RESEARCH ARTICLE

Genome Wide Assessment of mRNA inAstrocyte Protrusions by Direct RNA

Sequencing Reveals mRNA Localization forthe Intermediate Filament Protein Nestin

Rune Thomsen,1 Jonatan Pallesen,1,2,3 Tina F. Daugaard,1

Anders D. B�rglum,1,2,3,4 and Anders L. Nielsen1,2,3

Subcellular RNA localization plays an important role in development, cell differentiation, and cell migration. For a comprehen-sive description of the population of protrusion localized mRNAs in astrocytes we separated protrusions from cell bodies in aBoyden chamber and performed high-throughput direct RNA sequencing. The mRNAs with localization in astrocyte protru-sions encode proteins belonging to a variety of functional groups indicating involvement of RNA localization for a palette ofcellular functions. The mRNA encoding the intermediate filament protein Nestin was among the identified mRNAs. By RT-qPCR and RNA FISH analysis we confirmed Nestin mRNA localization in cell protrusions and also protrusion localization ofNestin protein. Nestin mRNA localization was dependent of Fragile X mental retardation syndrome proteins Fmrp and Fxr1,and the Nestin 3’-UTR was sufficient to mediate protrusion mRNA localization. The mRNAs for two other intermediate fila-ment proteins in astrocytes, Gfap and Vimentin, have moderate and no protrusion localization, respectively, showing that indi-vidual intermediate filament components have different localization mechanisms. The correlated localization of Nestin mRNAwith Nestin protein in cell protrusions indicates the presence of a regulatory mechanism at the mRNA localization level forthe Nestin intermediate filament protein with potential importance for astrocyte functions during brain development andmaintenance.

GLIA 2013;61:1922–1937Key words: intermediate filaments, Nestin, Gfap, Vimentin, cytoskeleton, glia

Introduction

Astrocytes constitute the most abundant cell type in the

CNS (Allen and Barres, 2009; Freeman, 2010; Sofro-

niew and Vinters, 2010). Astrocytes typically exhibit a highly

polarized morphology extending multiple pseudopodial pro-

trusions participating in (i) establishing scaffolds for crawling

neurons during CNS development, (ii) establishing a part of

the gliovascular structure and a part of the blood-brain-

barrier, and (iii) mediating interactions with synapses aiding

in optimal neuronal signal transduction (Allen and Barres,

2009; Morest and Silver, 2003; Sofroniew and Vinters,

2010). Many studies have been carried out in order to under-

stand the dynamics of astrocyte protrusions. During cell dif-

ferentiation and migration the astrocyte morphology changes

dramatically by formation of pseudopodial protrusions driven

by coordinated polymerization and depolymerization of the

cytoskeleton (Etienne-Manneville, 2004). The mammalian

cytoskeleton consists of three types of filaments: actin fila-

ments, microtubules, and intermediate filaments (IFs). The

highly diverse family of IF proteins are encoded by �70

genes, and the complexity of the IF family is increased by

generation of multiple protein isoforms (Herrmann et al.,

2009). Proteins of the IF family are subdivided into different

classes due to sequence homology and capability to

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22569

Published online September 5, 2013 in Wiley Online Library (wileyonlinelibrary.com). Received Jan 9, 2013, Accepted for publication Aug 5, 2013.

Address correspondence to Anders Lade Nielsen, Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark. E-mail: aln@hum-gen.au.dk

From the 1Department of Biomedicine, Aarhus University, Aarhus, Denmark; 2Center for Integrative Sequencing, iSEQ, Department of Biomedicine, Aarhus

University, Aarhus, Denmark; 3Lundbeck Foundation Initiative for Integrative Psychiatric Research, iPSYCH, Department of Biomedicine, Aarhus University, Aarhus,

Denmark; 4Center for Psychiatric Research, Aarhus University Hospital, Aarhus, Denmark.

Additional Supporting Information may be found in the online version of this article.

1922 VC 2013 The Authors. GLIA published by Wiley Periodicals, Inc.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in anymedium, provided the original work is properly cited.

co-assemble into IFs (Eriksson et al., 2009; Herrmann et al.,

2009). IF proteins have a common overall structure com-

posed of an amino-terminal head domain and a carboxy-

terminal tail domain linked together by a highly coiled

a-helical rod domain. The rod domain is highly conserved

among IF proteins whereas the head and tail domains exhibit

a large degree of variability (Eriksson et al., 2009; Herrmann

et al., 2009; Middeldorp and Hol, 2011). The coiled a-

helical rod domain mediates generation of parallel homo-

dimers, and the homo-dimers form anti-parallel tetramers

that are linked head to tail. Eight tetramers associate into

unit-length filaments by which the mature 7- to 11-nm thick

IFs are comprised (Herrmann et al., 2009). Apart from pro-

viding static mechanical support to the cell structure, IF pro-

teins are also involved in dynamic reorganization of the cell

morphology during growth and migration (Eriksson et al.,

2009; Michalczyk and Ziman, 2005).

Astrocytes of the mammalian brain express the IF pro-

teins Nestin, Vimentin, and Glial fibrillary acidic protein

(Gfap) (Middeldorp and Hol, 2011). Nestin is considered a

marker for undifferentiated progenitor cells, down regulated

in terminally differentiated cells, but become reactivated dur-

ing injury responses in a process termed reactive gliosis char-

acterized by cell hypertrophy and proliferation (Gilyarov,

2008). The Nestin protein has a short amino-terminal head

domain of only six amino acids (Herrmann et al., 2009).

Nestin is unable to form homomeric filaments but coassem-

bles with Vimentin and Gfap (Eliasson et al., 1999; Herr-

mann et al., 2009; Michalczyk and Ziman, 2005). Nestin is

believed to play a pivotal role in cell morphology changes

during mitosis through the dynamic assembly and disassem-

bly of Vimentin including IFs (Chou et al., 2003). Vimentin,

like Nestin, is expressed early during brain development.

Vimentin and Nestin becomes gradually replaced by Gfap in

terminally differentiated astrocytes but Gfap expression has

also been described in neuronal and astrocyte progenitor cells

(Doetsch et al., 1999; Imura et al., 2006; Michalczyk and

Ziman, 2005; Middeldorp and Hol, 2011; Middeldorp et al.,

2010; Zhu and Dahlstrom, 2007). Gfap expression is upregu-

lated along with Nestin and Vimentin during reactive gliosis

after CNS injury (Pekny et al., 2007). Gfap mRNA is alterna-

tive spliced to generate protein isoforms whereas alternative

splicing generating protein isoforms of Nestin and Vimentin

is not yet described, but alternative Vimentin mRNA splicing

exists (Blechingberg et al., 2007a; Condorelli et al., 1999;

Middeldorp and Hol, 2011; Nielsen and Jorgensen, 2004;

Nielsen et al., 2002; Quinlan et al., 2007; Zhou et al.,

2010). Attention is emerging to understand how IFs are regu-

lated during cell growth, migration and morphology changes.

RNA localization in cell protrusions was demonstrated

in a considerable number of cell types including oocytes,

crawling fibroblasts and in axons and dendrites of neurons

(Doyle and Kiebler, 2011; Mili and Macara, 2009). RNA

localization is important for development, cell differentia-

tion and cell functionality (Mili and Macara, 2009). A lim-

ited number of studies have described IF mRNA

localization in astrocytes. One study demonstrated subcellu-

lar localization of the Nestin mRNA in protrusions of radial

glia cells (Dahlstrand et al., 1995). Gfap mRNA localization

was demonstrated in the branch points and distal parts of

astrocyte protrusions (Landry et al., 1994; Medrano and

Steward, 2001). However, detailed studies of the subcellular

mRNA localization patterns in astrocytes remains to be con-

ducted. Comprehensive examinations of RNA molecules

localized in fibroblast, neuronal and cancer cell protrusions

were performed by microarray analysis of RNA purified

from pseudopodial cell protrusions separated from cell

bodies using the Boyden chamber assay (Feltrin et al.,

2012; Mili et al., 2008; Shankar et al., 2010). In this study

we took advantage of a recently presented high throughput

next generation sequencing (NGS) method, termed single

molecule direct RNA sequencing (DRS) (Ozsolak et al.,

2009). DRS analyses were applied on mouse astrocyte RNA

from protrusions and cell bodies isolated using a modified

Boyden chamber assay. We identified numerous mRNAs

with localization to cell protrusions including NestinmRNA. We further analyzed Nestin mRNA and protein

localization in astrocyte protrusions and the presented

results indicate that the function of Nestin in reorganization

of astrocyte morphology can be regulated through coordi-

nated mRNA and protein localization.

