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Supporting Online Material

Materials and Methods

In situ hybridization. Brains were removed from C57Bl6 mice from ages P1, P7, and

adults (>3 months). Antisense riboprobes containing the coding region of mouse PK2

(GenBank accession number AF487280; residues 1-528), the 3' UTR of mouse PKR2

(GenBank accession number AF487279; residues 1147-2211), or the 3' UTR of mouse

PKR1 (GenBank accession number AF487278) (1) were generated. The in situ

hybridization was processed as previously described (1). The distribution patterns of

mRNA were analyzed in autoradiogram and emulsion-dipped sections. Specific

hybridization was analyzed using a video-based computer image analysis system (MCID,

Imaging Research). All procedures regarding the care and use of animals are in

accordance with institutional guidelines

Transwell Assay. Transwell membranes (5 µm pores, Costar) were pre-coated on both

sides with 0.1% gelatin. Cells from the RMS and olfactory ventricle (OV) regions of P7

or adult rats were dissected out from sagittal sections (as described below) and incubated

with 2 mg/ml papain in B27/HibernateA (Invitrogen) at 30oC for 30 min. The cell

suspension was then triturated ten times and spun for 10 min at 3000 RPM. The cells

were subsequently washed and resuspended in B27/Neurobasal A (Invitrogen) with

penicillin and streptomycin. Dissociated cells were incubated in 5% CO2 at 37oC for 2 hr

before migration assays. 50,000 cells were placed in the upper chamber and 1nM or

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30nM of recombinant human PK2 were added to the lower chamber and incubated in 5%

CO2 at 37oC for 24 hr. Recombinant human PK2 and prokineticin receptor antagonist,

A1MPK1, were prepared as described (2). For antagonist studies, A1MPK1 was added to

the lower chamber at a concentration of 100nM. The membrane was subsequently fixed

with 4% paraformaldehyde and stained with Tuj1 (as described below). The upper side

was wiped off, and the cells that had migrated and attached the lower side were counted

blind, in five central fields with a 10x objective. After confirmation that almost all the

migrating cells stained positively with Tuj1, subsequent samples were stained with 50

µg/ml of propidium iodine and counted.

Explant co-culture. Brains from newborn Sprague-Dawley rats (P7) were removed and

coronal and sagittal sections of 400 µm were cut with a Brinkman McILWAIN tissue

chopper. Tissues from the SVZa and RMS were isolated and trimmed to explants of

300-400 µm in diameter. Tissues were embedded into collagen gels as described (3). The

explants were cultured in 5% CO2 at 37oC in serum-free F-12 media supplemented with

B-27 (Invitrogen), penicillin, and streptomycin. We have generated a stable T293HEK

cell line that expresses full-length human PK2. PK2 expression was confirmed by

western blot analysis with an anti-PK1 antibody that cross-reacts with PK2. Aggregates

of untransfected or PK2 expressing T293HEK cells were made by the hanging drop

method (4). These cells were placed in collagen with SVZa explants and cultured for

18-24 hr as described (5). As indicated, PK2 or A1MPK1 were mixed into the collagen

gel and the same concentrations were added to the culturing media. The time-delayed

co-culture assay was performed as described (6). Briefly after the first 24 hr incubation

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of SVZa explants in collagen, the media was removed and a cell aggregate was

positioned next to the explant. Additional collagen was added on top and allowed to

harden, followed by the addition of F-12 media supplemented with B-27. Photographs

were taken and the number of migrating neuronal progenitors counted in sampling

quadrants at two time points, immediately before (0 hr) and (24 hr) after the placement of

the cell aggregates.

For quantification of explants, samples were fixed in 4% paraformaldehyde and

permeablized with 0.1% Triton X-100. Cells were subsequently stained with 50 µg/ml

propidium iodine. Fluorescence images of samples were viewed with a Zeiss

microscope. We quantified the distribution of migrating cells by dividing the area

surrounding each explant into four quadrants and the number of propidium iodine labeled

cells in the proximal quadrant to the cell aggregates was compared with that in the distal

quadrant. For distance traveling experiments, images were taken and the distances the

cells traveled away from each explant was measured.

For immunohistochemistry, samples were fixed in 4% paraformaldehyde

overnight, then carefully removed from culture dishes and permeablized with 0.2%

Triton X-100. Samples were blocked with 4% horse serum in phosphate buffered saline

(PBS) (pH7.4) for 1 hr. Incubation with a primary mouse monoclonal antibody to Tuj1

(Convance) was carried out overnight at 4oC. Samples were then washed and incubated

with a FITC-Donkey anti-mouse IgG (Jackson ImmunoResearch Labs) for 2 hr at room

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temperature. They were subsequently washed before being mounted onto a slide.

