1

Supplemental Materials and Methods

Transgenic Mice

All animals were used according to protocols by our institution licensing and

the Italian Ministry of Health. Conditional p75lox/lox mice were provided by Brian

A. Pierchala (Department of Biologic and Materials Sciences, University of

Michigan School of Dentistry, 1011 N. University Avenue Ann Arbor, MI USA).

We generated p75lox/lox-Cre mice and TrkBlox/lox-Cre mice by crossing p75lox/lox

or TrkBlox/lox mice (provided by Rudiger Klein, Max Planck Institute,

Martinsried, Munich, Germany) with GlastCre::ERT2 mice (provided by

Magdalena Götz, Helmholtz Zentrum, Munich, Germany) and R26R mice

expressing the -galactosidase reporter gene.

Antibodies

Louis Reichardt (University of California, San Francisco, USA) and Moses

Chao (Skirball Intitute of Biomolecular Medicine, New York, USA) provided

specific blocking antibodies against p75NTR. The following antibodies were

purchased: rabbit -p75NTR (Promega); mouse -Smi312 (Covance); mouse

-Smi35 (Covance); rabbit and chicken -GFP (Invitrogen); rabbit -Dcx

(Abcam); rabbit -RFP (Rockland); rabbit - -galactosidase (Cappel); chicken

- -galactosidase (Abcam).

DNA Constructs and Viral Vectors

Plasmids. For in vitro experiments and in utero electroporation, we used the

murine Moloney leukemia virus (MoMulV)-based vector expressing GFP

under the cytomegalovirus early enhancer element and chicken β-actin

promoter, here referred as pCAG-GFP vector. Replacing the GFP encoding

sequence with one of the following fluorescent molecules created additional

2

plasmids: red-fluorescent protein (RFP), p75-GFP fusion protein, membrane-

tagged GFP (provided by H. Lickert, Helmholtz Zentrum Muenchen,

Neuherberg, Germany), and Cre-GFP fusion protein (provided by F.H. Gage,

Salk Institute, La Jolla, USA). pCAG-IRES (internal ribosomal entry site)-

eGFP and pCAG-IRES-tdTomato were used as internal controls for in utero

electroporation experiments.

The 29 bp scramble (5’-GCACTACCAGAGCTAACTCAGATAGTACT-3’) is a

random sequence checked for absence of targets in the mouse and rat

transcriptome. Mouse 5’-ACCGAGCCGTGCAAGCCGTGCACCGAGTG-3’;

5’-CCTGGCCGATGGATCACAAGGTCTACGCC-3’ and rat 5’-

CGACAACCTCATTCCTGTCTATTGCTCCA-3’; 5’-

TGAGGTTCCTCCAGAGCAAGACCTTGTAC-3' short-hairpin RNA (shRNA)

plasmids targeting the p75NTR sequence and scramble sequence were

purchased (Origene Technologies).

For the generation of lentiviruses, the same short-hairpin sequences targeting

the murine p75NTR mRNA and the scramble were separately inserted under

the U6 promoter in a bio-safe, HIV-based viral vector expressing eGFP as

reporter gene. Lentiviruses were produced with a final titer of about 7 x 109

particles/ml. MoMulV-based vectors encoding for GFP, RFP and p75-GFP

were used for generating replication-deficient retroviruses with a titer of about

5 x 108 particles/ml.

Cell Cultures and Nucleofection

Hippocampal cultures. Primary cultures of dissociated hippocampal and

cortical neurons were prepared from embryonic E17 C57BL/6 mice. In brief,

hippocampi were dissected, digested with 0.25% trypsin EDTA for 20 min at

3

37°C and dissociated in plating medium (DMEM low glucose; Invitrogen)

supplemented with 10% fetal bovine serum (GIBCO) and

penicillin/streptomycin, and plated on poly-L-lysine coated coverslips (0.1

mg/ml; Sigma). The medium was changed after 4 hours with Neurobasal

containing B-27 and pen/strep/glutamine (GIBCO). Neurons were

nucleofected using the Amaxa nucleofector kit for primary neural cells (Amaxa

Bioscience) according to the manufacturer's instructions. Single

nucleofections were optimized using 3 µg of total DNA for a total amount of 3-

4×106 cells. Briefly, cells were resuspended in 100 μl of transfection buffer

prior to addition of the selected plasmids and electroporated according to a

specific predefined program for primary neurons. Following electroporation,

cells were incubated in the culture medium at 37°C for 10 min and then

seeded into DMEM supplemented with 10% FBS. After 4 hr, the medium was

replaced with Neurobasal supplemented with B-27 and pen/strep/glutamine

(GIBCO).