Materials and Methods

Primary Cells, Cell Lines, and Brain TissuePrimary astrocytes cultures were prepared as described (Andres-

Barquin et al., 1994; Thomsen and Lade Nielsen, 2011). A pregnant

mouse of the NMRI mouse strain was purchased from Taconic,

Denmark and astrocytes were isolated from the cerebral cortex of

newborn (P0) mice. After 8 days in vitro (DIV) cells showed a

95% confluence and >80% of the cell were staining positive for

the astrocyte marker Gfap. Before experimental procedure cells

were trypsinized using 0.5% trypsin-EDTA (GIBCO). Under these

conditions, neurons, oligodendrocytes and microglia rapidly die or

do not adhere (Imura et al., 2006). The type 2 astrocyte mouse

cell line C8-S, the mouse embryo-derived teratocarcinoma cell line

P19, the mouse neuroblastoma cell line N1E-115, and the mouse

fibroblast cell line NIH/3T3 were purchased from the American

Tissue Culture Collection and cultured in Dulbecco’s Modified

eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS),

streptomycin, penicillin and glutamine, in a 5% CO2 humidified

atmosphere at 37�C. P19 cells were, if indicated, incubated with

retinoic acid (1 lM, R2625, Sigma) for 24 h. Whole brains were

collected from P0 and 21-month-old (P21m) NMRI mouse.

Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes

November 2013 1923

Boyden Chamber Isolation of Cell ProtrusionsCell protrusions were isolated using a modified Boyden chamber

assay as previously described (Thomsen and Lade Nielsen, 2011). To

obtain sufficient protein and RNA, six 9.6 cm2 cell culture inserts

(BD Falcon) with a 1-lm pore size polystyrene membrane, and three

75 cm2 growth flasks of cell culture were used for each experiment.

Cell culture inserts were coated with extra cellular matrix (ECM)

protein Collagen type-I (Sigma; C7661) (final concentration 10 lg

mL21 in phosphate buffered saline (PBS)) at 37�C for 2 h. The cell

cultures inserts were afterward directly transferred to a six-well tissue

culture dish containing serum free DMEM. Cells were grown to

90% confluence and medium changed to serum free medium with

antibiotics and glutamine before cells were grown for additional

24 h. Cells were detached using 0.5% Trypsin-EDTA, and the tryp-

sin was inactivated by dissolving cells in DMEM with 10% FBS.

Cells were pelleted and re-dissolved in serum free DMEM, and 2 3

106 cells were seeded for each cell culture insert. Cell protrusions

were growing through the membrane for 24 h at 37�C. Inserts were

washed in PBS and the cells were gently scraped off the membrane.

First the cell protrusion fraction (PF) was made by scraping the bot-

tom side of the membrane. Cells were lysed by carefully washing the

cell scraper in 1 mL TRI-Reagent (Sigma) for RNA or in 1 mL 1x

protein gel loading buffer (Fermentas) for protein isolation. After-

wards, the cell body fraction (CF) was made by scraping the upper

side of the membrane and dissolving the cell material in TRI-

reagent or 1x protein gel loading buffer.

RNA Purification, cDNA Synthesis and Real TimeQuantitative PCR (RT-qPCR)RNA from cells and tissues was purified by standard TRI-reagent

protocol (Sigma) using 1 lg of glycogen for precipitation (Thomsen

and Lade Nielsen, 2011). cDNA was made using an iScript cDNA

synthesis kit (Bio-Rad). Nearly 1 lL of total RNA solution was used

per reaction. RT-qPCR was performed using SYBR Green 480 mas-

ter mix (Roche). RT-qPCR analyses were performed using a Roche

LightcyclerTM 480, with a primer annealing temperature of 58�C.

Primers were designed as intron spanning and PCR amplicons were

verified by gel electrophoresis and melting curve peak analysis.

Primer sequences are shown in Supporting Information Table 1.

Direct RNA Sequencing (DRS), Data Processing andAnalysisA modified Boyden chamber assay was used for isolation of cell pro-

trusions and cell bodies as previously described (Thomsen and Lade

Nielsen, 2011). Single RNA molecules were sequenced by DRS

(Ozsolak et al., 2009) using the Helicos Biosciences platform (Heli-

cos Biosciences, Boston, MA). Sequencing reads were mapped to a 2

kb region surrounding the 30 distal part of 27,131 genes for poly-

adenylated RNA of the mm9 version of the mouse genome. Bioin-

formatics raw data analysis and sequence alignment was made by

Helicos Biosciences, and transcript reads were presented as an excel

spread sheet. Details of the RNA preparation and sequencing can be

found at http://www.helicosbio.com/. Expression values were proc-

essed as RNA transcripts per million reads (tpm). To avoid including

false positives due to stochastic counts in the lower range, transcripts

exhibiting counts of <5 tpm in both PF and CF were excluded. The

RNA enrichment in protrusions was presented as the relative expres-

sion value in PF compared to CF [tpm PF/tpm CF]. A list of the

250 transcripts with the highest ratio were submitted for ordination

of gene name, location, and type, followed by functional annotation

analysis using the IPA ingenuity online platform, URL: http://

www.ingenuity.com. For increased stringency a direct relationship

analysis was performed including pathways described both human

rat and mouse. Results were presented with P values calculated by

the Benjamini-Hochberg method, to control for multiple testing.

RT-qPCR amplifications were made in triplicates for each gene and

Ct values were converted into linear values using the Xo method

(Thomsen et al., 2010). The mRNA localization ratio was calculated

as the ratio between the mean expression level in CF and PF and

afterwards normalized to the determined mRNA localization ratio

for Arpc3. Differences in localization ratios were analyzed by a Stu-

dent’s unpaired two tailed t test. All experiments were repeated three

times.

siRNA TransfectionsFor siRNA experiments 1.000.000 C8-S cells were immediately

before the transfection plated into 10 cm dishes in DMEM with

10% FBS. In 640 lL serum free medium was mixed siRNA to a

final concentration of 2 lM and incubated for 5 min. Nearly 13 lL

Dharmafect was mixed with 1267 lL serum free medium and incu-

bated 5 min. The two solutions were mixed and incubated 20 min,

added to the cells, and incubated 24 h. The medium was changed to

serum free medium and cells incubated for further 24 h. The cells

were used in a standard Boyden chamber assay with 1 lm mem-

branes and RNA purified from three membranes for each transfec-

tion and pooled. siRNA sequences Fmr121063: GGAUCAAGA

UGCAGUGAAA; Fmr12447: GUGAUGAAGUUGAGGUUUA;

Fxr12219: GAGAUGAAGUAGAGGUAUA; Fxr12560: GCAACU

GUGAAGAGAGUAA; Fxr221269: GGAAAGAACGGGAAAG

UGA; Fxr221336: GAGAUAACGACAAGAAGAA; non-specific

control: AGGUAGUGUAAUCGCCUUG.

Cloning of 30-UTR Reporter Constructs andTransfectionsThe 30-UTR sequences were amplified from mouse C8S cDNA

(primer sequences are shown in Supp. Info. Table 1) and purified

bands digested with XmaI and XbaI. The pcDNA5-beta-globin-

6xMS2-SV40-LpA vector was XmaI and XbaI digested and after liga-

tion with UTR inserts and E. coli transformation, positive constructs

were verified by sequencing. To remove the 6xMS2-sites the vectors

were digested with NotI and SmaI and by Klenow polymerase treat-

ment blunt-ended. After ligation and E. coli transformation con-

structs were verified by sequencing. The constructs were pooled and

transfected into NIH/3T3 cells using 2 lg total DNA mixture. In

parallel was transfected the control construct pTAG4 (Blechingberg

et al., 2007b). For each transfection were used 150,000 cells in six-

well plates using 200 lL serum-free medium and 3 lL Xtreme

Gene 9 DNA transfection reagent version 03 (Roche). Cells were

incubated 24 h before a medium shift to serum free medium and a

subsequent incubation for 24 h. The cells were used in a standard

1924 Volume 61, No. 11

Boyden chamber assay with 1 lM membranes and RNA purified

from three membranes for each transfection and pooled.

