"The role of the nuclear factor TDP 43 in neurodegeneration" by Francisco E. Baralle

International Centre for Genetic Engineering and Biotechnology. Trieste (Italy)

The role of the nuclear factor TDP 43 in neurodegeneration.

The cellular RNA world:

Pre-mRNA splicing

• Essential step in gene expression

• >15% of human genetic diseases involve splicing errors

• Important regulatory step in gene expression

Alternative splicing

AUG

AUG UGA

UGA

Basic elements involved in the RNA splicing process

5’ splice site: recognized by U1snRNP

3’ splice site:

a) 3’ junction (AG): recognized by U2AF35

b) Poly-pyrimidine tract (PPT): recognized by U2AF65

c) Branch Point (BP): recognized by the Branch-point Binding Protein

AGguragu

U1snRNPmBBP

(SF1)

ynyurac ( y)nnyag

5’ss BP PPT 3’ss

3565

U2AF

subunits

U2snRNP

Exon 15’

pGU (Py)nA

Branch site

AGp

3’

5’-splice site 3’-splice site

SF1U2AF65 U2AF35

Regulatory Elements in pre-mRNA splicing

Exon 2

Regulatory sequence

Regulatory sequence

U1

Enhancer Silencer

Exon 15’

pGU (Py)nA

Branch site

AGp

3’

5’-splice site 3’-splice site

SF1U2AF65 U2AF35

Regulatory Elements in pre-mRNA splicing

Exon 2

Regulatory sequence

Regulatory sequence

U1

Enhancer Silencer

Over the years, a great number of Enhancer and Silencer factors have been identified:

Enhancers Silencers

SR protein family hnRNPs (such as A/B family, PTB,

CELF protein family hnRNP H…etc).

hnRNP L TDP-43

Tra2

YB-1

NOVA

In many cases exclusion/inclusion may thus determined by the resulting balance of power

Splicing mutations can be found in virtually any intron-containing gene. The frequency depends

on overall length and individual susceptibilities

Classical and non classical CF

Five “neutral” substitutions out of 19 induce exon skipping

The probability of inducing exon skipping is about 26%

In CFTR exon 12 synonymous sites are under selective pressure to maintain

the exon inclusion

The same sense mutations A49G, C40T and C52T are found in patients and

cause CFTR exon 12 skipping.

CERES 2

The coding sequences of CFTR exon 9 overlap with splicing regulatory sequences.

WT

exon 9

G424S

D443Y

I444S

A455E

V456F

9 +

9 -

Q414X

N418S

Q452P

per

cent

of

exon 9

incl

usi

on

exon 9

100

0

10

20

30

40

50

60

70

80

90

WT Q414X N418S G242S D443Y I444S Q452P A455E V456F

241 2 3 4 5 6a 6b 7 8 10 1112 14a14b 15 16 191817b17a 222120 23

TAGATG

13

mRNA

CFTR

Exons 9

Normal splicing

Aberrant splicing

CFTR

proteinMSD 1 MSD 2

NBD1

NBD2

Dominio

R

Plasmatic membrane

NH2 COOH

8 9 10

8 10

8 9 10Pre-mRNA

The degree of skipping of normal exon 9 is associated with the presence of polymorphic variants of the polypirimidine tract at the 3’ end of intron 8 (TG repeats from 9 to 13 and T9, T7, T5, T3)

TTTTGATGTGTGTGTGTGTGTGTGTTTTTTTTTAACAG

exon9

3’ss

(TG)m

Skipping of exon 9 is associated to monosymptomatic forms of CF (Congenital Bilateral Aplasia Vas Deferens, bronchiectasia, pancreatitis) and produce a non functional protein.

(T)n

Clinical

phenotype

Normal

Non-classical CF

Cystic Fibrosis

CFTR

genotype

T9

T7

T5

T5

T3

(TG)m (T)n

98 10

Inclusion

Skipping

This is one of the best clinical associations between a non coding genotype and a disease outcome:

TG9

TG10

TG11

TG12

TG13

TTTTGATGTGTGTGTGTGTGTGTGTTTTTTTTTAACAG

3’ssTG T9 9

TTG13 3

3’ss

TTTTGATGTGTGTGTGTGTGTGTGTGTGTGTGTTTAACAG

high inclusion

low inclusion

Minigene system applied to CFTR exon 9 splicing allowed us to replicate the effects of the UGmUn polymorphisms observed in patients.

