Contactin-2 Expression in the Cardiac Purkinje Fiber Network

1

Contactin-2 Expression in the Cardiac Purkinje Fiber Network Pallante et al. Contactin-2 in Purkinje Fibers

Benedetta A. Pallante, PhD, DVM,a, Steven Giovannone, MDa, Liu Fang-Yua,

Jie Zhanga, Nian Liu, MDa, Guoxin Kang, PhDa, Wen Dun, PhDb, Penelope A. Boyden, PhDb,

Glenn I. Fishman, MDa

aLeon H. Charney Division of Cardiology, NYU School of Medicine, New York, NY; bDepartment of Pharmacology, Center for Molecular Therapeutics, Columbia University, New

York, NY

Corresponding author:

Glenn I. Fishman, M.D.

The Leon H. Charney Division of Cardiology

New York University School of Medicine

522 First Avenue, Smilow 801

New York, New York 10016

Tel: 212-263-3967

Fax: 212-263-3972

Email: [email protected]

Journal Subject Codes: [132] Arrythmias-basic studies, [136] Calcium cycling/excitation-

contraction coupling, [143] Gene regulation, [89] Genetics of cardiovascular disease, [145]

Genetically altered mice, [152] Ion channels/membrane transport

h

M

e

t

hor:

M.D.

ey Division of Cardiology

ty School of Medicine

by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from

http://circep.ahajournals.org/











2

AbstractBackground - Purkinje cells (PCs) comprise the most distal component of the cardiac

conduction system and their unique electrophysiological properties and the anatomic complexity

of the Purkinje fiber network may account for the prominent role these cells play in the genesis

of various arrhythmic syndromes.

Methods and Results – Differential transcriptional profiling of murine Purkinje fibers and

working ventricular myocytes was performed to identify novel genes expressed in PCs. The

most highly enriched transcript in Purkinje fibers encoded Contactin-2 (Cntn2), a cell adhesion

molecule critical for neuronal patterning and ion channel clustering. Endogenous expression of

Cntn2 in the murine ventricle was restricted to a subendocardial network of myocytes that also

express -galactosidase in CCS-lacZ transgenic mice and the connexin40 gap junction protein.

Both Cntn2-lacZ knockin mice and Cntn2-EGFP BAC transgenic reporter mice confirmed

expression of Cntn2 in the Purkinje fiber network, as did immunohistochemical staining of single

canine Purkinje fibers. Whole-cell patch-clamp recordings and measurements of Ca2+ transients

in Cntn2-EGFP+ cells revealed electrophysiological properties indicative of PCs and distinctive

from those of cardiac myocytes, including prolonged action potentials and frequent

afterdepolarizations.

Conclusions - Cntn2 is a novel marker of the specialized cardiac conduction system.

Endogenous expression of Cntn2 as well as Cntn2-dependent transcriptional reporters provides a

new tool through which Purkinje cell biology and pathophysiology can now more readily be

deciphered. Expression of a contactin family member within the CCS may provide a

mechanistic basis for patterning of the conduction system network and the organization of ion

channels within Purkinje cells.

Key Words: cell adhesion molecules; electrophysiology; genetics, Purkinje fiber

g g

netwoworkkkkkrkk oofffffff mmmmmmmyyyyyyoyofffffff

d n

o

dase in CCS-lacZ transgenic mice and the Z connexin40 gap jun

ockin mice and Cntn2-EGFP BAC transgenic reporter mice P

in the Purkinje fiber network, as did immunohistochemical

by guest on May 1, 2016http://circep.ahajournals.org/Downloaded from


3

Introduction

Purkinje fibers (PFs) are the most distal component of the cardiac conduction system (CCS), first

described by Purkinje in 1839 as gray, flat, gelatin-like ramifications, running under the

endocardium1. Some seventy years later, Tawara more fully characterized the Purkinje system,

identifying the left (anterior, septal and posterior) and right fascicular strands, which served to

connect the distal PFs to the bundle branches proper2. Tawara was also the first to correctly

suggest the functional role of the Purkinje system in rapidly transmitting the electrical wave of

excitation to the ventricular muscle.

