patterning in Drosophila Neurons

of axons to guidance cues changes in the presence of the floor

plate (Shirasaki et al., 1998), and a ligand expressed at the floor

plate is required for the restricted expression of guidance recep-

tors (Long et al., 2004). On the other hand, not much work has

been devoted to unveiling the roles of cell-intrinsic programs in

regulating the intra-axonal localization of guidance receptors.

Cell-Intrinsic and -Extrinsic Pattern Generationwithin the AxonWe have described here a patterning phenomenon that takes

place within single axonal processes as a cell-intrinsic event.

This patterning involves compartmentalization of the axonal

membrane with a diffusion barrier located at a medial point of

the axon. Our data suggest that this patterning ability is a funda-

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LKI

J

M

PON

B E F Figure 4. Transmembrane Proteins Are Mobile

within the Compartment, but Are Less Mobile

across the Compartment Boundary

(A–D) FRAP experiments on ROBO3-EGFP (green) local-

ized to the distal axon segment (A). (B) The region indi-

cated with a yellow polygon was bleached. Recovery of

fluorescence was monitored in three regions indicated

by circles at 10 s intervals, and plotted in (C). (D) Average

recovery of fluorescence at 60 s after bleaching. The

recovery rates in distal-to-proximal (1) and proximal-to-

distal (3) directions were indistinguishable.

(E–H) FRAP experiments (same as those in A–D) on DRL-

EGFP (green). n = 32 (D), n = 23 (H).

(I–M) FRAP experiment of a transmembrane protein, CD8-

GFP, at the boundary of ROBO3-mRFP. (K) CD8-GFP was

bleached in the region outlined in yellow. Recovery of fluo-

rescence was monitored at 10 s intervals in the areas indi-

cated by circles, and plotted in (L). (M) Average recovery

of fluorescence at 60 s after bleaching. The movement

of CD8-GFP from the distal to proximal compartment

(location 2) and vice versa (location 3) is restricted.

(N–P) FRAP experiments of ROBO-EGFP (N), GFPgpi (O),

and GAP-GFP (P). Restricted mobility across the

boundary was observed with ROBO-EGFP (N) and

GFPgpi (O), but not with GAP-GFP (P). Numbers corre-

spond to the positions along axons as indicated in the

schematic neuron in (M), in which green and magenta

indicate the distributions of CD8-GFP, ROBO-EGFP,

GFPgpi, or GAP-GFP and ROBO3-mRFP, respectively.

For CD8-GFP (M), n = 18, for locations 1 and 2, and n =

25 for locations 3 and 4. For ROBO-EGFP (N), n = 22 for

locations 1 and 2, and n = 25 for locations 3 and 4. For

GFPgpi (O), n = 14 for locations 1 and 2, and n = 14 for

locations 3 and 4. For GAP-GFP (P), n = 19, for locations

1 and 2, and n = 14 for locations 3 and 4.

Error bars represent SEM. ***p < 0.00001, **p < 0.0001,

and *p < 0.005; Wilcoxon signed rank test. Scale bars,

10 mm.

mental property of Drosophila neurons,

because the compartment-specific localization

of GFP-tagged receptors can be observed in

the majority (>90%) of neurons. In the CNS of

Drosophila, more than 90% of neurons project

their axons to the contralateral side of the

nervous system, and the width of the commis-

sural segment or precrossing segment of those

neurons is 20–40 mm, which parallels the length of the proximal

compartment observed in vitro. This raises the possibility that

the intrinsic patterning ability of neurons may serve as the basis

of generating the intra-axonal localization of guidance molecules

in vivo.

In addition to these intrinsic abilities of neurons, our results

suggest that extrinsic factors may also contribute to the intra-

axonal patterning, because not all ROBO receptors examined

in this study recapitulated the localization patterns observed

in vivo. All three members of ROBO family receptors are localized

to distal axon in vivo (Kidd et al., 1998; Rajagopalan et al., 2000a,

2000b; Simpson et al., 2000a, 2000b). Whereas ROBO2 and

ROBO3 retained the ability to localize distally when isolated in

culture, ROBO was uniformly distributed along axons under

194 Neuron 64, 188–199, October 29, 2009 ª2009 Elsevier Inc.

Neuron

Intra-axonal Patterning in Drosophila Neurons

such conditions. Localization of ROBO may require extrinsic

signals that are absent in our culture system. One of the candi-

date extrinsic factors are the midline cells, which lie on an axonal

region where ROBO expression is downregulated in vivo (Dick-

son and Gilestro, 2006). It is also possible that the location of

the compartment boundary determined by the intrinsic mecha-

nisms is refined by extrinsic signals. It would be interesting to

test whether contact with midline cells in culture can induce

distal localization of ROBO, or alter the position of the boundary.