Fluorescence images of samples were viewed with a Zeiss microscope.

Matrigel Culture. Brains from newborn Sprague-Dawley rats (P7) were removed and

dissected as described above. Cultures of SVZ explants in Matrigel (Beckton Dickinson)

were performed as previously described (7). Briefly, explants were mixed with Matrigel

in the presence or absence of 60nM recombinant PK2 and cultured in six-well dishes.

After polymerization, the gel was covered with serum-free F-12 media with B-27,

penicillin, and streptomycin and PK2 embedded cultures were supplemented with the

same concentration of PK2. The explants maintained in a 5% CO2 incubator at 37oC for

24-30 hr. For the analysis of PK2 on chained-cells, we counted individual cells from

control and PK2 treated explants at high magnification.

Analysis of PK2-/- mice. PK2-/- mice were generated by the crossing of PK2+/- mice

and confirmed by Southern blot analysis. More than 20 PK2 -/- mice were examined for

this study. For morphological analysis, 20µm coronal and sagittal sections were stained

with cresyl violet and examined with a Zeiss microscope. OB diameter and GL thickness

were measured from every third sagittal section in series. The distances were measured

at the midpoint between the anterior tip of the OB and the flexure of the anterior border

of the cerebral cortex and dorsal surface of the OB. OV and RMS width were measured

at midpoint of the OV/RMS from every fourth coronal section from the start of the OB to

the middle of the forebrain. Three PK2 -/- and WT mice were used to calculate the

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average RMS/OV width. In situ hybridization of PK2 -/- mice with the PKR2 antisense

riboprobes were carried out as described above.

BrdU labeling. For the BrdU experiments, nine 6 weeks old PK2 -/- mice and

their WT controls were injected intraperitoneally with 100mg/kg BrdU and perfused 3hrs,

5 days or 28 days later. Animals were anesthetized with xylazine and ketamine and

transcardially perfused with 4% paraformaldehyde. Brains were cryoprotected with 30%

sucrose in 0.1 M phosphate buffer (pH 7.4), embedded in Tissue-Tek OCT (Sakura

Finetek), and sectioned on a cryostat in coronal or sagittal sections at 40 µm. To reveal

newly generated BrdU+ cells by diaminobenzidine (DAB) staining, sections were

pretreated with 0.6% H2O2 prior to a 30 min incubation in 2N HCl at 37oC.

Subsequently, the sections were incubated in blocking buffer, 3% horse serum in TBS

(pH 7.6) with 0.1% Triton-X100 for 30 min, and then for 72 hr with a mouse IgG anti-

BrdU in blocking buffer. Sections were subsequently visualized via Vectastain ABC

Systems (Vector Labs) and viewed under a Zeiss microscope at 20x and 40x objectives.

BrdU+ cells were quantified in every 5th section along the entire SVZ-OB pathway

(sagittal sections for 3hrs and 28days studies; coronal sections for 5 days study). The

number of BrdU+ cells was related to the area occupied by the cells in the SVZ, RMS,

OV and OB.

TUNEL staining. TUNEL staining was carried out in 40 µm coronal sections of

the OB to detect DNA fragmentation in situ. Tissues were treated with 10mM Sodium

Citrate pH 6.0 at 95oC for 15 min and allowed to cool to room temperature. Tissues were

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subsequently washed in PBS and equilibrated in terminal deoxynucleotidyl transferase

(TdT) reaction buffer (Roche) for 15 min. Sections were then incubated for 1 hr in a

humidified chamber at 37oC with the reaction buffer containing TdT and biotin-dUTP

(Roche Biosciences). After PBS washes, the sections were incubated with 1% H2O2 for 5

min to block endogenous peroxidases and washed. Sections were subsequently

visualized via Vectastain ABC Systems (Vector Labs) and observed under a Zeiss

microscope at 20x and 40x objectives. TUNEL+ cells were quantified as described above

for BrdU+ cells.

TH and PSA-NCAM immunostaining. Immunohistochemistry was carried out

in 40 µm coronal sections of the OB. After blocking, the sections were incubated

overnight at 4oC with one the following monoclonal antibodies: mouse anti-PSA-NCAM

(Chemicon) and mouse anti-Tyrosine Hyroxylase (Novus Biologicals). The fluorescent

second antibody used was FITC-Donkey anti-mouse IgG (Jackson ImmunoResearch

Labs). Sections were subsequently counterstained with DAPI (Vector Labs) and viewed

with a Zeiss fluorescent microscope.