PFs appear to play a prominent role in the genesis of ventricular arrhythmias, (reviewed in 3). PF

and anterior or posterior left fascicular triggers have been implicated in the initiation of

monomorphic ventricular tachycardia in post myocardial infarction patients, as demonstrated by

cure after focal ablation of Purkinje fiber or fascicular potentials4-6. PF-based triggers have also

been described in patients with ventricular tachycardia associated with dilated forms of

cardiomyopathy7, as well as idiopathic ventricular fibrillation (VF), in which ablation of

premature beats arising from the PF-network resulted in significant reductions in the recurrence

of VF8. PF-dependent triggering of arrhythmias has also been proposed in inherited syndromes

including catecholaminergic polymorphic VT9, Brugada syndrome and long QT syndrome10.

Despite growing evidence implicating PFs in ventricular arrhythmogenesis, our understanding of

the cellular mechanisms underlying PF-dependent diseases is hampered by the lack of

knowledge of the developmental biology of individual Purkinje cells (PCs), their patterning into

a network of highly coupled cells, as well as their adaptive and maladaptive responses to

pathologic stimuli. To some extent, this gap in knowledge reflects the anatomical complexity of

the PF-network, which includes branching cells that couple not only with neighboring PCs, but

arrhythmias, (r(r(r(r(r(r(ree

implplplplplplplicicicicicicicatatatatatatatededededededed i iiiiiin n nnnnn

i

t t

d

icular tachycardia in post myocardial infarction patients, as

tion of Purkinje fiber or fascn icular potentials4-6. PF-based t

patients with ventricular tachycardia associated with d



4

also with working myocytes at Purkinje-muscle junctions. Furthermore, PFs are thought to

include cells with functional and structural heterogeneity, such as the transitional J-cells11.

In recent years a number of “conduction-system” markers have been reported12-14. These include

transcription factors, gap junctional and ion channel associated proteins, with different

specificities and different roles in the development and function of the CCS. However, the

majority of these molecular markers, including HF-1b15 and Kcne116 preferentially identify cells

of the proximal CCS and to date, only two, the CCS-lacZ gene (resulting from insertion of an

Engrailed-2 reporter gene into the Slco3a1 locus17, 18) and gja5/connexin40 (Cx40), are robustly

expressed in PFs.

In the developing mouse ventricle, CCS-lacZ expression is first observed in trabecular

myocardium at embryonic day 10.5, a domain that appears to be the primary source of cells

contributing to the developing PF network. By E13.5 the reporter gene’s expression pattern is

similar to the neonate, with the appearance of a more discrete subendocardial fiber network19.

Expression is also observed in other components of the CCS, including both the sinoatrial node

(SAN) and atrioventricular node (AVN), as well as the His bundle and bundle branches20.

Unfortunately, expression of -galactosidase in the CCS-lacZ strain is the result of complex

insertional mutation associated with substantial genomic rearrangement, complicating

mechanistic studies of CCS-specific transcriptional regulation18.

Cx40 is a gap junction protein expressed in the developing and adult murine heart21 and Cx40-

EGFP reporter mice have also been used to visualize the ventricular conduction system 22. Cx40

expression is observed in other components of the CCS (AVN, His, Bundle Branches), but is

also abundantly expressed in atrial myocytes and coronary arteries. Examination of Cx40

deficient murine models has provided insight into its important role in development and function

obsererrrrrrveveveveveveved d d d d d ininininininin t ttttttrararararararabbbbebb

r u

t b

ryonic day 10.5, a domain that appears to be the primary sou

eveloping PF network. By E13.5 the reporter gene’s expres

te, with the appearance of a more discrete subendocardial fib



5

of the CCS. Loss of function of Cx40 results in an increased incidence of cardiac malformations

(double-outlet right ventricle, dilated and hypertrophic cardiomyopathy)23 as well as conduction

defects24. Interestingly, sequence variants within the human GJA5 locus have been described in

association with Tetralogy of Fallot25.