Mechanism of Pattern Generation and MaintenanceIt has been commonly suggested that axon guidance receptors

are targeted to the growth cone, and intra-axonal localization

A D

E

F

B

C

Figure 5. The Mobility of Transmembrane Proteins

at the Boundary Region Is Restricted by a Diffusion

Barrier Mechanism

(A and B) FRAP experiments performed on CD8-GFP with

�1 mm2 spot-size bleaching. ROBO3-mRFP was coex-

pressed with CD8-GFP to visualize its boundary of locali-

zation. (C) Representative images of FRAP at locations 1,

12, and 28 indicated in (A). Note that compared to location

1 and 28, recovery of fluorescence is significantly slower at

location 12, where the boundary of ROBO3-mRFP locali-

zation resides. Quantified data were plotted on (D), and

fitted to single exponential curves (see Experimental

Procedures). (E) Half-recovery time (t1/2) at each location

in (A) plotted as a function of the distance from the soma.

Barrier effect is found in the middle region of the axon,

but not at its base. (F) Average half-recovery time of

CD8-GFP plotted as a function of the distance from the

boundary of ROBO3-mRFP (positive is toward the axon

tip and negative is toward the cell body). Means and

SEM values for 7 mm windows were calculated at 2 mm

intervals along axons. The total number of data points is

196, derived from 15 different neurons. (*), larger than the

total average, p < 0.0003; Mann-Whitney U test. Scale

bars, 10 mm for (A) and (B), and 1 mm for (C).

A

B

C

Figure 6. Presynaptic Proteins Localize to the

Distal Axon Compartment

(A–C) Localization patterns of synaptic proteins in

cultured Drosophila neurons that show distal localiza-

tion of ROBO3-mRFP. Dlg-YFP (A), syt-eGFP (B), and

n-syb-eGFP (C) (green) are coexpressed with ROBO3-

mRFP (magenta) using elav-GAL4. Second-right-

most panels, fluorescence intensity profiles along the

axons. Right-most panels, the position of the

boundary for green and magenta channels of each

neuron is plotted as a distance from soma (mm) in the

ordinate and the abscissa, respectively. Scale bars,

10 mm.

patterns of guidance receptors reflect temporal

profiles of receptor expression at the growth

cone during axonal extension (Dodd et al.,

1988; Keleman et al., 2002). We demonstrated

here that intra-axonal localization patterns that are evident in

our culture condition can form regardless of the timing of

receptor expression. Although this result does not rule out the

involvement of temporal control of expression during axon navi-

gation in vivo (Rajagopalan et al., 2000b; Simpson et al., 2000b),

it suggests that critical mechanisms for the intra-axonal localiza-

tion of receptors described here are compartment-specific traf-

ficking pathways. One such trafficking mechanism could involve

local translation or targeted membrane transport, which can

specifically deposit membrane proteins to either the proximal

or distal membrane compartment. It is also possible that

membrane proteins are selectively retrieved from one compart-

ment through endocytic pathways.

Neuron 64, 188–199, October 29, 2009 ª2009 Elsevier Inc. 195

Neuron

Intra-axonal Patterning in Drosophila Neurons

Our time course experiment in shits1 mutant backgrounds

suggests that DRL is preferentially targeted to the proximal

compartment. We have also shown that the correct intra-axonal

localization of DRL requires Dynamin-dependent endocytosis;

however, at present we cannot distinguish whether or not the

endocytosis of DRL is compartment specific. Because our

FRAP experiments on CD8-GFP suggest that the barrier

between the proximal and distal axon compartments does not

completely block the movement of membrane proteins between

the compartments, it is possible that the Dynamin-dependent

endocytosis is required to remove DRL that leaks into the distal

compartment, serving to maintain the pattern generated by tar-

geting. Alternatively, endocytosis itself may be compartment

specific, contributing to the establishment of the pattern.