Western Blot AnalysisProteins were extracted for Western blot analysis by the Boyden

chamber assay. To normalize the protein amount from CF and PF

a-Tubulin and Actb were used as loading controls. Proteins were sep-

arated by SDS-PAGE in a 4–15% gradient polyacryl amide gel (Bio-

Rad). Proteins were transferred to a nitrocellulose membrane and

analyzed with following primary antibodies: rabbit anti a-Tubulin

(Rockland), rabbit anti b-Actin (Actb) (Sigma; A2103), rabbit anti

Histone H3 (Abcam; ab1791), mouse anti Nestin (Millipore;

MAB253), Mouse monoclonal anti Vimentin (Abcam; ab20346),

and goat anti Gfap (Santa Cruz; sc-6170). Nestin antibody was

diluted 1:1,000 and all other antibodies were diluted 1:10,000.

Horse radish peroxidase conjugated anti mouse, anti rabbit and anti

goat secondary antibodies (DAKO) were used for detection.

Single RNA Molecule FISH and ISHSingle molecule RNA fluorescence in situ hybridization (FISH) was

essential done as described (Femino et al., 1998). Probes consisting

of 50-mer single stranded DNA oligonucleotides were synthesized

and labeled with 4–5 Cy3 fluorophores. A total of eight various oli-

gonucleotides were hybridized to each target mRNA. Cells were

seeded onto 0.17-mm-thick coverslips (Marienfeld) either coated

with Collagen type 1 or uncoated and cultured in DMEM with

10% FBS, penicillin, streptomycin, and glutamine. At �60% con-

fluence cells were fixed in 4% paraformaldehyde for 20 min at room

temperature, and washed and stored in phosphate buffered saline

(PBS) at 4�C. Before hybridization, cells were permeabilized using

0.5% triton X-100 in PBS for 10 min at room temperature, washed

in PBS, and then incubated in pre-hybridization solution: (50%

formamide (Sigma; F4761) and 2 3 SSC (Ambion)) for 15 min. at

room temperature. The probes were hybridized in prehybridization

solution supplemented with 2 mg mL21 BSA (Roche), 0.2 mg

mL21 E. coli tRNA (Roche), and 0.2 mg mL21 sheared salmon

sperm DNA (Sigma; D7656) for 3 h at 37�C. About 10 ng DNA

probe was used per coverslip. Cells were washed twice with pre-

hybridization solution for 20 min at 37�C, then 10 min in 23 SSC

at room temperature, and in PBS for 10 min at room temperature.

Cell nuclei were counterstained with DAPI (0.5 mg L21 in PBS).

After a final wash in PBS, coverslips were rinsed in double distilled

water to remove excess salt, dried and mounted using ProLong gold

(InVitrogen). Actb probes were a kind gift from Dr. Robert H.

Singer. Probe sequences for Nestin and Gfap mRNA are shown in

Supporting Information Table 2. SSA4 oligonucleotides were previ-

ously described (Jensen et al., 2001).

Mouse P4 sagittal section mRNA in situ hybridization (ISH) data

for Nestin, Gfap, Actb, CamKIIa and Tubb3 were extracted from Allen

Developing Mouse Brain Atlas (http://developingmouse.brain-map.org)

and used for publication with permission.

ImmunofluorescenceCells were grown on 0.17 mm coverslips until 60% confluence, then

fixed in 4% paraformaldehyde for 20 min at room temperature, and

washed and stored in PBS at 4o C. Cells were permeabilized using

0.5% triton X-100 in PBS for 10 min at room temperature. A

blocking step was made using 1% BSA in PBS for 30 min at room

temperature. Primary antibodies were dissolved in blocking buffer

and incubated for 1 h at room temperature. Cells were washed three

times in PBS and incubating with secondary antibody dissolved in

blocking buffer. After three washes in PBS double immunofluores-

cence was performed as described above with a second treatment of

primary and secondary antibodies. After final secondary antibody

incubation cell were washed two times and cell nuclei were stained

with DAPI and washed once in PBS. Coverslips were rinsed in dou-

ble distilled water to remove salt, dried, and mounted with ProLong

gold. Primary antibodies used: Mouse anti Nestin (Milipore; MAB

253), mouse anti Vimentin (Abcam; ab20346), rabbit anti Gfap

(DAKO), rabbit anti a-Tubulin (Rockland), and rabbit anti Actb

(Sigma; A2103). All primary antibodies were diluted 1:500. Second-

ary antibodies were Alexa 488 conjugated goat anti rabbit IgG (Invi-

trogen A11034) and Alexa 555 conjugated goat anti mouse IgG

(Invitrogen A21127) both diluted 1:2000.

Microscopy and Image ProcessingAll images were made on a Zeiss axiovert 200m microscope, with a

plan apochromatic 633 1.4 NA objective, a HBO 100 W mercury

light source, and a CoolSNAP-HQ camera (Roper Scientific), oper-

ated by the MetaMorphVR software. Filters were from Chroma, Cy3

(41003), FITC (41001) and DAPI (31000). For RNA FISH analy-

ses were used a z-stack of 20 z-sections with 0.2-lm step size and

500 msec exposure. For FISH images z-stacks were collapsed to a

2D maximum intensity projection and for immunofluorescence a

single 2D image was selected from a 20 section z-stack. Images were

processed by background subtraction and normalization using the

open source software Image J (url: rsbweb.nih.gov/ij). To calculate

the mRNA localization ratio FISH images were processed by sub-

tracting the mean background value and the coordinates of the cell

center was determined manually in the DAPI channel, and the coor-

dinates of the apex of the cell protrusion was determined in the Cy3

channel. The intensities of each pixel and their respective coordinates

together with the coordinates of the cell center and protrusion were

imported into a custom made computer program. In this program, a

line was drawn from the center of the cell to the apex of the protru-

sion, and each point was perpendicularly projected to this line. A

pixel was categorized as localized if the position was more than 2/3

of the total length of the line toward the apex of the protrusion. A

localization score was finally calculated by dividing the sum of the

intensities of all localized pixels by the sum of the intensities of all

pixels projected on the line.

Statistical AnalysisRT-qPCR amplifications were made in triplicates for each gene and

Ct values were converted into linear values using the Xo method

(Thomsen et al., 2010). All the relative expression levels were nor-

malized to Arpc3 (for mRNA localization) or Gapdh (for mRNA

expression). Differences in the localization ratios and expression lev-

els were analyzed by a Student’s unpaired two tailed t test. All

experiments were repeated three times.

Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes

November 2013 1925

Results

Identification of Localized mRNAs in Mouse PrimaryAstrocyte Protrusions by DRSRNA localization has shown to play pivotal roles in cell sig-

naling, morphology, and migration during both embryonic

development, brain maintenance and in cancer metastasis

(Mili and Macara, 2009). Whereas mRNA localization in

neurons is extensively described, RNA localization in astro-

cytes is rather uncharacterized. To identify on a genome

wide scale mRNA species localized in astrocyte protrusions

we took advantage of the Boyden chamber cell fractionation

method to separate cell protrusions from cell bodies

(Thomsen and Lade Nielsen, 2011). Mouse primary astro-

cyte cultures were established from P0 mouse cortices and

grown for 8 DIV. The mRNA expression pattern in the

mouse primary astrocytes and two brain samples was by

RT-qPCR examined for expression of cell type specific

markers for microglia (Aif1), endothelial cells (Pecam1), oli-

godendrocytes (Mbp and Cnp), neurons (Nptx1), and astro-

cytes (Aldh1l1 and Gfap) (Imura et al., 2006; Stahlberg

et al., 2011) (Supp. Info. Fig. 1). The expression analysis

supported an in majority astrocyte lineage content of the

primary astrocyte cultures and this was further substantiated

by immunofluorescence staining showing that more than

80% of the cells were GFAP positive as also previously

described (Imura et al., 2006; Pekny et al., 1998; Stahlberg

et al., 2011; Thomsen and Lade Nielsen, 2011). It should

be emphasized that the heterogeneity of the primary astro-

cyte cultures still could result in identification by the Boy-

den chamber approach of mRNA species with expression

and RNA localization which cannot be confined to astro-

cytes. Compared to Stahlberg et al. (2011) presenting data

for 10–12 DIV primary astrocytes we note relative more

Nestin mRNA expression in our 8 DIV primary astrocyte

cultures (Supp. Info. Fig. 1). In accordance, by RT-qPCR

analysis of primary astrocyte cultures grown further DIV we

observed a decrease in Nestin mRNA expression (Supp.