Neuronal precursor cultures. C57Bl/6 mice were sacrificed by an overdose of

ketamine and brains were removed and placed in Hanks buffer (pH 7.3). The

dentate gyrus was quickly isolated and digested at 37°C for 40 min in an

enzyme solution containing 2.5 U/ml of Papain (Worthington), 1 U/ml of

Dispase (Roche), and 250 U/ml of DNase (Worthington). Cell suspensions

were then washed with D-PBS (GIBCO) and enriched for precursor cells

using a 22% Percoll gradient (Amersham). In some experiments, precursor

cells were nucleofected using the Amaxa nucleofector kit for adult mouse

stem cells (Amaxa Bioscience) according to the manufacturer’s instructions.

Nucleofection was performed using 3 µg of DNA and 3×106 cells for each

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electroporation. Cells were maintained in a serum-free medium containing

Neurobasal (Invitrogen), B27 without retinoic acid (Invitrogen), 2 mM

glutamax, 20 ng/ml EGF (Peprotech) and 20 ng/ml bFGF (Peprotech); cells

were plated on polystyrene surfaces coated with poly-lysine (Sigma; 10 μg/ml)

and laminin (Roche; 5 μg/ml). For cell differentiation, growth factors were

withdrawn for 48 hr (div 2). Cells were incubated overnight with FGF at 5

ng/ml; the day after, the medium was replaced with fresh medium lacking all

growth factors (div 3), and cells were allowed to differentiate for 7, 12 or 21

days.

Substrate Micropatterning

A silicon wafer was used to generate a template for the poly-

(dimethylsiloxane) (PDMS) mold and substrates were patterned in parallel

stripes of 50 μm width separated by 50 μm gaps. The PDMS cuboids were

prepared from Sylgard 184 base and curing agent (Dow Corning). Briefly,

glass coverslips were coated with poly-L-lysine (0.1 mg/ml), washed 3 times

in sterile water and air-dried. PDMS mold was reversibly sealed on poly-L-

lysine-coated glass coverslips, and the micro-channels formed between the

PDMS mold and coverslips were used for micro-fluidic patterning of the

substrates alone or together with BSA conjugated either to Alexa-647 (25

μg/ml) or Alexa-488 (5 μg/ml), allowing visualization of stripes. The substrate

solutions were prepared with the following concentrations of the factors:

BDNF, NGF and NT-3 (0.5 ng/ml). Solutions were maintained in the micro-

channels overnight and allowed to dry. Stripe-coated coverslips were

extensively washed before plating of dissociated neurons.

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Immunocytochemistry and Immunohistochemistry

For immunostaining of cultured hippocampal neurons, cells were fixed in cold

4% para-formaldehyde (PFA) for 20 min, washed with PBS, permeabilized in

0.1% Triton X-100 in PBS for 20 min and blocked with a buffer containing 3%

BSA in PBS for an additional 30 min. Neurons were then incubated with

primary antibodies at 4°C overnight. After washing, conjugated secondary

antibodies were used to detect the immunoreactive signal. Secondary

antibodies diluted in blocking buffer were incubated at room temperature for 2

hr. Cells were then washed with PBS, counterstained with DAPI (Invitrogen)

and mounted with Acqua Polymount (Polyscience Inc.). For staining of brain

slices, mice were deeply anesthetized and transcardially perfused with PBS

followed by 4% PFA. Brains were removed, fixed overnight in PFA and then

washed in PBS. Coronal or para-sagittal sections (70 to 100 µm thick) were

cut with a vibroslicer. Slices were rinsed in PBS, treated with 0.5% Triton X-

100 for 20 min, blocked with 3% BSA in PBS for 40 min and incubated

overnight free-floating with primary antibodies. Slices were then washed in

PBS and incubated for 2.5 hr at room temperature with secondary antibodies

diluted in blocking buffer. After washing, sections were incubated for 10 min at

room temperature with DAPI (Invitrogen) and then mounted with Acqua

Polymount (Polyscience Inc.).