In contrast to DRL, ROBO3 does not appear to require shibire

function for its localization, demonstrating the presence of differ-

ential trafficking mechanisms for DRL and ROBO3. Due to this

shi-independence of ROBO3, it is not possible to conclusively

demonstrate the presence of preferential targeting of ROBO3

by performing a time course experiment. Even if there is prefer-

ential targeting, it is likely that ROBO3 also needs to be removed

from the incorrect compartment, because ROBO3 shows a level

of lateral mobility on the axon similar to that of DRL. Since

ROBO3 localization is largely independent of Dynamin function,

such a retrieval pathway must be based on Dynamin-indepen-

dent mechanisms. While the complementary localization pat-

terns of DRL and ROBO3 suggests that intra-axonal com-

partments are fundamental units for localization of multiple

molecules, molecular mechanisms for generating or maintaining

their compartmental localization could be diverse.

Another critical issue raised in the previous studies in vivo is

how the intra-axonal localization of guidance receptors is main-

tained over time (Keleman et al., 2002). If the guidance receptors

are freely diffusible on the axonal membrane, they may spread

along the axon, leading to a uniform distribution. Our FRAP

experiments in cultured neurons revealed that localized recep-

tors (ROBO3-EGFP and DRL-EGFP) are indeed mobile within

the intra-axonal compartment. Although the mobility of these

localized receptors across the compartment boundary was not

directly measurable, the mobility of several transmembrane

proteins (ROBO-EGFP and CD8-GFP) and lipid-anchored pro-

tein (GFPgpi) that distribute along the entire axon length was

significantly restricted at the compartment boundary. This

restriction is likely due to the diffusion barrier that spans over

a 10 mm axon length around the boundary. We propose that

this barrier is a part of the mechanisms that maintain the pattern

of compartment-specific membrane proteins, as shown in

different subcellular regions such as the tight junction of epithe-

lial cells (Shin et al., 2006), the posterior ring of sperm (James

et al., 2004), the cleavage furrow of dividing yeast and mamma-

lian cells (Schmidt and Nichols, 2004; Takizawa et al., 2000), and

the initial segment of mammalian neurons (Kobayashi et al.,

1992; Nakada et al., 2003; Winckler et al., 1999). We did not

detect significant barrier effect on GAP-GFP, which resides in

the inner leaflet of the plasma membrane. We have also

observed that vesicles containing membrane proteins pass

through the barrier region (data not shown). Thus, we favor

a model in which the barrier becomes effective only after the

196 Neuron 64, 188–199, October 29, 2009 ª2009 Elsevier Inc.

molecules are inserted into the axonal membrane. It would be

important to test whether or not a diffusion barrier exists

in vivo, and whether or not it plays a role in the development of

the nervous system.

Potential Roles of Intra-axonal PatterningAn important but yet poorly explored question is the role of the

guidance receptors localized on axon shafts. A straightforward

explanation can be offered based on non-cell-autonomous func-

tions of guidance receptors or membrane proteins in general;

they may ‘‘label’’ axon pathways through specific adhesion

(fasciculation), or through presenting their ligands, thereby

providing instructive spatial cues for the navigation of other

axons (Goodman et al., 1984; Hiramoto et al., 2000; Lin et al.,

1994; Raper et al., 1984). For example, Fasciclin cell-adhesion

molecules have been suggested to provide pathway labels for

guiding other growth cones (Goodman et al., 1984; Lin et al.,

1994; Raper et al., 1984). Drosophila Netrin receptor Frazzled/

DCC relocates its ligand Netrin to strategic positions in the

nervous system, thereby generating guidance information for

a longitudinal pioneer neuron (Hiramoto et al., 2000). Other

studies reported that guidance receptors can also play non-

cell-autonomous roles in cell migration and synaptogenesis

(Ango et al., 2004; Kraut and Zinn, 2004). Thus, spatial patterns

of molecules on axon shafts likely have direct roles in neuronal

circuit formation.

Lastly, we propose that the compartmentalization of the

axonal membrane could be a common basis for the structure

and function of the nervous system. In the Drosophila ventral

nerve cord, formation of the longitudinal axon tracts depends

on the expression of ROBO receptors. On the other hand, longi-

tudinal axon tracts are considered as the site for synapse forma-

tion, because synaptic proteins such as synaptotagmin and syn-

apsin accumulate on the longitudinal tracts (Littleton et al., 1993;

Lohr et al., 2002). We found that in cultured neurons both ROBO

receptors and synaptic proteins localize to the distal axon

compartment. This may suggest that the spatial distribution of

guidance molecules and synaptic proteins can be collectively

governed by the compartmentalization of the axonal membrane.