The first well documented role of TDP-43 was the inhibition ofCFTR exon 9 splicing and interactions with other hnRNPs.

TDP-43 cellular functions are mediated by its binding to GU rich

RNA sequences and its interactions with other hnRNPs.

Its structure and biological properties are evolutionary conserved from

Drosophila to Man

RNA binding is essential for TDP-43 function in splicing

Transgenic TDP 43 expression in HEK 293 cell lines down regulates the

endogenous gene:

Transgene TDP-43 expression in stably transfected HEK293 cell lines cause

reduction in endogenous TDP 43 gene expression:

GAPDH

Induced TG TDP-43

WT F147/149L

72h Induction

2 3 4 51 6 7 8

2.8 kb

2 3 4 51 6 7coding sequence

untranslated sequence

TDPBR

TDP-43 mRNA species in stably transfected HEK293 cell lines are downregulated following transgene

TDP 43 cDNA induction:

2 3 4 5 6

pA4

pA1

TG-TDP43

TDP-43 RNA binding function is essential for self regulation

coding sequence

untranslated sequence

TDPBR

x7Tet

7

7

- +

endog.

2 3 4 51 6 7 8

int6 int7

Tet - +

7

7

a

anchor

TTTT

1

2

a-anchor

endog.

GFPfw-anchor

tag2.8 kb

GFPreporter

– +Tet – +

DiGFP

X7 7 8GFP

X7-TDPBR 7 8GFP

gt ag

gt ag

tag

tag

The TDPBR in the 3-UTR is needed for the regulatory feedback loop

GFPfw-anchor

7

7

valu

es n

orm

aliz

ed t

o U

6sn

RN

A

- Tet

+ TetBrUTP nuclear

Run-on

X1

X4int4

X6c pA1

h2pA4

3’pA4

10000

0

20000

30000

40000

50000

X1 X4int4

2 3 4 51 6 7

h2pA43’pA4

X6c pA1

8

endog.coding sequencenon-coding sequenceTDPBR

RNA polymerase II stalls in correspondence to the

TDPBR region:

Low TDP-43 concentrations(-Tet)

High TDP-43 concentrations(+Tet)

What events are associated with stalling?

Tet - + - + N C

1

2

1

g

2

71

72

6

TDPBR AAAAAAA

+

Two events: intron 7 skipping and a pA switch from pA1 to pA2

TDPBR

6

X7-sup5’-3’

GFP

GFP

TDP-43 TDP-43

int7

Cytoplasm

Nucleus

Improved 3’ss-5ss

Rapid nucleardegradation

Reduced amounts ofmRNA and protein

produced

GFP

Cytoplasm

Nucleus

No degradation

High amounts ofmRNA and protein

produced

X7-in7 cDNA

Intronless construct

GFP

int7

* *

Int7removal

Nuclearretention

GFP * *

Unproductivespliceosomal

complex

Model of TDP-43 autoregulation:

The splicing process in a 3’UTR may be recognized by the cell as anomalousand marked as an intron incompletely processed that must be degraded

Protein aggregation in neurodegenerative diseases

A wide variety of neurodegenerative diseases are characterized by the

accumulation of intracellular or extracellular protein aggregates.

Ross C.& Poirier M., 2005

Huntington Parkinson Alzheimer

Progressive neurodegenerative disease, that affects motor neurons that provide voluntary movements and muscle power.

When muscles no longer receive the messages from the motor neurons that they require to function, the muscles begin to atrophy (become smaller).

Some numbers:

The disease prevalence is ~5 people each 100,000.Most people develop ALS between 40 and 70years old (average 55).Life expectancy of an ALS patient averages from 2 to 5 years from the time of the diagnosis.

Amyotrophic Lateral Sclerosis

Pol II

Transcriptional

regulation

miRNA processing

pre-mRNA

splicing

Stress granule

formation

AAAAAAm7G

AAAAAAm7G

Translation

and protein

homeostasis

m7G Autoregulation

AAAAAA

m7G

mRNA stability

Nuclear/cytoplasmic

shuttling

mRNA transport

TDP Binding

region

(TDPBR)

Stress, oxidizing

agents, ageing

autophagy,

ubiquitin-proteasome

system (UPS)

Normal degradation

pathways

Chromatin

hnRNPs

FUS/TLS

P

P

P

PP

Ub

Ub

Ub

P

PUb

Ub

P

Aggregation

TAR DNA Binding Protein (TDP 43) is a splicing factor that belongs to the

hnRNP family and plays a role in many aspects of RNA metabolism.