Supplemental Figures Legend

Figure S1. Expression of PKR1 mRNA expression in adult mouse brain (coronal

sections). (left) Autoradiogram of PKR1 mRNA in the ependyma and subependymal

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layers of the olfactory ventricle (E/OV), (middle) PKR1 mRNA expression in SVZ of the

lateral ventricle (LV), and (right) PKR1 mRNA expression in the dentate gyrus (DG) of

the hippocampus. Scale bar, 1mm.

Figure S2. Migration of neuronal progenitors in Transwell assays. Cell migration

from adult rat RMS and OV regions is shown as a percent of the control. RMS and OV

cells showed chemotaxis to 1nM PK2 and 30nM PK2. 100nM A1MPK1 was used to

antagonize PK2 mediated migration. Values represent the mean ± SEM. *P < 0.001 and

**P < 0.05 represent significant differences between 1nM and 30nM PK2 versus Control,

respectively (n = 6).

Figure S3. Effects of PK2 on the migration of neuronal progenitors over time by a

collagen co-culture assay. Images depict distribution of cells migrating from a SVZa

explant co-cultured with stable PK2 cell-line at 16 hr (left) and 30 hr (right). Scale bar,

500 µm.

Figure S4. Neuronal nature of the cells migrating from SVZa explants. Image depicts

the distribution of cells migrating from a SVZa explant co-cultured with stable PK2 cell-

line (left). The same cells were stained for Tuj1, a marker for immature neurons (right).

Under different focal planes, essentially all migrating cells are Tuj1 positive. Scale bar,

500 µm.

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Figure S5. The effect of PK2 in a time-delayed co-culture assay (6). SVZa explants

were first cultured in collagen for 24 hr in the absence of control or the stable PK2

expressing cell line. Then either control or PK2 expressing cell aggregates were

positioned next to these explants, and the explants were cultured for an additional 24 hr.

Photographs were taken and the number of migrating neuronal progenitors were counted

in sampling quadrants (B) at two time points, immediately before (0 hr) and (24 hr) after

the placement of the cell aggregates. (A) The cells aggregates are at the top of the

photos in the 24 hr column. Twenty-four hours after the placement of PK2 cells (bottom

right) the number of neuronal progenitors on the proximal side of the explant is

significantly increased compared with 0 hr explant and compared the 24 hr control

explants (top right). (B) Diagram of the sampling quadrants used to quantify the time-

delayed co-culture experiment. (C) Quantification of the time-delayed experiment.

Individual cells were counted at 0 hr and 24 hr in the sampling quadrants shown in (B)

and mean number of cells per explant are shown. Error bars represent SEM. There was

a significant increase in the mean number neuronal progenitors cells migrating into

quadrants in I and II after the addition of the cell aggregates compared to the controls

(quadrant I: 80.1 ± 3.0 vs. 135.7 ± 8.3, control vs. PK2, respectively, *P<0.0001, n = 15;

quadrant II: 78.2 ± 2.1 vs. 124.0 ± 8.0, control vs. PK2, respectively, **P<0.0001, n

=15).

Figure S6. Morphological examination of olfactory bulbs (OB). (A) Left panel

displays the external morphology of WT OBs (left) and PK2 -/- OBs (right). The PK2 -/-

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OBs is greatly reduced compared to the WT OB. Right panel shows that the PK2 -/- OBs

are small and asymmetric (left). (B) Left panel illustrates the measurements taken to

quantify changes in OB diameter and GL thickness. For every 3rd Nissl-stained sagittal

section, the distances were measured at the midpoint between the anterior tip of the OB

and the flexure of the anterior border of the cerebral cortex and dorsal surface of the OB.

Middle and right panels respectively show decreased OB diameter and GL thickness in

PK2 -/- mice.

Figure S7. Immunohistochemical characterization for a subset of interneurons in the

periglomerular cell layer (PGL). Tyrosine hydroxylase (TH), the first enzyme in the

biosysthesis of dopamine, was used as a marker for dopaminergic interneurons. (A) Top

row shows DAPI (blue) stained coronal sections of WT OB (left) and PK2 -/- OB (right).

Middle row shows the corresponding sections (from the top row) immunostained for TH

(green). PK2 -/- OB exhibited a significant reduction in TH staining in the PGL

compared to WT OB. Bottom row shows a higher magnification of the images in the

middle row. The boxed region in the middle right image is shown in the bottom right

displaying very few TH positive cells. (B) Quantification of TH positive cells in the PGL

of WT and PK2 -/- OBs. (*P < 0.05, n = 3). Error bars represent SEM. Scale bars, 250

µm in top and middle row; 125 µm in bottom row.