There are also reports12, 14 describing PF-enriched expression of two cardiac homeobox

transcription factors: Nkx2-5 and Homeodomain only protein (Hop). Nkx2-5+/- mice are

characterized by ventricular conduction defects specifically attributable to a hypoplastic PF

network, thought to be the result of impaired postnatal differentiation of myocytes12. However,

Nkx2-5 is broadly expressed in many cell-types within the developing and adult heart, which

diminishes its utility as a PF-specific marker. Hop is a cardiac homeobox transcription factor

which acts downstream of Nkx2-514, 26. Hop-lacZ knockin mice show broad reporter gene

expression in the developing heart, which becomes somewhat more restricted to the atria and

proximal CCS in the adult.

Accordingly, with the goal of developing additional specific markers of the distal CCS that

might be useful to facilitate studies elucidating the molecular circuitry regulating CCS

specification, patterning and function, we performed transcriptional profiling of the murine PF

network.

Methods

Methods are described in brief here (see the online-only Data Supplement for a full description).

Mutant Mice. All experiments were performed according to protocols approved by the NYU

Institutional Animal Care and Use Committee and conformed to the National Institutes of Health

oping and aduultltltltltltlt h

omeeoboboboboboboboxoxoxoxoxoxox trtrtrtrtrtrtranananananananscsssss

e o

v

e

eam of Nkx2-514, 26. Hop-lacZ knockin mice show broad repoZ

veloping heart, which becomes somewhat more restricted to rr

e adult.



6

(NIH) guidelines for the care and use of Laboratory Animals. CCS-lacZ+/- transgenic

(backcrossed into the CD-1 background for at least nine generations)19 and Cntn2-lacZ knockin

mice (on a C57BL/6x129SvEv mixed background)27 have been previously described. Cntn2-

EGFP BAC transgenic mice [Tg(Cntn2-EGFP)344sat] were obtained from MMRRC and

maintained on a CD-1 background.

Microarray Profiling. Purkinje fiber (PF) enriched samples were obtained by micro-dissecting

the subendocardial layer of 6-8 month old CCS-lacZ murine hearts and PF depleted samples

were micro-dissected from the sub-epicardial layer of the ventricular walls.

One-step Reverse Transcription and Polymerase Chain Reaction (RT-PCR) and

Quantitative real time RT-PCR (QRT-PCR). Total RNA was processed using QuantiTect

SYBR Green One-Step RT-PCR kit (Qiagen) or One-Step SuperScript III (Invitrogen). In QRT-

PCR experiments quantification of transcripts was performed using GAPDH as an internal

reference and the 2- CT method 28. For gene specific primers see Data Supplement.

Isolation of adult canine Purkinje Fibers (PFs) and individual Purkinje Cells (PCs). PCs

were enzymatically dispersed from PFs of the canine heart as previously described29, 30.

Immunohistochemistry and Immunocytochemistry, Antibodies. Mouse hearts or canine PFs

were fixed in 4% PFA, then equilibrated in 10% sucrose and embedded in OCT. Cryosections,

6-10 M thick were processed for staining with individual antibodies as detailed in the Data

Supplement.

Electrophysiological recordings in isolated ventricular myocytes. Ventricular myocytes were

isolated using an established enzymatic digestion protocol31 yielding to 60-80% rod-shaped,

calcium-tolerant myocytes. Action potential duration (APD) was measured at 90% and 50% of

repolarization (APD90 and APD50). DADs were defined as phase 4 positive (depolarizing)

tion (RT-PCCR)RRRRRR

procococococococesesesesesesesseseseseseseed d d d d d d usususususususiiiiinini g

t r

u a

e

tep RT-PCR kit (Qiagen) or One-Step SuperScript III (Invitr

uantification of transcripts was performed using GAPDH as a

CT method 28. For gene specific primers see Data Supple



7

deflections of the membrane potential. EADs were defined as positive (depolarizing)

oscillations occurring during phase 2 or 3 of action potential.

Ca2+ imaging. Isolated myocytes from Cntn2-EGFP transgenic mice were loaded with the

membrane-permeant Ca2+ indicator dye Rhod-2/AM and imaged by confocal microscopy (Leica

SP5). In some experiments, the Ca2+ transients were measured by microfluorimetry (IonOptix,

Milton, MA) using Indo-1/AM.