Its aggregation/dysfunction is central to ALS and FTLD pathogenesis

Buratti and Baralle, 2012, TiBS 37: 237.

Ubiquitinated, misfolded and hyper phosphorylated TDP 43 was identified as the major

component of the pathological inclusions found in the brain of FTLD and ALS patients

Neumann et al., 2006 Science 314: 130-136

Deletion of the Drosophila homologue of human TDP-43 )TBPH) leads to a paralytic phenotype:

But cannot get out of the pupal

cage without external help, are

deficient in locomotion and have a

very short life span

Flies apparently

develop normally

W1118; TBPH/TBPHW1118; +/+ control

Deletion of TBPH does not affect the external

morphology of mutant flies

But flies develop locomotive effects

W111

8;

TB

PH

+/+

Genetic Expression of Human TDP-43 in

Motoneurons can rescue fly motility

TBPH23/-; D42-G4/ UAS-hTDP-43

TBPH23/TBPH23

TBPH23/-; Elav-G4/ UAS-hTDP-43

TBPH LOF affects Synaptic Growth and Bouton Shape

W1118 TBPHΔ23 TBPHΔ142

W1118 TBPHΔ23 TBPHΔ142 siRNA Tg-hTDP43

Microtubule Organization

TBP

HΔ23/D

23

TBP

HΔ142/Δ

142

W1

11

8R

escu

e

anti-HRP anti-MAP-1B merge

Ubiquitinated, misfolded and hyper phosphorylated TDP 43 was identified as the major

component of the pathological inclusions found in the brain of FTLD and ALS patients

Neumann et al., 2006 Science 314: 130-136

Transcriptional

regulation

pre-mRNA

splicing

miRNA

processing

Autoregulation

mRNA

transport and

stability

Aggregation

TAR DNA Binding Protein (TDP 43) is a splicing factor that belongs to the

hnRNP family and plays a role in many aspects of RNA metabolism.

Its dysfunction is central to ALS and FTLD pathogenesis

RRM1 RRM2

C-terminal tail

NLS

N C

NES

RRM domainsN-terminal tail

Q/N

1 414342 366

TDP Binding

region (TDPBR)

AAAAAA

m7G

Translation

A peptide containing the 321-366 region of TDP-43

can efficiently compete with hnRNP A2 for the

binding to TDP-43

321AMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQA366

C-terminal tail of TDP-43 contains a

Q/N domain that mediates its

interaction with hnRNPs.

p342-366p342AAAAp363AAAAp352AAAAA

(UG)6

(UG)6

(UG)6

peptide

THE Q/N RICH REGI0N IS ESSENTIAL A2 INTERACTION AND AGGREGATION

--

+ p321-366 (2,5M)

5 10 20 GST-A2 288-341 (M)5 10-TDP-43 (1M)+ + + + + + + +

-

% o

f si

gnal

rem

ain

ed

in t

he

wel

ls

4 5 62 31 7 8

0

20

40

60

80

100

120

1

+ + + + + + + +GST-A2 288-341 (M)

TDP-43

5 10 20 405 10

+ p321-366 (2,5M)

GST288 341

GST- A2-(288-341)

A2

Aggregate formation induced by the p321-366 peptide can be competed by addition

of an excess amount of hnRNP A2 C-terminal sequence

0

10

20

30

40

50

60

70

1FLAG-TDP-43+ EGFP

FLAG-TDP-43+ EGFP-12xQ/N

IN

S

P

% o

f FL

AG

-TD

P-4

3

-C

-C

331-369

-C

-C

Tandem repeats of the Q/N rich sequence induce in vitro and in vivo aggregation

of TDP 43

IN S P IN S P

FLAG-TDP-43+ EGFP-12xQ/N

WB

: an

ti-F

LAG

WB

: an

ti-G

FP

FLAG-TDP-43+ EGFP

30

46

58

46

1 2 3 4 5 6

SDS

0,5%0,25 uM

+ + + + +

WB

: an

ti-T

DP

-43 GST-TDP-43

EGFP-constructs

0,125 uM

FU

S-V

5

(an

ti-V

5)