Figure S8. Histological analysis for the enlargement of the olfactory ventricle (OV)

and rostral migratory stream (RMS) in PK2 -/- mice. (Top) A diagram shows the

approximate positions from which coronal sections were taken. (Middle) Top panels

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show the Nissl-stained sections depicting the OV, OV/RMS, and RMS from WT mice.

Along each section from the RMS to the OV, PK2 -/- mice have substantially wider

pathway and a denser number of cells compared to WT. (Bottom) A graph shows the

quantification of the width of OV-RMS pathway in WT and PK2 -/- mice. ** (P < 0.001)

indicates that there was a significant increase in the width of OV-RMS pathway at every

section in the PK2 -/- mice compared to WT (n = 3). Error bars represent SEM. Scale

bar, 250 µm.

Figure S9. Normal cell proliferation in the SVZ and RMS of PK2 -/- mice. The brain

was examined three hours after a single injection of BrdU was given to label the

mitotically active cells. (A, B) Sagittal section through the forebrain of WT (left) and

PK2 -/- mice (right) showed a similar distribution of BrdU+ cells in the SVZ (A) and in

the RMS (B). (C) Quantification of BrdU+ cells shows no difference in cell proliferation

in the SVZ and RMS of WT and PK2 -/- mice. (6577 ± 1152 vs. 6754 ± 1104 BrdU+

cells/mm2 in the SVZ and 6938 ± 542.9 vs. 6343 ± 1071 BrdU+ cells/mm2 in the RMS

for WT and PK2 -/- mice, respectively; n = 3) CC, corpus callosum; LV, lateral

ventricle; ST, striatum. Error bars represent SEM. Scale bar, 125 µm.

Figure S10. Accumulation of BrdU+ cells in the RMS of PK2 -/- mice. A coronal

section through the forebrain of WT (left) and PK2 -/- mice (right) shows an increased

number of BrdU+ cells in the RMS of PK2 -/- mice. Scale bar, 125 µm.

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Figure S11. Increase apoptosis in the OB of PK2 -/- mice. Cell death was examined

with TUNEL in WT OB (left) and PK2 -/- OB (right). TUNEL + cells are shown by the

arrows. TUNEL + cells were quantified in the OB of WT and PK2 -/- mice (bottom).

PK2 -/- OB exhibited a significant increase in apoptosis. (* P < 0.05, n = 3). Scale bar,

125 µm.

Figure S12. PKR2 mRNA expression in the OB and RMS of adult WT and PK2 -/-

mice OB. Left panels show autoradiograms of PKR2 mRNA expression in the RMS and

OB (WT, Top and PK2 -/-, Bottom). Center panels depicts PKR2-positive cells in low

and high magnifications in emulsion-dipped sections (WT, Top and PK2 -/-, Bottom).

Note the numerous PKR2-positive cells migrate away from the OV of WT OB and into

the GL and PGL (Top center panels). A notable decrease in PKR2-positive cells

migrating away from the OV was evident in PK2-/- OB (Bottom center panels). Right

panels show corresponding Nissl-stained sections of WT (Top) and PK2-/- (Bottom).

Scale bar, 500µm in left and left center panels; 250 µm in right and right center panels.

Figure S13. Absence of chemotaxis activity from the OB of PK2 -/- mice. The

granular cell layers (GL) were dissected from WT and PK2-/- OBs and a co-culture assay

with SVZa explants from neonatal rats was performed. Top left panel shows an

asymmetric distribution of neuronal progenitors from a SVZa explant directed towards

the WT GL. Bottom left panel shows that neuronal progenitors migrate symmetrically

around the SVZa explant when cultured with PK2 -/- GL. The bar graph shows the

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quantification of the proximal/distal distribution of SVZa cells in the co-culture assay.

(*P < 0.05, n = 6). Error bars represent SEM. Scale bar, 500 µm.

References

1. M. Y. Cheng et al., Nature 417, 405 (May 23, 2002). 2. C. M. Bullock, J. D. Li, Q. Y. Zhou, Mol Pharmacol 65, 582 (Mar, 2004). 3. A. G. Lumsden, A. M. Davies, Nature 306, 786 (Dec 22-1984 Jan 4, 1983). 4. C. M. Fan, M. Tessier-Lavigne, Cell 79, 1175 (Dec 30, 1994). 5. W. Wu et al., Nature 400, 331 (Jul 22, 1999). 6. M. Ward, C. McCann, M. DeWulf, J. Y. Wu, Y. Rao, J Neurosci 23, 5170 (Jun

15, 2003). 7. H. Wichterle, J. M. Garcia-Verdugo, A. Alvarez-Buylla, Neuron 18, 779 (May,

1997).