Statistics. Data are presented as mean ± SEM. Values were compared with a two-tailed t-test

and P values less than 0.05 were considered statistically significant.

Results

Cntn2 Transcripts are Preferentially Expressed in Cardiac Purkinje Fibers. Using CCS-

lacZ hearts, in which the distal conduction system is readily visualized, we performed

comparative transcriptional profiling of mRNA prepared from Purkinje fiber (PF)-enriched

subendocardial fractions harvested distal to the bundle branches proper and from PF-deficient

subepicardial layers (Fig. 1A). Enrichment of PFs was confirmed by XGal staining of dissected

tissue (Fig. 1B Top) and by RT-PCR, which showed preferential expression of transcripts

encoding both -galactosidase as well as connexin40 (Fig. 1B Bottom). Comparison of global

gene expression patterns from three independent datasets of PF-enriched and PF-depleted

samples by microarray analysis (Dataset Supplement) identified 163 probe sets that were

significantly up-regulated (5.87-1.33 fold increase) and 251 that were down-regulated (0.77-0.26

fold decrease). The transcript encoding Contactin2 (Cntn2; UniGene Mm.34775), showed the

highest level of enrichment (5.87). RT-PCR was used to confirm the microarray results

a .

h o

are Preferentially Expressed in Cardiac Purkinje Fibers.

h the distal conduction system is readily visualized, we perfo



8

identifying Cntn2 expression in cardiac PFs (Fig. 1C). Of note, two members of the Iroquois

gene family, Irx5 (4.18) and Irx2 (2.89), were also enriched in PFs, as was Sema3b, a putative

molecular partner of contactins. Interestingly, Nav1.8/SCN10A, which encodes the -subunit of

a TTX-resistant sodium channel isoform, was also enriched in the PF fraction (2.15). Sequence

variation in the SCN10A gene has recently been associated with human conduction system

disease32.

Cntn2 is Expressed on the Sarcolemma of Murine Purkinje Cells. We performed

immunohistochemical staining to specifically localize expression of Cntn2 in the heart.

Cryosections of adult murine hearts were co-stained for Cntn2 as well as the cardiac-specific

marker sarcomeric actinin, which revealed that Cntn2+ cells belong to the cardiac lineage (Fig.

2A Left). Co-staining of CCS-lacZ hearts for Cntn2 and -galactosidase ( Gal) or staining of

serial sections for Cntn2 or XGal, confirmed that Cntn2+ cells were also Gal+ (Fig. 2B Left) or

XGal+ (Fig. 2B Right). Furthermore, these images also demonstrated that the Cntn2 positive

cells were localized just beneath the thin endocardial cell layer (Fig. 2A Right), similar to what

we previously reported using the CCS-lacZ marker20. Taken together, these data indicate that

Cntn2 is specifically expressed in adult murine PFs.

Cntn2 is Expressed in the Proximal and Distal CCS. To more fully examine the spatial

pattern of Cntn2 in the adult murine heart, we utilized two independent reporter gene mice:

Cntn2-lacZ knockin mice and Cntn2-EGFP BAC transgenic mice. Examination of the

endocardial surfaces of the ventricular cavities of whole mount Cntn2-lacZ hearts (Fig. 3A Top,

Left) and Cntn2-EGFP (Fig. 3A Top, Right) hearts revealed dense networks of XGal+ and EGFP+

fluorescent PFs, which were also observed after sectioning (Fig. 3A Bottom). Detailed histologic

examination of Cntn2-EGFP hearts also demonstrated expression in cells of the sinoatrial node

s well as the cacacacacaaardrrrrrr

ong tototototototo t ttt t t thehehehehehehe cacacacacacacardrdrdrdrdrdrdiaiii

n o

n F

n

ng of CCS-lacZ hearts for SS Cntn2 and -galactosidase ( Gal) o

ntn2 or XGal, confirmed that Cntn2+ cells were also Gal+ (F

t). Furthermore, these images also demonstrated that the Cn



9

(SAN; Fig. 3B Top)33, including cells close to the crista terminalis in the posterior part of the