Info. Fig. 2). We note a decrease in cell culture growth

capability and lack of efficient plating in the Boyden cham-

ber setting by using older DIV primary astrocyte cultures

and selected eight DIV astrocytes for the subsequent

analysis.

By RT-qPCR purification by the Boyden chamber

assay of protrusion and cell body RNA from eight DIV

mouse primary astrocytes was consolidated as described

(Thomsen and Lade Nielsen, 2011). Absence of nuclear

contamination of the PF was further controlled by the lack

of DAPI staining and lack of histone proteins. Reproducible

NGS analysis by single molecule DRS using minute RNA

quantities were recently presented (Ozsolak et al., 2010). In

the DRS procedure cDNA synthesis and amplification are

evaded and NGS analysis can be performed directly on pol-

yadenylated RNA (Ozsolak et al., 2009). RNA samples

from primary mouse astrocyte PFs and CFs isolated by the

Boyden chamber assay were analyzed by DRS. The resulting

output of 36 bases average size sequences were mapped to a

2 kb region surrounding the distal 30 end of 27131 genes of

the mm9 mouse genome. The total number of mapped

sequence counts in primary astrocytes was 5444770 for the

CF and 2147050 counts for the PF. The individual

sequence number for each transcript was normalized to

transcripts per million reads (tpm). The tpm values repre-

sent the relative abundance of a given mRNA in the total

population of mRNA molecules present in the CF or the

PF. In all subsequent analyses we included transcripts with

count numbers �5 tpm in both CF and PF to avoid insig-

nificantly expressed mRNAs and false positives due to even-

tual stochastic fluctuations in the lower range sequence

number. The total number of transcripts having �5 tpm in

both CF and PF was 8894. The localization ratio was calcu-

lated as the ratio between the tpm values from the PF and

CF, and 2298 mRNAs were determined to have a localiza-

tion ratio >1 (Fig. 1A). Complete list is shown in Support-

ing Information Table 3A and the 250 mRNAs with

highest localization ratios are shown in Supporting Informa-

tion Table 4. The localization ratio for a given mRNA will

be a variable depending on cell morphology and experimen-

tal settings, but the hierarchical order for localization ratios

is envisaged to be relative independent of these variable fac-

tors. Moreover we note that a mRNA localization ratio> 1

is not describing that a majority of this mRNA is localized

in protrusions but instead describes the relative enrichment

in protrusions and can thereby represent localization of only

a minor fraction of the total amount of a particular mRNA

within the cell. DRS determined mRNA localization ratios

for Rab13 mRNA (ratio 16.7), p0071/Pkp4 mRNA (ratio

15.7), and Kank2/Ankrd25 mRNA (ratio 10.9) are in

accordance with previous mRNA localization observations

(Mili et al., 2008; Thomsen and Lade Nielsen, 2011).

These results demonstrate that the Boyden chamber assay

combined with single molecule DRS are applicable to iden-

tify localized RNAs on a genome wide scale. Comparing

the 250 most localized mRNAs with mRNA expression data

from in vivo mouse P7 astrocytes (Cahoy et al., 2008) con-

firmed expression of the localized mRNAs in astrocytes invivo (Data not shown). Annotation analysis revealed that

the most localized mRNAs encode a broad variety of pro-

tein families (Supp. Info. Tables 4, 5 and Fig. 3). The most

localized mRNAs encode proteins with a localization pat-

tern strikingly similar to the total population of expressed

mRNAs and in this line we notice that a significant fraction

of protrusion localized mRNAs encode nuclear proteins.

1926 Volume 61, No. 11

mRNA Localization Analysis in the Mouse C8-SAstrocyte Cell LineWe next included the mouse astrocyte like cell line C8-S

that originally was isolated from the cerebellum of a post

natal (P8) mouse and resembles type II astrocyte Bergmann

glia cells (Alliot and Pessac, 1984). C8-S is a homogenous

and non-cancerous cell line with a highly polarized mor-

phology making it a qualified model for RNA localization

studies. We note that the RNA localization analysis in

mouse primary astrocytes represent an analysis of a hetero-

geneous cell population whereas C8-S RNA localization

analysis would be confined to a more homogenous cell type

of astrocyte lineage. To identify mRNAs localized in C8-S

cells, we again separated protrusions from the cell bodies by

the Boyden chamber assay and purified total RNA for

DRS. Sequences were mapped to a 2 kb region of the 30

distal part of 27131 genes from the mm9 mouse genome

sequence. The total counts number for CF was 2269453

and for PF 2090020, and 7697 transcripts had read num-

bers �5 tpm in both PF and CF. The normalized reads

were plotted as the ratio between PF and CF (Fig. 1C). The

number of transcripts with a localization ratio> 1 was

1436. The 250 mRNAs with highest localization ratios are

shown in Supporting Information. Table 6 and the com-

plete list in Supporting Information Table 3B. Annotation

analysis revealed that the most localized mRNAs encode a

FIGURE 1: Transcriptome analysis by DRS. (A) Graphical summary of the Boyden chamber assay for isolation of cell protrusions. ECMindicates coating of the membranes with Collagen type-1 prior to the seeding of cells to stimulate protrusion migration through the1 lm pore size membrane. Protrusions were growing for 24 h before isolation. The protrusions and cell bodies were isolated in two sep-arate fractions before subsequent analysis. (B,C) DRS transcriptome analysis of Boyden chamber purified RNA from mouse primary astro-cyte protrusions (B) and mouse C8-S cell protrusions (C). Bar plots displaying the RNA localization ratios between the normalizednumbers of DRS identified transcripts in protrusions over normalized number of transcripts in cell bodies in a hierarchical order. RNAswith �5 tpm in both protrusions and cell bodies are included. (D) Plot of the RNA localization ratio in primary astrocytes relative to theRNA localization ratio in C8-S cells. For visualization a red line indicates equal localization ratios, a dashed green line indicates the 26relative most localized RNAs in C8-S cells and a dashed yellow line indicating the 28 relative most localized RNAs in primary astrocytes.(E) RT-qPCR analysis of the RNA localization ratio in C8-S cells compared with the RNA localization ratio in primary astrocytes. RNAlocalization ratios are normalized to Arpc3. P values were calculated by an unpaired student’s t test. [Color figure can be viewed in theonline issue, which is available at wileyonlinelibrary.com.]

Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes

November 2013 1927

broad variety of protein families (Supp. Info. Table 7 and

Fig. 3).

Comparative mRNA Localization Analysis of MousePrimary Astrocytes and C8-S CellsA total of 7119 mRNA species had transcript reads �5 tpm

in PF and CF for both C8-S cells and mouse primary astro-

cytes. To examine whether we could identify cell type specific

localized mRNAs, we plotted the localization ratio of primary

astrocytes against C8-S cells (Fig. 1D). The analyses revealed

that majority of the localized mRNAs in mouse primary

astrocytes also localized in C8-S cells but also identified vari-

ability in localization patterns (Supp. Info. Tables 8 and 9).

Moreover, we note that the annotation analysis of localized

mRNAs also revealed differences for C8-S and primary astro-

cytes (Supp. Info. Fig. 3, Supp. Info. Tables 5 and 7).

To verify observed mRNA localizations in primary

astrocytes and C8-S cells we conducted RT-qPCR analysis of

representative candidates using independent biological RNA

samples from Boyden chambers. We selected Tensin3 (Ten3)

mRNA as it is highly expressed in both primary astrocytes

and C8-S cells but exhibited a larger RNA localization ratio

in primary astrocytes than C8-S cells, sperm flagellar 1

(Spef1) mRNA which is highly expressed in both primary

astrocytes and C8-S cells but with a higher RNA localization

ratio in C8-S cells than primary astrocytes, cytochrome c oxi-

dase subunit IV isoform 1 (Cox4i1) mRNA which is highly

expressed but displays no RNA localization in primary astro-

cytes and C8-S cells, and Rab13 and p0071/Pkp4 mRNAs

which are localized in both cell types. The RNA localization

ratios were normalized to Arpc3, a component of the Arp2/3

complex (Mili et al., 2008). Arpc3 mRNA showed localiza-

tion in neither the DRS analysis and nor in RT-qPCR analy-

ses using NIH/3T3 cells, primary astrocytes and C8-S cells

(Feltrin et al., 2012; Mili et al., 2008; Thomsen and Lade

Nielsen, 2011). RT-qPCR analysis revealed a significant local-

ization of Tensin3 mRNA in primary astrocytes compared to

C8-S cells and a significant localization of Spef1 mRNA in

C8-S cells compared to primary astrocytes (Fig. 1E). Cox4i1mRNA had no RNA localization in primary astrocytes and

C8-S cells, whereas Rab13 and p0071 mRNAs have RNA

localization in both cell types (Fig. 1E). The results of the

RT-qPCR analysis were similar to the DRS analysis, support-

ing that the Boyden chamber method combined with a DRS

analysis also is applicable for identification of specific RNA

localization patterns between different cell types.