FL

AG

-TIA

-1

an

ti-F

LA

G

EGFP-12xQ/N Merge

FU

S-V

5

(an

ti-V

5)

FL

AG

-TIA

-1

an

ti-F

LA

G

EGFP-12xQ/N Merge

EG

FP

EG

FP

-1x

Q/N

EG

FP

-4x

Q/N

EG

FP

-12

xQ

/N

EGFPEndo TDP-43

(anti-TDP-43) DAPI

Merge

EGFP/anti-TDP-43

Enlarged image

FLAG-TDP-43 WT + GFP-

12xQ/N

FLAG-TDP-43 321-366 + GFP-12xQ/N FLAG-TDP-43 F147,149/L + GFP-

12xQ/N

an

ti-F

LA

GG

FP

-12xQ

/NM

ER

GE

En

larg

ed

im

ag

e f

rom

each

co

nd

itio

n

*

*

*

FLAG-TDP-43 321-366 + GFP-12xQ/N

The Q/N rich TDP 43 region is essential for the recruitment of TDP 43 to the

aggregates while the RNA binding function is not needed

+Tetracycline

-Tetracycline

TDP-12xQ/N

TDP-43

Aggregate formation:

Red- TDP-12xQ/N

Green – (End. TDP-43)

Yellow (merge)

Blue (nuclei)

EGFP-12xQ/N Anti-TDP-43 Merge

Anti-Flag Anti-TDP-43MERGE

anti-Flag/anti-TDP-43

Aggregation can be enhanced by linking 12xQ/N to TDP 43 itself

Tet + + --

variant 1variant 2 47.5

α-POLDIP3

variant 1variant 2

SPLICING CHANGES AFTER AGGREGATION ARE SIMILAR TO THOSE OBSERVED

AFTER TDP 43 DEPLETION BY RNAi

Climbing

Solubility

IHC

Movement

IHC

Adult fly 3rd instar larvae

Looking for a phenotype…Driver: Elav, nSyb, GMR, etc.Temperature: 25 or 29°CGrowth cycle: Adult fly or 3rd instar larvae

TDP-43 aggregation model in flies

Climbing during aging

29°C

ela

v/W

1118

ela

v/E

GF

P#3

ela

v/G

FP

12x#3

0

2 0

4 0

6 0

8 0

1 0 0

2 9 C D 1 E L A V

G e n o ty p e

% T

op

cli

mb

ing

fli

es

ela

v/W

1118

ela

v/E

GF

P#3

ela

v/G

FP

12x#3

0

2 0

4 0

6 0

8 0

1 0 0

2 9 C D 7 E L A V

G e n o ty p e

% T

op

cli

mb

ing

fli

es

**

*

ela

v/W

1118

ela

v/E

GF

P#3

ela

v/G

FP

12x#3

0

2 0

4 0

6 0

8 0

1 0 0

2 9 C D 1 4 E L A V

G e n o ty p e

% T

op

cli

mb

ing

fli

es

*

**

CLIMBING ASSAY ELAV 12XQ/N

Larval brain immunohistochemistry

• Temperature: 25°C

• Growth cycle: 3rd instar larvae

• Antibodies:

Anti ELAV (rat): 1:250

Anti TBPH (rabbit): 1:300

Anti GFP (mouse): 1:200

+/Elav; +/EGFP2

29°C

Anti GFPAnti ELAV

Merge

+/Elav; +/GFP12x

29°C

Merge

Genetic interaction TBPH and GFP-12xQ/N

Genetic interaction TBPH and GFP-12xQ/N

Larval eye disc IHC

• Temperature: 25°C

• Growth cycle: 3rd instar larvae

• Antibodies:

Anti ELAV (rat): 1:250

Anti Flag-TBPH (mouse): 1:200

Anti GFP (rabbit): 1:250

GMR/+; TB1/+

25°C

Anti ELAVAnti Flag-TB1

Merge

Merge Merge

Anti ELAVAnti GFPAnti Flag-TB1

GMR/+; TB1;12X/+

25°C

Finding drugs to clear TDP-43 aggregates

One possible approach could be to enhance degradation of the aggregated protein.