SAN; at the base of the vena cava in the central part of the SAN; and in the walls of the vena

cava in the most anterior part of the SAN. Co-expression of Cntn2 and the nodal marker Hcn4

further confirmed that Cntn2 is indeed expressed in a sub-population of elongated Hcn4+ cells in

the SAN. Detailed examination of the His bundle/atrioventricular (AVN) region revealed

expression of Cntn2-EGFP in Hcn4+ cells of the AVN, localized posteriorly to the His Bundle

(Fig. 3B Center). Co-staining for connexin40 (Cx40) also confirmed expression of Cntn2-EGFP

in Cx40+ PCs of both ventricles (Fig. 3B Bottom). Staining with anti-Cntn2 antibodies confirmed

that EGFP reporter gene expression in Cntn2-EGFP transgenic mice correctly reflected

endogenous gene expression, as shown by co-localization of the two markers in components of

the CCS (Fig. 3C).

Cntn2 expression in canine Purkinje fibers. Immunohistochemistry of isolated canine PFs

showed preferential expression of Cntn2 on the sarcolemma of individual Purkinje cells (PCs;

Fig. 4A). Myocytes expressing Cntn2 on their cell membrane also expressed the PC-marker

Cx40 at their intercalated disks, as observed in both longitudinal (Fig. 4B Left) and tangential

(Fig. 4B Right) cell sections. Confocal images of dissociated canine PCs (Fig. 4C) confirmed the

expression of Cntn2 on the sarcolemma of Cx40+ cells with a rod-shaped, Purkinje-like

phenotype. The cardiac pore-forming sodium channel subunit, Nav1.5/Scn5a, was concentrated

at the intercalated disks and along the cell membrane in PCs. Confocal images confirmed that

Cntn2 is expressed in Nav1.5+ PCs; surface (Fig 4D) and sub-surface (Fig 4E) optical slices

showed that, while co-expressed in the same cell, Cntn2 and Nav1.5 appeared to be targeted to

distinct membrane domains with limited co-localization, most clearly seen in the sub-surface

view.

mice correctlyyyyyyy r r rrrrre

two mamamamamamamarkrkrkrkrkrkkererererererers s s s s ss ininininininin

n

n

n canine Purkinje fibers. Immunohistochemistry of isolate

expression of Cntn2 on the sarcolemma of individual Purkin



10

Isolation and Functional Analysis of Cntn2-EGFP+ myocytes. To further analyze the

properties of the Cntn2-expressing myocytes, we dissociated myocytes from the ventricles of

adult Cntn2-EGFP+ transgenic mice. Cntn2-EGFP+ myocytes were readily seen under

epifluorescence microscopy, with an elongated, rod-shaped phenotype typical of PCs, easily

distinguishable from the non-fluorescent working ventricular myocytes (Fig 5A Left). Cntn2-

EGFP+ cells did not express Cx43 (Fig 5A Center), the most abundant connexin of working

myocytes, but were strongly positive for Cx40. This specificity of Cx40 expression was most

obvious in a cluster of myocytes that included both EGFP+ and EGFP- cells (Fig 5A Right).

Action potentials (APs) from Cntn2-EGFP+ myocytes were characterized by a distinct plateau in

phase 2 (Fig. 5B). APD50 and APD90 values were both significantly longer than those recorded

from Cntn2-EGFP- myocytes (Table 1). Cntn2-EGFP+ myocytes also demonstrated,

spontaneous electrical oscillations in phase 2, which resulted in early-after depolarizations

(EADs) (Fig. 5C Top). In some instances (Fig. 5C Bottom), Cntn2-EGFP+ myocytes also

developed delayed afterdepolarizations (DADs) at faster pacing rates (5 Hz); in contrast, none of

Cntn2-EGFP- cells exhibited either EADs or DADs.