In utero Electroporation and Stereotaxic Surgery

Timed-pregnant Sprague-Dawley rats (Harlan Italy SRL, Correzzano, Italy)

and p75 lox/lox were anesthetized with isoflurane (induction, 3.5%; surgery,

2.5%) at E17 and E15.5 respectively, and the uterine horns were exposed by

way of a laparotomy. Saline solution containing the expression vector of

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interest (2-4 mg/ml) together with the dye Fast Green (0.3 mg/ml; Sigma) was

injected (4-5 µl) through the uterine wall into one of the lateral ventricles of the

embryo, and the embryo’s head was electroporated using tweezer-type

circular electrodes across the uterine wall delivering square-wave electrical

pulses (50 V, 50-ms duration at 100-ms intervals for rats; 30 V, 50-ms

duration at 1-s intervals for mice) with a electroporation generator

(CUY21EDIT; Nepa Gene). The uterine horns were then returned to the

abdominal cavity, the wall and skin sutured, and the embryos allowed to

continue development. Control embryos were electroporated with control

vectors (GFP or scramble-RFP) and experimental embryos were

electroporated with Cre-GFP, p75-GFP or shRNA targeting the rat p75NTR

transcript. In most of the experiments, constructs were co-transfected with a

separate vector pCAG-IRES-tdTomato expressing a red fluorescent protein

tdTomato or pCAG-IRES-EGFP construct. For the analysis of cortical neural

development, embryonic (E21 for rats or E18.5 for mice) or postnatal (P7)

brains were dissected out and fixed with 4% paraformaldehyde. Brains were

sectioned coronally with a vibratome (1000plus Sectioning System; vibratome,

St. Louis, MO) at the level of the somatosensory cortex and slices (40-80 µm)

were mounted in Vectashield (Vector Laboratories, Inc.). For virus delivery

into the dentate gyrus, adult (2-3 months old) mice were anesthetized (100

mg/ml ketamine plus 20 mg/ml xylazine in saline solution) and a total volume

of 0.5 µl of retro- or lentivirus was infused into each hemisphere (coordinates

from Bregma: anteroposterior -2 mm, lateral ±1.5 mm, ventral 1.9 mm)

through the insertion of capillary glasses (WPI) connected to a manual syringe

pump (Narishige). Mice were allowed to recover and housed in standard

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cages until the day of sacrifice. At 7, 12 or 21 days post-injection, mice were

perfused with PBS (0.1M) followed by cold 4% PFA for 30 min, then brains

were dissected.

Time-lapse Experiments

Maintained hippocampal neurons or adult hippocampal progenitor cells were

seeded onto poly-L-lysine-coated (0.1 mg/ml) or poly-L-lysine (0.01 mg/ml)

and laminine (0.05 mg/ml) coated glass-bottom dishes (MatTek, USA),

respectively, and maintained in growth or differentiating medium. For live

imaging, cell culture dishes were mounted on a dedicated stage incubator

(OkoLab, Italy) onto an Eclipse Ti-E inverted microscope (Nikon) equipped

with a perfect focus system. NIS software (Nikon) was used to select suitable

areas according to cell density and the presence of fluorescent neurons. A

20x Nikon objective was used for these experiments by setting the exposure

times were adjusted on the basis of the initial fluorescence of the cells.

Confocal Microscopy and Quantitative Image Analysis

Confocal imaging was performed using a laser-scanning motorized confocal

system (Nikon A1) equipped with an Eclipse Ti-E inverted microscope and

four laser lines (405, 488, 561 and 638 nm).

Analysis of neurons in vitro. Z-series images were taken with an inter-stack

interval of 0.5 m using 40x (NA 1.30) or 60x (NA 1.4) oil objectives. Neurons

were classified into three groups depending on their axonal phenotype, which

was assessed for the presence or absence of axon-specific markers such as

Smi312 or Tau after immunostaining: single axon (single), no axon (none) and

multiple axons (multiple). Cells were analyzed using the National Institutes of

Health software ImageJ. Two types of analyses were carried out:

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quantification of specific marker immunoreactivity along single processes of

individual cells and analysis of the fluorescence intensity localized at the

process tips. For each type of quantification, laser intensities and camera

settings were maintained identically within the same experiment to allow

comparison of different experimental groups and treatments. The mean and

maximal values of signal intensities were calculated with ImageJ by manually

drawing precise ROIs either along the cell neurites (segmented line) or

around the neurite tips (circular) and compared to the signal intensity of the

cell body. In order to establish the number of positive or negative neurites and

tips for any specific marker, the intensity ratio of each process was compared

to that of the axon (Smi312 positive), and the resulting value ± one standard

deviation was considered the reference threshold level. For cells analyzed at

time points earlier than the appearance of axonal markers, the classification in

single, none or multiple positive neurites/tips was assessed by evaluating the

presence of neurites/tips that showed at least double the intensity of other

processes in the same cell. For acquisition of neurons seeded on stripes, z-

series of large image fields were taken with an inter-stack interval of 1.5 m

thickness and by using 20x (NA 0,75) or 40x (NA 0.75) air objectives. The

analysis of hippocampal neurons plated on stripes was carried out by

specifically focusing on those cells for which the cell body was located on a

stripe boundary. In these cells the initiation of the axon on or off the stripe was

analyzed, and the preference index (PI) was calculated according to the

following formula: (percentage of cells on - percentage of cells off the

stripe)/100.

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Analysis of adult generated granule cells. To analyze newly generated

neurons in the adult dentate gyrus upon virus injection, confocal acquisitions

were carried out with a 40x (NA 1.30) or 60x (NA 1.40) oil-immersion

objectives by setting a 1 m thick interval between stacks. Only cells for which

the morphology was maintained upon brain slicing were included in the

quantifications. The analysis of the phenotype was conducted by classifying

neurons for their orientation with respect to the granular cell layer (horizontal

or radial orientation) and either (i) the presence of one single basal process

extending through the hilus for a distance of at least 50 m (single axon), (ii)

the absence of processes or (iii) the presence of multiple short basal

processes terminating within 20-30 m from the cell body (no axon) in radially

oriented neurons. For the colocalization of p75NTR immunoreactivity in newly

generated neurons, acquisitions were conducted with a 63x oil objective (NA

1.40) (2x digital magnification) with an inter-stack interval of 0.3 m. ImageJ

was used to extract colocalized pixels and superimpose them on the resulting

deconvolved image (Huygens Professional 3.0, Scientific Volume Imaging) of

the GFP immunoreactivity.

Analysis of in utero electropration. For the analysis of cortical neurons, large

confocal images were acquired with 20x (NA 0.75) or 40x (NA 0.75) air

objectives and an inter-stack interval of 1.5-2 m. At E21 for rats and E18.5

for mice, reporter positive cells located either in the IZ or CP were classified

for the presence of one long basal trailing process (single axon) or the

absence of axon (no axon), the latter phenotype corresponding either to the

lack of trailing process or the presence of multiple basal short processes. At

P7, large confocal images of entire brain slices were acquired, maintaining

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fixed laser intensities and camera settings. Reconstructed images were

processed using ImageJ to calculate the integrated mean intensity of selected

ROIs such as a band in layer III where the proximal axonal fibers of layer II

electroporated neurons were located and selected regions of the corpus

callosum in the ipsi- and contralateral hemisphere to the electroporated side.

Magnifications of individual reporter positive neurons were acquired with a

63x objective.

7 dpi 12 dpi 21 dpi

retr

o-G

FP

A

axon

sgz

CA3

sgz axon

CA3

axon

sgz

retro-GFPinjection

3 5 7 12 21 dpi0

NEWBORN NEURONSDIFFERENTIATION

DA

PI G

FP

DENTATEGYRUS

CA3hl

ml

gcl

ml

21 dpi

mossyfibers

axon

dendrites

retro-GFP

perfusion

dendrites

dendrites

dendrites

2

3D z-depth

21 dpip75wt/wt -Cre21 dpi

axon

noaxon

ml

gcl

ml

ml

gcl

hl

p75lox/lox -Cre

retr

o-G

FP

C2D reconstruction

3D z-depth

z

D

10 m

20 m

30 m

40 m

50 m

60 m

z

60 m60 m

0 m 0 m

30 m 30 m

x

yz

x

yz

GFP

p75wt/wt -Cre p75lox/lox -Cre

axon

dendrites

dendrites

no axon

B virus injection

0

4

8

12

0

4

8

12

p75N

TR

/ G

FP

colo

caliz

atio

n (%

)

3 dpi

7 dpi

5 10 15 20 25

Newborn neurons

p75N

TR

/ G

FP

colo

caliz

atio

n (%

)