Next we searched for candidate mRNAs having protru-

sion localization and in addition being enriched for astrocyte

expression. A comprehensive identification of mRNAs rela-

tively enriched in in vivo mouse astrocytes, mature astrocytes,

and developing astrocytes compared to other brain cell types

was described by Cahoy et al. (Cahoy et al., 2008). The study

also included identification of mRNAs enriched in in vitrogrown astroglia cells compared to in vivo astrocytes (Cahoy

et al., 2008). Of the 250 most localized mRNAs in mouse

primary astrocytes 28 were also identified to be enriched in at

least one of these four defined astrocyte populations (Supp.

Info. Table 10). Twenty two of the mRNAs have enriched

expression in astrocytes in vivo and 15 have enriched expres-

sion in astroglia cells grown in vitro (Supp. Info. Table 10).

We note that all identified mRNAs to be both localized in

astrocyte protrusions and expression enriched in developing

astrocytes in vivo also are relatively enriched in primary astro-

glia cells in vitro [Supp. Info. Table 10 and (Cahoy et al.,

2008)]. Moreover, the comparative analysis pointed that albeit

some mRNAs with localization in astrocyte protrusions have

enriched expression in astrocytes only few were assigned to be

astrocyte specific (Dio2, Ppp1r3c and Gfap). Recently, Feltrin

et al. described 80 mRNAs localized in mouse N1E-115 neu-

roblastoma cells (Feltrin et al., 2012). Of the 25 most protru-

sion localized mRNAs in mouse primary astrocytes and C8-S

cells 13 mRNAs were also identified to be significantly local-

ized in N1E-115 cells and of the 80 most protrusion localized

mRNAs in mouse primary astrocytes and C8-S cells, 28 and

26, respectively, were also significantly localized in N1E-115

cells (Supp. Info. Tables 11 and 12) (Feltrin et al., 2012).

Such mRNAs could represent a group of commonly expressed

and protrusion localized mRNAs and several of these were

also identified to be localized in mouse fibroblast protrusions

(Mili et al., 2008). Of the 80 N1E-115 localized mRNAs 4

were overlapping with the astrocyte enriched and localized

mRNAs (Cyb5r3, Ddr2, Arhgap11a, and Kctd10) (Supp. Info.

Table 10).

mRNA Localization for the IF Protein NestinWe observed that the mRNA for the IF protein Nestin had a

high localization ratio in both mouse primary astrocytes and

in C8-S cells and a preferential expression in developing

astrocytes (and other neural cell type progenitors in vivo)(Fig. 2A, Supp. Info. Tables 4, 6, 10) (Cahoy et al., 2008).

One of our long term research aims is to identify mechanisms

for IF regulation and we accordingly focused the subsequent

studies on Nestin mRNA. In primary astrocytes NestinmRNA had a localization ratio of 42 and was identified as

the relatively most protrusion localized mRNA. In C8-S cells

the localization ratio was 19. The RNA localization ratios for

the two Nestin related IF proteins in astrocytes, Gfap and

Vimentin, were 5 and 0.8 in primary astrocytes and 1 and

0.7 in C8-S cells, respectively. The RNA localization outcome

of the DRS analysis was confirmed by RT-qPCR analyses

using independent biological samples including primers

against Nestin, Vimentin, and Gfap cDNA. As references we

1928 Volume 61, No. 11

included analyses of Actb and Arpc3 cDNA. The RNA local-

ization ratios were normalized to Arpc3 given the value 1

(Fig. 2B). The RT-qPCR results from mouse primary astro-

cytes showed localization of Nestin mRNA in the cell protru-

sions with a localization ratio of 130. Likewise, we noted that

the Gfap mRNA was localized in the protrusions with a 13

times higher ratio than Arpc3. The Vimentin mRNA had no

localization, with a ratio of 0.6, which was concurrent to the

ratios of Arpc3 and Actb mRNAs. In a similar RT-qPCR

experimental setting using RNA material from C8-S cells Nes-tin mRNA showed a significant localization in cell protru-

sions, whereas neither Vimentin nor Gfap mRNAs displayed

localization (Fig. 2C). We note that the Gfap mRNA expres-

sion in C8-S cells was approximately 500-fold lower than for

primary astrocytes. In a time course experiment for C8-S cell

protrusion growth Nestin RNA localization was at maximum

FIGURE 2: Nestin mRNA localization in cell protrusions from mouse primary astrocytes and C8-S cells. (A) Localization ratios determinedby DRS for mRNA for IF proteins and controls. (B,C) RT-qPCR analysis of RNA localization ratios in primary astrocytes (B) and C8-S cells(C). cDNA was made from RNA from the protrusion and the cell body fractions. The ratios are normalized to Arpc3. (D) RT-qPCR analysisof RNA localization ratios in C8-S cells after growth in Boyden chamber assays for the indicated times. The RNA localization ratios arenormalized to Arpc3 and subsequently to the RNA localization ratio after 24 h growth time given the value 100. (E) RT-qPCR analysis ofRNA localization ratios in C8-S cells after growth in Boyden chamber assays with Laminin and Fibronectin membrane coating. (F) RT-qPCR analysis of RNA localization ratios in P19 cells without (2RA) and with (1RA) retinoic acid incubation for 24 h and the localizationratios normalized to Arpc3.

Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes

November 2013 1929

after 24 h (Fig. 2D). Nestin RNA localization was also

observed in C8-S cells after coating membranes in the Boy-

den chamber with the extracellular matrix proteins Laminin

or Fibronectin (Fig. 2E), whereas absence of coating hindered

quantitative experiments because only few cell protrusions

penetrated the membrane.

To examine if the observed Nestin mRNA localization

was restricted to cells of astrocyte lineage we next examine

mouse embryo-derived teratocarcinoma P19 cell line in the

Boyden chamber setting. P19 cells are pluripotent and can

differentiate into cell types of all three germ layers and in

response to retinoic acid treatment P19 cells first become

neural stem-like cells with Nestin expression and finally dif-

ferentiate to neural cells (neuron, glia, etc.) (Jones-Villeneuve

et al., 1982; Tan et al., 2010). After the Boyden chamberassay P19 cells without incubation with retinoic acid had alow level of Nestin mRNA expression but RT-PCR analyseswere able to determine Nestin mRNA localization to the P19cell protrusions (Fig. 2F). After retinoic acid incubation for24h Nestin mRNA expression was increased �25-fold (datanot shown) and again Nestin mRNA localization to cell pro-trusions was determined (Fig. 2F). We were unable to detectGfap mRNA expression to a significant level in the P19 cellsamples (data not shown). We conclude that the NestinmRNA present in mouse P19 pluripotent embryonic carci-noma cells also have protrusion localization capability.

Cis-elements determining mRNA localization are typi-

cally enclosed in the 3’-UTR (Mili and Macara, 2009). To

determine if the 30-UTR was involved in mediating localiza-

tion of Nestin mRNA to cell protrusions we cloned the 419

bp 30-UTR sequence including the intrinsic poly-adenylation

signal into a modified pcDNA5-beta-globin-6xMS2-SV40-

LpA vector downstream of the spliced b-globin transcription

unit. We also cloned the Rab13 30-UTR for positive localiza-

tion control and the Vimentin 30-UTR for negative control

together with the vector without an UTR insert. We were

unable to transfect mouse primary astrocytes and C8-S suffi-

ciently for quantitative ectopic RNA localization measure-

ments and instead used mouse NIH/3T3 cells which

previously were used as model in mRNA localization studies

(Mili and Macara, 2009). After cell transfection and Boyden

assay purified RNA was examined by RT-qPCR for expression

of the ectopic mRNAs by a forward primer recognizing b-

globin exon 1 included in the chimeric mRNAs and a reverse

primer corresponding to the inserted UTR fragments or aspecific primer for the vector without an UTR insert. cDNAfrom the PF and the CF from cells transfected with the con-

trol vector, pTAG, resulted in no significant RT-qPCR ampli-fication. Insertion of the Nestin 30-UTR resulted in anapproximately fourfold increase in localization ratio comparedto the Vimentin 30-UTR and vector control (Fig. 3A). The

Rab13 30-UTR resulted in a further fourfold increase in

localization ratio (Fig. 3A). From the transfection experimentswe conclude that the Nestin 30-UTR includes sequence deter-minants which alone can mediate RNA localization.