Confocal microscopy and fluorescence photometry with calcium-sensitive dyes were also

performed to study intracellular calcium dynamics. Compared to the Cntn2-EGFP- working

ventricular myocytes, Cntn2-EGFP+ cells displayed significantly slower kinetics of activation

and relaxation (Fig. 5D, E), with longer time to peak fluorescence, reduced peak rate of rise of

fluorescence and slower decay (Table 1). EGFP+ myocytes were also significantly more likely

to develop spontaneous Ca2+ events (Fig 5F, G).

acterized by a a a a aa a dddddd

ntly l llllllononononononongegegegegegeger r r r r r thththththththananananaaa

m a

l

p c

myocytes (Table 1). Cntn2-EGFP+ myocytes also demonstra

al oscillations in phase 2, which resulted in early-after depol

p). In some instances (Fig. 5C Bottom), Cntn2-EGFP+ myoc



11

Discussion

We identified Contactin2 (Cntn2) as a novel marker that is preferentially expressed in the cardiac

conduction system, including myocytes of the murine and canine Purkinje fiber networks.

Contactins are a subgroup of cell adhesion molecules of the immunoglobulin (Ig) superfamily.

There are at least six contactins: Cntn1, Cntn2, BIG-1, BIG-2, NB-2 and NB-3; all include six Ig-

like and four fibronectin III-like domains that are anchored to the plasma membrane by a

glycosyl phosphatidylinositol (GPI) moiety. Cntn2, also known as transiently-expressed axonal

glycoprotein 1 (TAG-1), in mouse and rat 34, transient axonal glycoprotein 1 (TAX1) in human

and axonin-1 in chicken, has been most fully characterized in the central (CNS) and peripheral

nervous systems (PNS) where it is expressed in the membrane of neurons and myelinating glial

cells.

We used several complementary strategies to confirm the transcriptional profiling data

demonstrating expression of Cntn2 within the Purkinje fiber (PF) network. These included

immunohistochemical studies of murine and canine heart, as well as analyses of two genetically

engineered reporter mice, i.e., Cntn2-lacZ knockin mice and Cntn2-EGFP BAC transgenic mice.

Interestingly, while our initial transcriptional screen was focused on identifying transcripts

enriched in Purkinje fibers, our studies demonstrated expression of endogenous Cntn2 (and

reporter gene expression) throughout the entirety of the adult CCS, including nodal as well as

fast-conducting cells of the His-Purkinje network. This observation, coupled with our previous

description of widespread expression of the CCS-lacZ reporter gene throughout both the

proximal and distal CCS, suggests that despite the functional diversity that exists within the

heterogeneous elements of the CCS, these disparate components of the conductive network share

some commonality with respect to transcriptional regulation.

e central (CNSSSSSSS) ) ))))) a

f neururururururu ononononononons ssss s ananananananand d d d d d d mymmmmm

m n

e s

mplementary strategies to confirm the transcriptional profilin

ession of Cntn2 within the Purkinje fiber (PF) network. Thes



12

In this study, we took advantage of Cntn2-EGFP reporter mice 35 since they provided us with a

powerful approach to identify and functionally characterize individual Cntn2-expressing murine

myocytes. These data, including patch clamp recordings and measurements of intracellular

calcium dynamics, were consistent with a PC phenotype. Importantly, our functional studies

indicated a significantly greater propensity of single murine PCs to develop spontaneous

intracellular calcium release events (Fig. 5F) and afterdepolarizations (Fig. 5G). These

observations are consistent with the purported role of PCs in the genesis and maintenance of

various arrhythmic syndromes, including inherited diseases such as catecholaminergic

polymorphic ventricular tachycardia 9 and idiopathic ventricular fibrillation36, as well as common

acquired pathologies, such as post-myocardial infarction ectopy29, 37. Importantly, the Cntn2-

EGFP mice will allow us to characterize the ventricular conduction system at varying levels of

resolution, including studies of single cells (as in the current study), as well as analyses of the

complex branching structure of the Purkinje fiber network. These data should be of benefit in

the formulation of increasingly sophisticated computational models of cardiac excitation38, 39.

These studies do not yet address the specific function of Cntn2 in the cardiac conduction system,

although there are tantalizing hints from studies in the developing and adult nervous system.

During development, Cntn2 participates in neural cell adhesion and migration, neurite outgrowth

and fasciculation, axon pathfinding and myelination40. Even more relevant for the interpretation

of our findings in PCs is the role of Cntn2 in adult neurons where it is involved in ion clustering

with important implications for their electrophysiological properties.