Endogenous TDP-43

Newly produced TDP-43

Will be less sequestered

= “TDP-43 sink”

Aggregates induced by transient transfection of EGFP12Q/N display

autophagosomal markers

Hek293 GFP-12xQ/N

Tet induction

for 24 hrs

Wash

Compound

treatment for

48 hrs

Cell lysis

SDS-PAGE with urea and DTT

WB anti GFP

Clearance assay using the cellular model of TDP-43 aggregation

EGFP 12Q/N aggregates are partially resolved by poly anions effectors

C 40uM 100uM

actin

GFP-12xQ/N

TDP-43 aggregation model in cells

C 0.1uM 1uM 10uM 20uM

actin

GFP-12xQ/N

actin

GFP-12xQ/N

C 20uM C 0.1uM 1uM 10uM

FDA approved

drug 1

FDA approved

drug 2

FDA approved

drug 3

Possible disease connections following alterations at the TDP-43 autoregulatory loop level

INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGYTrieste, Italy

Emanuele BurattiAmit Bhardwaj

Mauricio BudiniLaura De Conti

Valentina RomanoMaureen Okuku

Jeremias HerzogFatemeh Mohagheghi

Zainuddin QuadriSimona LangellottiMaurizio Romano

Cristiana StuaniChiara Chiavelli

Frederick AllainPeter LukavskyZurich CH

Ashish DhirNick ProudfootOxford UK

Fabian FeiguinLucia Cragnaz

Raffaella Klima

IP HEK-TDP-12X-Q/N stable cell line

(HSP70)

Is there anything else in the aggregates?

Anti-TDP-43(Flag-TDP-12xQ/N and endogenous TDP-43)

Anti-HSP70 MERGEanti-TDP-43/anti-HSP70

Anti-HSP70

83

Tet + + + + + +

83

47.5

Anti-TDP-43

Anti-TDP-43

Hsp 70 is present in the early stage aggregates

FLAG-TDP-43 EGFP-12xQ/N Merge HA-Ubiquitin

FLAG-TDP-43 EGFP-12xQ/N Merge p409/p410

AGGREGATES ARE UBIQUITINATED AND PHOSPHORYLATED

AS IN THE PATIENT’S BRAIN INCLUSIONS

GFP

reporter

– +Tet

coding sequence

untranslated sequence

TDPBR

endog.

2 3 4 51 6 7 8

int6 int7

DiGFP

tag2.8 kb

A

X7-Δin7cDNA 7GFP

7GFP

7GFP

B

X7 7 8GFP

8

8

X7-pA2

X7-2pA1

X7-TDPBR 7 8GFP

gt ag

7GFP 8X7-gt-ag

gt ag

gt ag

gt ag

tag

gt ag

tag

tag

tag

tag

tag

gt ag

– + – + – + – + – +

Flag-TDP-43

TDP-43 end.

Tub.

X7X7-

TDPBR

X7-gt-ag

X7

-pA2

X7

-Δin7

cDNA

X7-

2pA1

1 2 3 4 5 6 7 8 9 10 11 1

2

C

D

1 2 3 4 5 6 7 8 9 10 11 1

2

GF

P/D

iGF

P n

orm

aliz

ed d

ata

1.0

0.8

0.6

0.4

0.2

0.0

Analysis of the relative importance for self regulation of pAs quality and splicing

- + X7

- +X7-pA2 X7-2pA1

- + TetTet Tet

77

7

8´ 8´´7Cryptic

pASV40

77

87

1

2

1

3

46

1

X7 7 8GFP

GFPfw

A

B C D E

anchor

TTTT

7

Autoregulation Yes Yes Yes

X7 gt-ag

71

- + Tet

No

F

5

G

- + Tet

No

X7 TDPBR

- + X7-Δin7cDNA

Tet

72

No

7

77

8

Autoregulation

mRNA isoforms in self regulation

TDP-43 continuosly shuttling

between the nucleus and the

Cytoplasm.

Physiological conditions

TDP-43 localization

Pathological conditions

Exported to the cytoplasm to

form insoluble aggregates.