Future work to identify the molecular basis of the compartmen-

talization, and to establish the link between cellular identity and

this intracellular pattern, will aid in determining how intra-axonal

patterning contributes to tissue organization.

EXPERIMENTAL PROCEDURES

Drosophila Strains

The following stocks were used: y w, shits1(Grigliatti et al., 1973), elav-

GAL4(c155) (Lin and Goodman, 1994), UAS-GAP-GFP (generated by Akira

Chiba), UAS-myr-mRFP (generated by Henry Chang), UAS-mCD8-GFP (Lee

and Luo, 1999), hs-hid Y (generated by Mark Van Doren), UAS-syt-eGFP

(Zhang et al., 2002), UAS-n-syb-eGFP (Zhang et al., 2002) (from Bloomington

Stock Center), hs-GAL4 (Halfon et al., 1997), UAS-GAL4 (Hassan et al., 2000),

UAS-Nod-lacZ (Clark et al., 1997), UAS-Kin-lacZ (Clark et al., 1997), NP5280

(Hiyashi et al., 2002) (from DGRC, Kyoto), UAS-GFPgpi (Greco et al., 2001)

(kindly provided by Suzanne Eaton), UAS-Dlg-YFP (Kohsaka et al., 2007)

(kindly provided by Akinao Nose), elav-GeneSwitch (Osterwalder et al.,

2001) (kindly provided by Thomas Osterwalder), and UAS-HA-ROBO3

(Rajagopalan et al., 2000a) (kindly provided by Barry Dickson). hs-hid Y was

used for virgin collection of elav-GAL4. Targeted gene expression in transgenic

Neuron

Intra-axonal Patterning in Drosophila Neurons

Drosophila was achieved using the binary GAL4/UAS system (Brand and

Perrimon, 1993). Transgenes UAS-ROBO-EGFP, UAS-ROBO2-EGFP, UAS-

ROBO3-EGFP, UAS-ROBO3-mRFP, UAS-DRL-EGFP, and UAS-myr-EBFP2

were created using standard PCR-based methods from cDNA clones of

ROBO, ROBO2, and ROBO3 (Simpson et al., 2000a) (kindly provided by Julie

Simpson); DRL (Callahan et al., 1995) (kindly provided by Shingo Yoshikawa);

and EBFP2 (Ai et al., 2007) (Addgene). EGFP was fused to the C termini of

receptors, removing their 30-UTRs. Myristoylation signal for UAS-myr-EBFP2

was cloned from the UAS-myr-mRFP transgenic line.

Drosophila Primary Cell Cultures

Primary cell cultures of Drosophila embryonic neurons were prepared as

described previously (Patel et al., 1987), with slight modifications. Postmitotic

neurons before axonogenesis were obtained by homogenizing embryos at

stages 9–11. The cells obtained were suspended in Schneider’s Drosophila

medium (GIBCO) supplemented with 10% FBS, and cultured on glass bottom

dishes (MatTek) coated with 1 mg/ml poly-DL-ornithine (Sigma). Cell density

was adjusted to minimize cell-cell contacts. Cultures were kept at 25�C for 1

to 2 days without additional CO2. Axons and dendrites were defined by the

localization of Kin-bgal and Nod-bgal, respectively (Clark et al., 1997). Kin-

bgal accumulates to the tip of the longest neurite, while Nod-bgal localizes

to the cell body or to shorter neurites directly extending from the cell body

(Figure S1). The longest neurite is therefore referred to as an axon in this report.

A temporal shift in induction was achieved through heat-shock-triggered

induction, using a combination of hs-GAL4 (Halfon et al., 1997) and UAS-

GAL4 (Hassan et al., 2000) strains. A brief heat-shock induction of hs-GAL4

at 37�C for 30 min maintains GAL4 expression through the UAS-GAL4 autor-

egulatory loop. Neurons were imaged just before induction; the same neurons

were identified 24 hr after induction with the help of a grid-coverslip (MatTek),

and their morphology and fluorescence signals were compared.