Cntn2 is specifically involved in the clustering of delayed rectifier Shaker-type K+ channels,

Kv1.1 and Kv1.2, and their Kv 2 subunits at the juxtaparanodal region (JXP) of myelinated

axons 27. Concentration of Kvs at the JXP and their segregation from domains enriched in

fibrillation , , asasaaaaa

9, 377. I I IIIImpmpmpmpmpmpmpororororororortatatatatatatantntnnnnn ll

o a

g n

s e

ow us to characterize the ventricular conduction system at va

g studies of single cells (as in the current study), as well as an

structure of the Purkinje fiber network. These data should be



13

voltage-gated Na+ channels (Navs), at the nodes of Ranvier plays an important role in limiting

neuronal excitability by maintaining neuronal internodal resting potential or preventing rapid re-

excitation41. Molecular mechanisms of Cntn2-mediated Kv clustering in neurons involve trans

interaction with other Cntn2 molecules on the glial membrane and heterophilic cis interactions

with Caspr2, a member of the neurexin superfamily in the neuronal membrane (axolemma).

Caspr2 expression in the heart has not yet been examined, however in the nervous system, it

influences the function of Cntn2 as demonstrated by studies showing that in Caspr2 deficient

mice Cntn2 is unable to localize at the JXP and Kvs diffuse to the internodal region 27.

Conversely, in Cntn2-/- mice, there is mislocalization of Caspr2 and Kvs at the internodal

regions, demonstrating the interdependence of Cntn2 and Casp242. These observations suggest

that the function of Cntn2 in PCs could also be to regulate Kv clustering through its interaction

with Caspr2, thereby modulating PC excitability. Our data showing that Cntn2 and Nav1.5

appear to be targeted to distinct membrane domains support this functional parallel in PCs and

neurons. Additional co-localization studies using Cntn-EGFP mice will be required to explore

potential molecular interactions between Cntn2, Kvs, Navs, and Caspr2 in PCs.

Previous studies have also shown that Cntn2 is able to induce cis activation and binding of L1-

CAM to the adapter protein ankyrin43, which, in neurons, is implicated in Nav clustering at the

nodes (ankyrin-G) 44. In cardiomyocytes, mutations in ankyryn-G binding domains of Nav1.5

that result in impaired Nav1.5 localization to the intercalated disks were recently implicated in

the pathogenesis of ventricular arrhythmias45-47. Furthermore, in the canine model of arrhythmia

post-infarction, ankyrin-G has been implicated in sodium channel remodeling of epicardial

border zone cells48. In light of its interaction with ankyrin it is conceivable that Cntn2 may play

and Kvs at the e eee iniiniiii

42. TTTTTTThehehehehehehesesesesesesese oo o oooobsbsbsbsbsbsbserereeeee v

C h

y a

d l

Cntn2 in PCs could also be to regulate Kv clustering through

y modulating PC excitability. Our data showing that Cntn2 a

d to distinct membrane domains support this functional paral



14

a mechanistic role in ankyrin-dependent regulation of ion channel targeting and

arrhythmogenesis.

In conclusion, in the present study we have identified Cntn2 as a novel marker that is robustly

expressed throughout the specialized conductive system of the heart, including the distal

Purkinje fiber network. This discovery has enabled us to visualize the CCS, to isolate individual

Purkinje cells, and to characterize their distinct physiological properties. One can now envision

numerous strategies to decipher the mechanistic role of the Purkinje fiber network in the genesis

and maintenance of clinically significant cardiac rhythm disturbances. Moreover, by analogy to

neuronal systems, we suggest that Cntn2 may regulate CCS network formation and the

excitability of its component cells.

Acknowledgements: We thank Dr. Andrew Furley (University of Sheffield) for making the

Cntn2-lacZ mice available to us and Dr. Takeshi Sakurai (Mount Sinai School of Medicine) for

providing them.

Funding Sources: This work was supported by NIH R01HL64757, R01HL081336 and a New

York State Stem Cell Science Award to G.I.F., a Glorney-Raisbeck Fellowship in Cardiovascular

Disease to S.F.G, and NIH R01 HL58860 to P.A.B.