GOF LOF

Climbing during aging

25°C

ela

v/W

1118

Ela

v/e

gfp

2

Ela

v/1

2X

3

0

2 0

4 0

6 0

8 0

1 0 0

g e n o ty p e

% T

op

cli

mb

ing

fli

es

2 5 C D 1 E L A V

ela

v/W

1118

Ela

v/e

gfp

2

Ela

v/1

2X

3

0

2 0

4 0

6 0

8 0

1 0 0

2 5 C D 7 E L A V

g e n o ty p e

% T

op

cli

mb

ing

fli

es

*

*

ela

v/W

1118

Ela

v/e

gfp

2

Ela

v/1

2X

3

0

2 0

4 0

6 0

8 0

1 0 0

g e n o ty p e

% T

op

cli

mb

ing

fli

es

2 5 C D 1 4 E L A V

***

**

ela

v/W

1118

Ela

v/e

gfp

2

Ela

v/1

2X

3

-2 0

0

2 0

4 0

6 0

8 0

1 0 0

g e n o ty p e

% T

op

cli

mb

ing

fli

es

2 5 C D 2 1 E L A V

****

****

Buratti and Baralle, 2012, TiBS 37: 237.

TAR DNA Binding Protein (TDP 43) is a splicing factor that belongs to the

hnRNP family and plays a role in many aspects of RNA metabolism.

Its aggregation/dysfunction is central to ALS and FTLD pathogenesis

RRM1

linker

C-terminus

N-terminus

RRM2

P187

L188

Q184

K102

N259

C244

M132

I253

Q134

I249

L139

E200

R151

D247

S254

Interactions between the TDP-43 RRMs

RRM1 RRM2GUA5

D105

F149

S254

GUGUGAAUGAAU

K145

R197

linker

GUA5 interacts at the interface of both RRMs

RRM1

RRM2

GUA1

N179

W113

D174

GUA3

URA2R171

URA4

L177

K176

F147

I107

GUGUGAAUGAAU

linker

Specific interactions on RRM1

RRM1

RRM2

E261

URA8

GUA9

N259

S258

F229

F194

K192

K263

F231

F221

GUGUGAAUGAAU

Specific interactions on RRM2

Functional studies on the CFTR exon 9 splicing model

of relevant TDP 43 amino acid variants

+ + + + + + + + + +

Add back of si-resistant TDP-43 wt and mutants (X>A)

+ +

GUA5 GUA1 GUA3

siRNA

Ex 9+

Ex 9-

URA4 URA8 GUA9

+ + + + + +

Single mutants

+

+ + + + + + + +

Double mutants Add back of si-resistant TDP-43 wt and mutants (X>A)

Strong impairement of splicing inhibition ability

Moderate impairement of splicing inhibition ability

No/Weak impairement of splicing inhibition ability

siRNA

Ex 9+

Ex 9-

WT F4

L

M132

I249

R151

D247D174

R171

K176

N179

K192

E261D105

S254

WT F4L D105 F149 K181 R197 S254 H256 W113 R171 D174 K176 N179 F194 S258 K192 E261 K263

TDPBR

7 87

6

6

AAAAAAA

6

Low TDP-43 concentrationsHigh TDP-43 concentrations

Efficient TDP-43 production

coding sequence non-coding sequence

AAAAAAA

TDPBR

Reduced TDP-43 production

AAAAAAA

+AAAAAAA

Model of TDP-43 autoregulation:

A

B

7GFP

7GFP

28S

18S

Two identical mRNAs ending in pA2have different cellular distribution if they are generated from splicing or from direct transcription of an intronless gene

X7 7 8GFP

X7-sup5’-3’ 7 8GFP

gt ag

gt ag

A

C

Fig.4

gacg/gtgggtgt

gCAg/gtAAgtgt

tctttgttttgcag/ccctgaa

tTtttTttttgcag/Gcctgaa

- +Tet

X7- sup5’-3’

28S

18S

7GFP

7GFP

B

D

WMM=6.61

WMM=13.07

NNSp=0.86

NNSp=1.00

X7- sup5’-3’

+CHX

F

GFP

reporter

DiGFP

Tub.

Flag-TDP-43

TDP-43 end.

X7 X7- sup5’-

3’

E

- +

- +Tet - +

7

Improved splice sites drive splicing Independently ofTDP 43 levels but the mRNAproduced is inefficient in protein production.

GFP Anti-digox-rhod merge

Fig.5

X7

X7-Δin7cDNA

X7-sup5’-3’

A

B

C

TO-PRO 3

9μ

m

9μ

m

9μ

m

9μ

m

9μm 9μm 9μm 9μm

9μ

m

9μ

m

9μ

m

9μ

m