We noted the presence of a high Guanine (G) content

and algorithm predicted putative G-quadruplex motifs in

the Nestin 30-UTR (http://bioinformatics.ramapo.edu/

QGRS). The fragile X mental retardation protein family

composed of Fmrp, Fxr1, and Fxr2 can through the RGG-

box associate with G-quadruplex motifs and mediate trans-

port to neuronal dendrites (Bagni and Greenough, 2005;

Darnell et al., 2001; Mili et al., 2008; Schaeffer et al.,

2001). To determine if these factors are involved in NestinmRNA localization we depleted C8-S cells of these factors

individually or in combinations by transient siRNA treat-

ments and by Boyden chamber following measured NestinmRNA localization. Semi-quantitative measurements

showed that the Fxr2 mRNA expression level in C8-S was

several fold lower than Fxr1 (data not shown). Fmr1mRNA, which encodes the Fmrp protein, was also lower

expressed than Fxr1 (data not shown). Fmr1, Fxr1 and Fxr2

siRNA mediated co-depletion resulted in an approximately

twofold reduction in the Nestin RNA localization ratio (Fig.

3B). For the individual siRNAs we observed that depletion

of Fmr1 and Fxr1 resulted in an approximately twofold

reduction in the Nestin RNA localization ratio supporting

that the proteins, directly or indirectly, are involved in Nes-

tin mRNA localization (Fig. 3B).

FISH Analysis of Nestin mRNATo substantiate the Nestin mRNA localization studies we

examined for Nestin mRNA localization in mouse primary

astrocytes by FISH. For optimal cytoplasmic RNA detection

we took advantage of the single RNA molecule FISH tech-

nique (Femino et al., 1998). We designed probes complemen-

tary to mRNAs of Nestin and Gfap. For the FISH assay we

used 50-mer DNA oligonucleotide probes each labeled with

five covalently coupled Cy3 fluorophores and single mRNA

molecules were detected by hybridizing a pool of eight differ-

ent probes targeting each mRNA species. For control we

included probes against Actb mRNA. Primary astrocytes (8

DIV) plated on Collagen ECM protein coated coverslips

were used for analysis and the results of the FISH assays were

monitored by blinded cell counting. Approximately 50% of

the primary astrocytes were scored positive for Nestin mRNA

expression due to the number of Nestin mRNA FISH signals.

We note that Nestin mRNA FISH signals also could be

detected in cells initially scored negative for Nestin mRNA

expression by the blinded cell counting indicating that the

actual number of Nestin expressing cells is higher than 50%.

In approximately half of the positively scored cells numerous

Nestin mRNA FISH signals could be detected in cell

1930 Volume 61, No. 11

protrusions in accordance with Nestin mRNA protrusion

localization (Fig. 4A). FISH analysis showed that GfapmRNA was more uniformly distributed in the cytoplasm

compared to Nestin mRNA. However, Gfap mRNA could be

observed in the outermost regions of the cytoplasm in a

majority of the Gfap positive cells, whereas Actb mRNA pre-

dominantly was confined to the peri-nuclear part of the cells

(Fig. 4A).

We also examined subcellular RNA localization in C8-S

cells by RNA FISH, using probes against Nestin, Actb and

mRNA for the S. cereviseae heat shock protein SSA4 as nega-

tive control. Gfap mRNA expression is very low in C8-S cells

and accordingly not included in the analysis. C8-S cells were

plated on collagen ECM protein coated coverslips and the

results of the FISH assay were analyzed by blinded cell count-

ing. Most C8-S cells (>99%) were scored positive due to the

number of Nestin mRNA FISH signals. In approximately half

of the positively scored cells numerous Nestin mRNA FISH

signals could be detected in cell protrusions in accordance

with protrusion localization of Nestin mRNA (Fig. 4B, upper

panels). Actb mRNA was predominantly confined to the peri-

nuclear region of the cell body with a low amount of mRNA

also observed in cell protrusions (Fig. 4B, middle panels).

The negative control probe showed no signal (Fig. 4B, lower

panels). To determine if there is an effect of ECM compo-

nents on Nestin mRNA localization we performed a similar

RNA FISH analysis using C8-S cells plated on ECM

uncoated coverslips (Supp. Info. Fig. 4). We observed no

changes in Nestin and Actb mRNA localization patterns

(Supp. Info. Fig. 4). To further examine the difference of

mRNA localization between Nestin and Actb in C8-S cells,

we randomly selected 10 cells with Nestin mRNA signals and

9 cells with Actb signals (Fig. 4C). Signal intensities were

summarized and signals that displayed a relative distance

more than two thirds from the center of the nucleus were

regarded as localized. Localized signal intensities were divided

by the total signal intensities within the cell to obtain a local-

ization ratio. The ratios were statistically analyzed by a stu-

dent’s t test, which showed that the localization of Nestin

FIGURE 3: (Continued)

FIGURE 3: Cis- and trans-factors involved in Nestin mRNA local-ization. (A) The Nestin 30-UTR include cis-sequences for mRNAlocalization to protrusions. The 30-UTR sequences of Nestin,Vimentin and Rab13 were transfected as a pool into mouse NIH/3T3 cells together with the expression vector without insert.RNA fractions from protrusions and cell bodies were purified bythe Boyden chamber assay. By RT-qPCR the RNA localizationratios were determined and normalized to the localization ratiofor the vector without insert (control) given the value 1. (B)Involvement of the FMRP protein family for Nestin mRNA local-ization. In C8-S cells Fmr1, Fxr1, and Fxr2 were either depletedtogether using a pool of siRNA (sifpool, left panels) or by indi-vidually siRNA (right panels). At 24 h after transfection C8-S cellswere used in Boyden chamber assay and Nestin RNA localizationratio determined and normalized to 100 for the control siRNAtransfection. The efficiency of siRNA treatments were measuredby RT-qPCR analysis of Fmr1, Fxr1, and Fxr2 and normalized to1 for the control siRNA transfection.

Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes

November 2013 1931

mRNA was significantly higher than Actb (Fig. 4D). In con-

clusion, the observations from the FISH assays were in con-

cordance with the biochemical analysis showing localization

of a fraction of the Nestin mRNA in cell protrusions.

Nestin Protein Localization in Astrocyte ProtrusionsWe examined whether the Nestin protein display localization

analogous to the mRNA. In this line we note that it was pre-

viously shown that mouse Nestin protein localizes in growth

cones of P19 derived neurons and cerebellar granule cells

(Yan et al., 2001). We isolated protein extracts from the PF

and CF of mouse primary astrocytes using the Boyden

chamber assay and subsequently performed Western blot anal-

ysis. To normalize the protein content between the PF and

the CF we included analysis of a-Tubulin and Actb represent-

ing controls for uniform protein distribution. Histone H3

was included to control that cell nuclei were confined to the

CF. Western blot analysis showed that Nestin and Vimentin

were relatively more present in the PF whereas Gfap was

equally detected in the CF and the PF (Fig. 5A). Western

blot analysis also showed that in the C8-S cell PF Nestin and

Vimentin proteins were relatively enriched (Fig. 5B). Notably,

the absence in the presented Western blot analysis of detecta-

ble Vimentin and Nestin in the CF fraction shall not be

interpreted as absence of the proteins in this fraction but

solely reflects a relative increased localization in protrusions

compared to a-tubulin and Actb.