Retro-GFPRetro-Cre-GFPMean - SD

dpi0

Perfusion

73

D

GFP

p75-

GF

PG

FP

polarity phenotype

single axonno axon

GFP p75-GFP

axon

iz

cp

svz

iz

cp

svz

E21

*

**

0

40

60

80

20

100

pola

rity

phen

otyp

e (%

)

no axonaxon

GFP

p75-GFP

p75-

GF

PG

FP

P7proximal fibers(area 1)

ipsilateral fibers(area 2)

GF

P f

luor

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nce

inte

nsity

p75-GFPGFP

prox

imal

contralateral fibers

ipsi

late

ral

cont

rala

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l

0

100

150

200

50

250

GFP p75-GFP

****

**

GFP p75-GFP

1

2

2

1

C

noaxon

no axonaxon

BG

FP

12

3

3

3

2

3

retro-p75-GFPretro-GFP

12 21 1

1

DA

PI G

FP

Sm

i35

21 dpi 21 dpi

axon

noaxon

retro-GFP retro-p75-GFP

noaxon

21 dpi

GF

P

axon

21 dpi

A

pola

rity

phen

otyp

e (%

)

0

40

60

80

20

100

retro

-GFP

retro

-p75

-GFP

retro

-GFP

retro

-p75

-GFP

12 dpi

horizontal

radial (axon)

radial (no axon)

21 dpi

** **

retro-GFP or retro-p75-GFP injection

21 dpi

perfusion

TrkBlo

x/lo

x -Cre

Pol

arity

phe

noty

pe (%

)0

40

60

80

20

10012dpi 21dpi

TrkBwt/w

t -Cre

horizontal

radial (axon)

radial (no axon)

virus injection

21 dpi0-5

Tamoxifenperfusion

12

TrkBwt/wt-Cre TrkBlox/lox-Cre

retr

o-G

FP

ga

l

TrkBlo

x/lo

x -Cre

TrkBwt/w

t -Cre

21 dpi

axon

21 dpi

axon

Supplemental Figure Legends

Figure S1. Knock out p75NTR abolishes axon formation in adult-generated neurons

in vivo, Related to Figure 6.

(A) Coronal section of the mouse hippocampus showing adult-generated neurons

transduced with GFP-retrovirus (green) in the granule cell layer (gcl), extending

dendrites in the molecular layer (ml) of the dentate gyrus and axons through the hilus

(hl) toward the CA3 area. Scale bar is 100 m. Diagram on the right illustrates the

experimental setting used to label newborn neurons in the adult hippocampus. Mice 2

months of age were injected with retro-GFP and perfused at 3, 5, 7, 12 and 21 dpi.

Sample tracings of newborn neurons are shown for each investigated time. Panels

show representative confocal images of transduced neurons at the subgranule zone

(sgz) of the dentate gyrus, extending the axon (red arrowheads) toward the CA3 region

of the hippocampus at 7, 12 and 21 dpi. Scale bar is 20 m.

(B) Diagram illustrating the experimental protocol used to knock out p75NTR in newborn

neurons of the adult dentate gyrus. p75lox/lox mice were injected with retro-GFP or retro-

Cre-GFP and perfused at 3 and 7 dpi. Graphs show p75NTR and GFP colocalization in

single cells transduced with GFP and Cre-GFP at 3 and 7 dpi. The lower graph (7 dpi)

shows fluorescence cut-off levels defined as mean p75NTR/GFP colocalization in

transduced GFP-cells, with SD subtracted. Data depict the average percentage (± SEM)

from 3 different preparations (~ 80 cells each).

(C) Two-dimensional reconstruction of newborn neurons from p75wt/wt-Cre or p75lox/lox-

Cre mice injected with retro-GFP 10 days after tamoxifen induction (5 days) and

perfused 21 dpi.

(D) Three-dimensional reconstruction of a newborn neuron from p75wt/wt-Cre mice

(dashed area in C). Newborn neuron displayed one distinguishable axon extending from

the cell body that exceeds z-axes (z= 60 m). Right panel show three-dimensional

reconstruction of two newborn neurons from p75lox/lox-Cre mice. Neurons displayed

short processes extending from the cell body that failed to exceeds xyz-axes. z-axe

distance is visualized in pseudocolor.

Figure S2. Ectopic p75NTR expression abolishes axon formation in vivo, Related to

Figures 6 and 7.