Temperature shift experiments for the shits1 mutant were performed as

follows. Cells were cultured at 22�C for 24 hr before the temperature was

shifted to restrictive conditions. The expression of receptors under the control

of elav-GeneSwitch was initiated by adding 20 nM RU486 (Sigma) to the

culture medium immediately before shifting the temperature to the permissive

or nonpermissive conditions. Temperature shift was achieved by keeping

glass bottom culture dishes on a water bath placed in an incubator set at

a desired temperature. Fixation of cells was also done on the water bath in

the incubators after 6 hr (for 29�C, 31�C, and 33�C conditions) or 12 hr (for

22�C condition) from the temperature shift.

For the pulse chase experiments described in Figure 3G, fixation was done

after 2 hr, 4 hr, and 6 hr from the temperature shift (shifted to 29�C). Detection

of newly synthesized proteins was done by surface labeling (see Immunostain-

ing section below). We have confirmed that the labeling and detection condi-

tion we used for this experiment does not produce any signal in the absence of

RU486. Reversibility of the effects of temperature up-shift on DRL-EGFP local-

ization was verified as follows. After 24 hr of culture at 22�C, cells were incu-

bated for 6 hr at a restrictive temperature (29�C), and then transferred to

22�C for 5 hr before fixation.

Immunostaining

The following primary antibodies were used: rabbit anti-ROBO2 (Simpson

et al., 2000a) (1:1000 dilution), rat anti-DRL (Callahan et al., 1995) (1:1000 dilu-

tion), anti-HRP (Jackson Laboratories), rat anti-GFP (nacalai tesque), mouse

anti-HA (nacalai tesque), and rat anti-bgal. We also used the following anti-

bodies obtained from the Developmental Studies Hybridoma Bank, developed

under the auspices of the NICHD and maintained by The University of Iowa,

Department of Biological Sciences, Iowa City, IA 52242: anti-ROBO (Kidd

et al., 1998) (mAb 13C9) and anti-ROBO3 (Simpson et al., 2000a) (mAb

14C9 and 15H2), BP102 anti-CNS axons [1:10–1:20, developed by Corey

S. Goodman], and anti-tubulin (mAb 12G10) [1:20, developed by Joseph

Frankel and E. Marlo Nelsen]. Immunostaining of whole embryos was per-

formed as described previously (Patel, 1994). For staining cultured cells, cells

were fixed with 4% paraformaldehyde/PBS for 20 min at room temperature

and then permeabilized with 0.1% Tween-20/PBS. For surface labeling exper-

iments, cells were fixed for 10 min with 4% paraformaldehyde/PBS, and PBS

was used for all the following steps. Blocking was carried out using Block Ace

(Dainihon Pharmaceutical) for 30 min. Cells were incubated overnight with

primary antibodies in 10% Block Ace, 0.1% Tween-20/PBS at 4�C and then

stained with secondary antibodies for 1 hr at room temperature. The following

secondary antibodies were used: Alexa 405 conjugated goat anti-mouse IgG;

Alexa 488 conjugated goat anti-rabbit IgG (Invitrogen, used at 1:1000 dilution);

and DyLight488-, Cy2-, Cy3-, or Cy5-conjugated donkey anti-mouse, anti-

rabbit, or anti-rat IgG (Jackson Laboratories, used at 1:1000 dilution). Zenon

Alexa Labeling Kit (Invitrogen) was used for the double staining with mAb

BP102 and mAb 14C9. All double staining experiments were done by sequen-

tial labeling with two antibodies.

Quantitative Analysis of Localization

To avoid subjective differences between observers and to improve reproduc-

ibility between analyses, localization patterns and the positions of boundaries

were determined by quantitative criteria, except for Figure 3G (see below). All

the images were collected at an 8-bit depth, and axonal trajectories were

traced by using the ImageJ plug-in NeuronJ (Imagescience). Either fluores-

cence signals of membrane-targeted fluorescent protein (GAP-GFP or myr-

mRFP) or color-inverted bright field images (phase contrast or DIC) were

used for tracing. Pixel intensity along the trace was analyzed by the following

algorithm, using a custom program written in R software (The R Foundation).