To examine subcellular protein distributions in closer

details we performed immunofluorescence analysis. Nestin

and Vimentin were detected using mouse primary antibodies

and an Alexa 555 conjugated secondary antibody and Gfap

was detected using a rabbit primary antibody and an Alexa

488 conjugated secondary antibody. In primary mouse astro-

cytes in the order of 80% of the cells were scored positive for

Gfap, Vimentin and Nestin but we note very heterogeneous

expression levels. Most Gfap positive cells showed filamentous

Gfap distribution throughout the cytoplasm (Fig. 5C,D). The

largest fraction of the Vimentin positive cells has filamentous

staining throughout the cytoplasm (Fig. 5C, upper panels)

but some cells (�30%) have Vimentin accumulation in cell

protrusions with simultaneous Vimentin filaments mostly in

the central part of the cell (Fig. 5C, lower panels). In Vimen-

tin and Gfap positive cells we observed co-localization includ-

ing colocalization in protrusions (Fig. 5C). Most Nestin

positive cells showed filamentous Nestin distribution

FIGURE 4: (Continued)

FIGURE 4: Subcellular detection of Nestin mRNA by single mole-cule RNA FISH. (A) Single molecule RNA FISH analysis of endog-enous Nestin, Actb and Gfap mRNAs in cultured primaryastrocytes isolated from P0 mice (left panels). A region of inter-est (ROI) is highlighted and magnified by a 53 zoom factor (mid-dle panels). Cell nuclei were stained by DAPI (right panels). Scalebar 5 10 lm. (B) Single molecule RNA FISH of Nestin and ActbmRNAs in C8-S cells (left panels). SSA4 served as negative con-trol. Middle panels show a ROI representing the tip of a cell pro-trusion enlarged five times (middle panels). Cell nuclei werestained by DAPI (right panels). Scale bar 5 20 lm. (C) Heat plotdisplaying C8-S Nestin and Actb mRNA localization analysis out-put (right panels) and their respective raw images (left panels).Cell center is marked by a red spot en the length of the cell pro-trusion is marked by a green line. Examples of cells with ActbmRNA signals (upper two panels) and Nestin mRNA signals(lower two panels) are shown. (D) The outcome of a paired twotailed t test for Nestin and Actb mRNA localization according tothe procedure in (C). The cell counting numbers were for Actbn 5 9 and for Nestin n 5 10. [Color figure can be viewed in theonline issue, which is available at wileyonlinelibrary.com.]

1932 Volume 61, No. 11

throughout the cytoplasm including the cell protrusions (Fig.

5D, upper panels). In Nestin and Gfap positive cells we

observed co-localization including colocalization in protru-

sions. Some Nestin positive cells (�20%) showed more pro-

nounced Nestin accumulation in protrusions with Gfap

colocalization and simultaneous Nestin in the central part of

the cell (Fig. 5D, lower panels). In C8-S cells Gfap expression

could not be detected whereas Vimentin and Nestin were

detected in nearly all cells (Fig. 5E). In �50% of the C8-S

cells Nestin accumulation was present throughout the protru-

sions (Fig. 5E). In nearly all C8-S cells Vimentin accumula-

tion was present throughout protrusions (Fig. 5E). The

control proteins a-Tubulin and Actb were uniformly dispersed

throughout the cytoplasm (Fig. 5E). Thus, both in primary

astrocytes and C8-S cells a fraction of the Nestin protein, as

well as the Nestin mRNA, was present in protrusions.

Discussion

The number of mRNAs that exhibit a distinct subcellular

localization pattern has increased dramatically as genome

wide technologies are improved (Lecuyer et al., 2007). More

than 1,000 mRNAs are identified to be relatively localized in

neuronal protrusions (Eberwine et al., 2001; Feltrin et al.,

2012; Taylor et al., 2009; Willis and Twiss, 2010; Willis

et al., 2007). Our presented DRS-based results strongly indi-

cate that numerous polyadenylated RNAs also are localized in

protrusions of primary astrocytes and the astrocyte cell line

C8-S. The heterogeneity of the used primary astrocytes could

in principle result in identification of mRNA localization

which cannot be confined to astrocytes but may represent

expression or localization in a minor cell subpopulation of

different lineage within the cell culture. We note that non-

Gfap positive cells within primary astrocyte cultures most

probably are primarily meningeal cells of fibroblast lineage

(Imura et al., 2006). However, comparing the 250 most local-

ized mRNAs from primary astrocytes with expression data for

in vivo astrocytes (Cahoy et al., 2008) we could verify astro-

cyte expression of the identified localized mRNAs. Annota-

tion analysis revealed that the 250 most localized mRNAs in

primary astrocytes and the C8-S astrocyte cell line encode a

broad variety of protein families. Moreover, both the 50 and

the 250 most localized mRNAs encode proteins which exhibit

a localization pattern similar to the total population of

expressed mRNAs (Supp. Info. Fig. 3). In this line, a signifi-

cant fraction of the protrusion localized mRNAs encode

nuclear proteins, which indicates that RNA localization to

protrusions might not necessarily be predictive for a direct

function of the encoded protein in cellular protrusions or

that such protein has moonlighting capacity. A previous study

showed that a majority (�70%) of all expressed RNAs are

localized in Drosophila embryos (Lecuyer et al., 2007) and

FIGURE 5: Nestin protein localization analyses. (A) Western-blotanalysis of protein isolated from the cell body fraction (CF, Boy-den chamber upper side) and cell protrusion fraction (PF, Boydenchamber lower side) using mouse primary astrocytes. Proteinsare detected by Western blotting using primary antibodiesagainst Nestin, Vimentin, Gfap, a-Tubulin, Actb, and Histone H3.a-Tubulin and Actb served as load controls and Histone H3 tocontrol for the lack of cell body migration through the Boydenchamber membrane. (B) Western blot analysis of protein isolatedfrom CF and PF by the Boyden chamber assay using C8-S cells.(C,D) Double immunofluorescence analysis of Vimentin and Gfap(C) and Nestin and Gfap (D) in mouse primary astrocytes. Pro-teins were detected using primary antibodies against Nestin,Vimentin and Gfap. Nuclei were stained with DAPI and shown inmerged pictures (Gfap, green; Nestin, red; Vimentin, red; DAPI,Blue) of representative cells. Scale bar 5 20 lm. (E) Immunofluo-rescence analysis of C8-S cells showing subcellular protein local-ization of Nestin, Vimentin, Actb and a-Tubulin. Cell nuclei werestained with DAPI (blue). Scale bar 5 20 lm. [Color figure can beviewed in the online issue, which is available atwileyonlinelibrary.com.]

Thomsen et al.: Nestin mRNA Localization in Mouse Astrocytes

November 2013 1933

points toward the notion that RNA localization is a norm

rather than the exception. We determined a higher number of

localized mRNAs in primary astrocytes than in C8-S cells, a

result that could be dependent on the different morphologies

of the cells. To further verify the determined RNA localiza-

tion patterns we conducted RT-qPCR analysis of representa-

tive mRNAs. The RT-qPCR analyses verified that Tensin3

mRNA displays a larger RNA localization ratio in primary

astrocytes than C8-S cells, and Spef1 mRNA shows a higher

RNA localization ratio in C8-S cells than primary astrocytes

(Fig. 1D). The result of the RT-qPCR analysis was similar to

the DRS analysis, further supporting that the Boyden cham-

ber assay combined with a DRS analysis is applicable to com-

pare and identify both commonly and cell type specific

localized RNAs. The Spef1 protein was previously determined

to have a developmental dependent subcellular localization in

mouse sperm cells (Chan et al., 2005). Tensin proteins act as

mediators between the extracellular matrix and the cytoskele-

ton, and studies of human cancer cell lines showed that Ten-

sin3 act as a negative regulator of cell migration

(Martuszewska et al., 2009). The results suggest that the two

examined astrocyte cell types have different intrinsic capacity

for Spef1 and Tensin3 mRNA localization which could be

mediated through nonidentical expression patterns of mRNA

localizing trans-factors or inclusion of different yet unidenti-

fied cis-sequences in the transcripts through alternative RNA

processing.

Directional transport by cytoskeletal motors is the pre-

dominant mechanism for delivering mRNA to the destination

(Bullock, 2011). mRNA and associated protein trans-factors

are cotransported as messenger ribonucleoprotein (mRNP)

particles (Mili and Macara, 2009). The mRNP transport is

facilitated by myosin motor proteins on actin filaments or via

kinesin and dynein motor proteins on microtubules (Shav-Tal

and Singer, 2005). Different motors can be active in sorting

mRNP in the same cell at the same time leading to differen-

tial patterns of mRNA localization (Bullock, 2011). Assembly

of the cytoskeletal filaments used for mRNP transport can be

self-regulated through mRNA localization dependent mecha-

nisms which are well established for actin filaments (Shav-Tal

and Singer, 2005). Actb mRNA is localized to the leading

edge of fibroblasts in an actin filament dependent manner

and disruption of Actb mRNA localization results in slower

cell motility, loss of directionality, delocalization of actin poly-

merization and altered adhesion dynamics (Katz et al., 2012;

Mingle et al., 2005; Shestakova et al., 2001). The actin-

related protein 2/3 (Arp2/3) complex is a crucial actin poly-

merization nucleator and is localized to the leading protru-

sions of migrating fibroblasts. mRNAs for the seven subunits

of the Arp2/3 complex (Arpc1a, Arpc2, Arpc3, Arpc4, Arpc5,

Actr2, and Actr3) are localized to fibroblast protrusions in

both actin filaments and microtubules dependent manners

supporting that the Arp2/3 complex is targeted to the site of

function by mRNA localization (Mingle et al., 2005). In the

Boyden chamber approach we neither observed mRNA local-

ization for Actb mRNA nor the seven mRNAs for the Arp2/3

complex (Supp. Info. Table 3). This observation is in accord-

ance with other reports describing the lack of mRNA localiza-

tion for these actin filament components and may reflect the

use of different experimental settings to detect mRNA local-

ization (Feltrin et al., 2012; Mili et al., 2008).