(A) Schematic diagram illustrates the experimental setting used to express control GFP

or p75-GFP in newborn neurons of the adult hippocampus. Mice 2 months of age were

injected with retro-GFP or p75-GFP and perfused at 21 dpi. Confocal images (in 2D

projection) of newborn neurons transduced with GFP-encoding retrovirus at 21 dpi.

GFP-transduced cells expressed one distinguishable axon. p75-GFP-transduced cells

expressed multiple short processes (white arrows) but no axon. Scale bar is 10 m. The

polarity phenotype is quantified as percentage of radial cells showing a single axon or

no axon after 12 and 21 dpi. Data depict average percentage (± SEM) from 3 different

preparations (~ 30 cells each) for each time point. (** p < 0.01). Right panels show GFP

fluorescence of newborn neurons stained for the axonal marker Smi35. Insets are high

magnification (3 times) images showing a Smi35 positive axon (boxed region 1; white

arrows) and two Smi35 negative processes (boxed region 2 and 3; white arrowheads).

Scale bar is 10 m.

(B) GFP fluorescence of E21 rat cortices transfected by in utero electroporation at E17

with GFP or p75-GFP vectors. Scale bar is 200 µm.

(C) 2D projection of confocal images (GFP fluorescence) of typical cortical neurons.

Scale bar is 10 µm.

(D) Coronal sections of the somatosensory cortex of P7 rat cortices electroporated as in

B. Scale bar is 10 µm.

Figure S3. Knock out of TrkB receptor does not impair axonal initiation in

newborn neurons of the adult hippocampus

Schematic diagram illustrates the experimental protocol used to knock out TrkB in the

adult dentate gyrus. TrkBwt/lwt-Cre and TrkBlox/lox-Cre mice were treated with tamoxifen

for 5 days, injected with GFP-transducing retrovirus the last day of tamoxifen treatment

and perfused after 12 or 21 dpi. Representative confocal images of control neurons or

neurons in which TrkB gene was deleted by tamoxifen-induced expression of Cre

recombinase, 21 days after retroviral injection. After tamoxifen treatment, recombination

was driven by the Glast promoter and monitored by the reporter gene gal. Neurons

expressing the recombination ( gal positive) and transduced by retrovirus (GFP

positive) were analyzed depending on the orientation (horizontal or radial) and

extension of the axon (axon or no axon). Data depict average percentage (± SEM) from

4 different mice (~ 50 cells each). Scale bar is 10 m.

Supplemental Movie Legends

Movie S1, Related to Figure 1. Time-lapse showing a neuron before and after axon

cut. After 48 hr, one of the neurites that was short at the time of the axon cut had grown

into a new axon, which showed high p75NTR immunostaining (pseudocolor image) after

72 hr. Scale bar is 20 m.

Movie S2, Related to Figure 2. Time-lapse showing neurons cultured on alternate

patterns of poly-L-lysine (grey) and BDNF (blue) stripes in the absence or presence of

α-p75 blocking antibody. On the right is shown a neuron at the stripe boundary growing

the axon on a BDNF stripe. On the left is shown a neuron growing the axon off the

BDNF stripe. Scale bar is 10 m.

Movie S3, Related to Figure 3. Time-lapse showing neurons transfected with sh-p75

imaged together with nearby untransfected neurons for 72 hr. Only control neurons

developed one axon that underwent a period of protracted growth. Fluorescence signal

confirmed sh-p75 (red) at the end of the recording. Scale bar is 15 m.

Movie S4, Related to Figure 4. Time-lapse showing neurons transfected with p75-GFP

imaged together with nearby untransfected neurons for 72 hr. Only control neurons

developed one axon that underwent a period of protracted growth. Fluorescence signal

confirmed p75-GFP (green) at the end of the recording. Scale bar is 15 m.

Movie S5, Related to Figure 5. Time-lapse showing a typical progenitor cell that

generated two immature neurons after 3 days in vitro (div). The red newborn neuron

extended/retracted undifferentiated neurites from 3 to 6 div, until it acquired a neuronal

phenotype (Dcx positive), and characterized by one (Smi312 positive) axon after 7 div.

Scale bar is 20 m.

Movie S6, Related to Figure 5. Time-lapse showing one newborn neuron transfected

with sh-p75 imaged together with nearby untransfected neuron for 7 days in vitro (div).

Fluorescence RFP-signal confirming sh-p75 expression is shown at the end of

recording. Only control neurons developed one (Smi312 positive) axon. Scale bar is 20

m.