Images were processed with a 5 3 5 median filter, and background subtrac-

tion was applied. If the maximum pixel intensity along the trace was less

than 20% of the maximum value of the dynamic range, the neuron was sorted

as ‘‘not detectable’’ (N.D.). To obtain intensity per membrane area, pixel inten-

sity of the molecule was divided by the amount of a membrane marker, GAP-

GFP or myr-mRFP, where possible. Intensity was normalized so that 1 and

0 represented the maximum and minimum intensity, respectively. Localization

was determined by a statistical significance test where the average normalized

intensities for the proximal and distal halves of an axon were compared. If a p

value was larger than 1.0e�13 (Wilcoxon signed rank test), the neuron was

classified as having a uniform distribution of markers. The position of a

boundary was defined as the point where normalized intensity becomes

continuously higher than 0.2 over a 4 mm length. When there was no point

matching the above criteria of a boundary, the neuron was again classified

as having a uniform distribution of markers. Thus ‘‘uniform’’ distribution here

should be considered as having no clear localization with a boundary.

Table 1 and Table 2 list a classification of neurons using the algorithm

described above. The percentages of neurons judged to have localized pattern

(e.g., distal for ROBO2 and ROBO3, proximal for DRL) differ between the

endogenous proteins (Table 1) and EGFP-tagged transgenes (Table 2). There

are several factors that contribute to this difference. While the transgenes are

expressed in all neurons using elav-GAL4 driver (Lin and Goodman, 1994),

endogenous genes are driven by their own promoter and are expressed only

in a subset of neurons. Thus any potential cell-type-specific differences in

the intra-axonal patterning process could cause a difference in the frequency

of neurons with localized pattern. The classification is also influenced by the

signal intensity and the signal-to-noise ratio, which in turn depends on the

method of observation (antibody versus GFP fluorescence, the quality of anti-

body, expression levels). Thus the percentages of neurons with localized

patterns are not directly comparable, especially between endogenous

proteins (Table 1) and GFP-tagged reporters (Table 2).

In Figure 3G, the fluorescence signal of the membrane marker myr-mRFP

was barely detectable at 2 hr after temperature shift, and thus we analyzed

all the data for Figure 3G visually.

Photobleaching Experiments

All photobleaching experiments were done by expressing fluorescent reporter

genes using elav-GAL4 driver. A Zeiss LSM 510 META confocal microscope

was used for all imaging, using a 100 X 1.45 N.A. objective lens at 25�C.

Bleaching was performed at the maximum laser intensity of a 488 nm argon

laser. Image acquisition was typically performed at 0.3%–0.8% intensity.

Image analysis was performed using ImageJ software (NIH) or a custom

program written in R software (The R Foundation). Recovery was calculated

according to the following formula: Recovery (%) = 100[It� I0]/[Ipre� I0], where

I = (Ia � Ib), Ia is the average fluorescence intensity of the ROI, Ib is the average

fluorescence intensity of the background region of equal size, It is the intensity

Neuron 64, 188–199, October 29, 2009 ª2009 Elsevier Inc. 197

Neuron

Intra-axonal Patterning in Drosophila Neurons

at each time point, I0 is the postbleach intensity, and Ipre is prebleach intensity.

The mobile fraction and the half-recovery time, which is the time required to

reach half of the intensity of the mobile fraction, were calculated by fitting

the curves obtained by a least-squares regression against the following equa-

tion: Recovery (%) = A[1� exp(�t/t)], where A is the mobile fraction and t is the

time constant. Statistical analysis was performed using R software.

All experiments presented in this study were replicated at least two times

with independent experiments and their results were reproducible.

SUPPLEMENTAL DATA

Supplemental data for this article include two Supplemental Figures and two

Supplemental Tables and can be found at http://www.cell.com/neuron/

supplemental/S0896-6273(09)00631-X.

ACKNOWLEDGMENTS

We thank J. Simpson, S. Yoshikawa, S. Eaton, A. Nose, B. Dickson, the Bloo-

mington Drosophila Stock Center, DGRC Kyoto, and DSHB for providing

materials. We thank N. Patel for advice on culture experiments; K. Fukatsu

for advice on FRAP experiments; T. Takano-Shimizu for advice on statistical

analyses; T. Yoshimori for advice on shi experiments; T. Hirata and T. De Falco

for comments on the manuscript; members of the Suzuki, Emoto, Hirose, and

Hiromi laboratories for discussions; and M. Aono, C. Asaka, E. Suzuki, Y. Ike-

tani, H. Hatanaka, and Y. Noguchi for technical assistance. This work was sup-

ported by the grants from the JSPS (T.K.), the PREST program of JST (M.H.),

the CREST program of JST, and the Takeda Science Foundation (Y.H.). The

authors declare no competing financial interests.

Accepted: August 20, 2009

Published: October 28, 2009

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