Only few studies have addressed mRNA localization in

astrocytes in relation to IFs. In this study we have identified

mRNA localization of Nestin. Compared to control Arpc3mRNA, Nestin mRNA exhibited a significant localization in

astrocyte protrusions as well as in protrusions of P19 embryo-

derived teratocarcinoma cells. Gfap mRNA displayed a more

moderate localization. Vimentin mRNA exhibited no signifi-

cant localization, neither by RT-qPCR nor DRS analysis. For

detailed subcellular analysis of Nestin mRNA we performed

single RNA molecule FISH. This revealed that Nestin mRNA

was localized to protrusions in �50% of the primary astro-

cytes and C8-S cells scored positive for Nestin mRNA expres-

sion. Moreover, when we applied a computer program to

analyze FISH images of Nestin mRNA positive cells and com-

pared these with Actb mRNA positive cells, we found that

Nestin mRNA had significantly more localization to cell pro-

trusions than Actb mRNA. The Nestin mRNA localization

results sustain previous results obtained from the developing

mouse brain indicating that Nestin mRNA is localized in

columnar neuroepithelial cells and radial glial cells (Dahl-

strand et al., 1995), and studies made on tissue sections of

chicken brain and cell cultures demonstrating mRNA localiza-

tion for Transitin, a Nestin like IF protein, in developing

radial glia cells from chickens (Lee and Cole, 2000). To

examine if Nestin mRNA localization also could be detected

in vivo we analyzed mouse P4 brain sagittal section mRNA in

situ hybridization (ISH) data for Nestin, Gfap, Actb,

CamKIIa, and Tubb3 (extracted from Allen Developing

Mouse Brain Atlas) (Supp. Info. Figs. 5 and 6). Nestin ISH

signals were sparsely detected and based on the data we were

not able to conclusively determine if Nestin mRNA in vivo is

localized in astrocyte protrusions.

The presented Nestin mRNA localization observations by

DRS, RT-qPCR and FISH in astrocyte cells are concordant

with the results showing presence of Nestin protein in the cell

protrusions (Fig. 5). Together the data supports that Nestin

mRNA can be localized and most likely also locally translated

in astrocyte protrusions. Although local proteins synthesis was

not shown directly, our results suggest that Nestin protein is

subcellular localized, at least partly, as a consequence of local

mRNA translation. Changes of cell morphology depend on

1934 Volume 61, No. 11

coordinated assembly and disassembly of cytoskeleton proteins.

Notably, it has been demonstrated that Nestin can inhibit

Vimentin filament formation in a concentration dependent

manner (Steinert et al., 1999). Thus, localization and local

translation of the Nestin mRNA can be used by the cell to cre-

ate the necessary local environment to provide optimal condi-

tions for IF modulation in the early onset of protrusion

formation. The notion that local assembly of Nestin and

Vimentin could participate in modulation of astrocyte mor-

phology is further indicated by the results showing significant

Vimentin protein localization in protrusions. Interestingly, we

showed no significant Vimentin mRNA localization and

Vimentin is accordingly most likely localized by protein trans-

port mechanisms. This is supported by numerous studies dem-

onstrating mictrotubule and dynein dependent transport of

non-filamentous IF protein particles (Perlson et al., 2005; Prah-

lad et al., 1998; Yoon et al., 1998). Moreover, studies of

migrating endothelial cells have shown Vimentin localization to

focal adhesions sites (Tsuruta and Jones, 2003). Gfap expression

is graduate up-regulated as Nestin and Vimentin expression

decreases and it is proposed that Vimentin and Nestin filaments

are scaffolds for the establishment of long term Gfap filaments

(Dahlstrand et al., 1995). Our data indicates that different

localization of Vimentin, Gfap, and Nestin mRNAs and the

resulting proteins altogether participates in the control of IF

dynamics in cell protrusions.

Several studies have demonstrated that mRNA localiza-

tion depends on cis-elements that typically, but not exclu-

sively, are enclosed in the 30-UTR and associated RNA

binding trans-factors (Mili and Macara, 2009). In a reporter

assay we determined that the Nestin mRNA 30-UTR was suf-

ficient to mediate localization to cell protrusions whereas the

Vimentin mRNA 30-UTR lacked this functionality. Fmrp and

the autosomal paralogues, Fxr1 and Fxr2, compose a family

of functional homologous RNA-binding proteins including

two ribonucleoprotein K homology domains and a cluster of

arginine and glycine residues in the RGG box (Bassell and

Warren, 2008; Tan et al., 2009). These domains are impor-

tant for RNA binding and polyribosome association. The

FMRP-family has an important role in translation control,

both in vivo and in vitro, and FMRP regulates protein syn-

thesis at sites where mRNAs are locally translated (Kindler

and Kreienkamp, 2012). The FMRP-family shuttles between

the nucleus and the cytoplasm and after mRNA association

in the nucleus forms a ribonucleoprotein complex transported

to dendrites and spines (Kim et al., 2009; Kindler and

Kreienkamp, 2012). An mRNA target sequence for the

FMRP-family consists of a G-quadruplex motif recognized by

the RGG box (Melko and Bardoni, 2010). Isolation of

FMRP containing ribonucleoprotein complexes from mouse

brains identified 432 mRNA species whereof 70% included

putative G-quadruplex motifs (Brown et al., 2001). Moreover,

30% of an examined group of mRNA localized in neuronal

dendrites, such as Psd95 and CamkIIa, contain G-quadruplex

motifs in the 30-UTR (Subramanian et al., 2011). We showed

that Fmrp and Fxr1, either directly or indirectly, are involved

in Nestin mRNA localization. The Nestin mRNA contains

several putative G-quadruplex motifs in the 30-UTR which

could indicate that these elements in association with Fmrp

and Fxr1 participates in Nestin mRNA localization. In

genome wide identification analysis of mRNAs associating

with Fmrp the Nestin mRNA does not appear as a highly sig-

nificant Fmrp target (Ascano et al., 2012; Brown et al., 2001;

Darnell et al., 2011). It should be emphasized that NestinmRNA expression is relatively low in the examined cell lines

and mouse brain samples which could be hindering identifi-

cation of Fmrp and Nestin mRNA interactions. In this line

Darnell et al. found interactions between Fmrp and Nestin

mRNA but the interaction was not scored significantly posi-

tive (Darnell et al., 2011).

In summary, we identified that IFs potentially can be

regulated at the level of mRNA localization mechanisms. We

demonstrated that Nestin mRNA and Nestin protein can have

localization in cell protrusions proposing that at least some

Nestin protein can be localized as a consequence of local

translation of the Nestin mRNA. The Vimentin mRNA dis-

played no significant protrusion localization whereas the

Vimentin protein is present. Finally, Gfap mRNA is moder-

ately localized in protrusions whereas the Gfap protein is dis-

tributed relatively evenly in the cytoplasm. The identified

mRNA localization patterns might reflect that IF proteins use

different sets of localization mechanisms with potential to reg-

ulate astrocyte morphology and migration during brain devel-

opment and maintenance.

Acknowledgment

Grant sponsors: The Lundbeck Foundation; Fonden til Læge-

videnskabens Fremme; NANONET—COST Action

BM1002; The Health Faculty, Aarhus University, Denmark.

The authors thank Robert H. Singer, Albert Einstein College

of Medicine, Bronx, New York, USA, for introducing them to

the FISH technique and the donation of FISH control probes.

The grant funders had no role in study design, data collection

and analysis, decision to publish or preparation of the manu-

script. The authors declare no conflicts of interests.

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