Conflict of Interest Disclosures: None

work formationnnnnnn a a



15

References

1. Jay V. The extraordinary career of Dr Purkinje. Arch Pathol Lab Med. 2000;124:662-

663.

2. Suma K. Sunao Tawara: a father of modern cardiology. Pacing Clin Electrophysiol.

2001;24:88-96.

3. Scheinman MM. Role of the His-Purkinje system in the genesis of cardiac arrhythmia.

Heart Rhythm. 2009;6:1050-1058.

4. Bogun F, Good E, Reich S, Elmouchi D, Igic P, Tschopp D, Dey S, Wimmer A,

Jongnarangsin K, Oral H, Chugh A, Pelosi F, Morady F. Role of Purkinje fibers in post-

infarction ventricular tachycardia. J Am Coll Cardiol. 2006;48:2500-2507.

5. Hayashi M, Kobayashi Y, Iwasaki YK, Morita N, Miyauchi Y, Kato T, Takano T. Novel

mechanism of postinfarction ventricular tachycardia originating in surviving left posterior

Purkinje fibers. Heart Rhythm. 2006;3:908-918.

6. Morishima I, Nogami A, Tsuboi H, Sone T. Verapamil-sensitive left anterior fascicular

ventricular tachycardia associated with a healed myocardial infarction: changes in the

delayed Purkinje potential during sinus rhythm. J Interv Card Electrophysiol.

2008;22:233-237.

7. Sinha AM, Schmidt M, Marschang H, Gutleben K, Ritscher G, Brachmann J, Marrouche

NF. Role of left ventricular scar and Purkinje-like potentials during mapping and ablation

of ventricular fibrillation in dilated cardiomyopathy. Pacing Clin Electrophysiol.

2009;32:286-290.

8. Haissaguerre M, Shoda M, Jais P, Nogami A, Shah DC, Kautzner J, Arentz T, Kalushe

D, Lamaison D, Griffith M, Cruz F, de Paola A, Gaita F, Hocini M, Garrigue S, Macle L,

Weerasooriya R, Clementy J. Mapping and ablation of idiopathic ventricular fibrillation.

Circulation. 2002;106:962-967.

9. Cerrone M, Noujaim SF, Tolkacheva EG, Talkachou A, O'Connell R, Berenfeld O,

Anumonwo J, Pandit SV, Vikstrom K, Napolitano C, Priori SG, Jalife J. Arrhythmogenic

mechanisms in a mouse model of catecholaminergic polymorphic ventricular tachycardia.

Circ Res. 2007;101:1039-1048.

10. Haissaguerre M, Extramiana F, Hocini M, Cauchemez B, Jais P, Cabrera JA, Farre J,

Leenhardt A, Sanders P, Scavee C, Hsu LF, Weerasooriya R, Shah DC, Frank R, Maury

ininnnnnnatatatatatata inininininining g g g g gg ininininininin s s s s s s surururururururvivivivivivivivvvvvvv

, e

a h

k y

-

, Nogami A, Tsuboi H, Sone T. Verapamil-sensitive left ante

achycardia associated with a healed myocardial infarction: ch

kinje potential during sinus rhythm. J Interv Card Electrophy

-237.



16

P, Delay M, Garrigue S, Clementy J. Mapping and ablation of ventricular fibrillation

associated with long-QT and Brugada syndromes. Circulation. 2003;108:925-928.

11. Li ZY, Wang YH, Maldonado C, Kupersmith J. Role of junctional zone cells between

Purkinje fibres and ventricular muscle in arrhythmogenesis. Cardiovasc Res.

1994;28:1277-1284.

12. Hatcher CJ, Basson CT. Specification of the cardiac conduction system by transcription

factors. Circ Res. 2009;105:620-630.

13. Myers DC, Fishman GI. Molecular and functional maturation of the murine cardiac

conduction system. Trends Cardiovasc Med. 2003;13:289-295.

14. Zhang S-S, Bruneau BG. Transcriptional control of the cardiac conduction system.

Advances in developmental biology. Vol 18: Elsevier B. V.; 2008:219-258.

15. Nguyen-Tran VT, Kubalak SW, Minamisawa S, Fiset C, Wollert KC, Brown AB, Ruiz-

Lozano P, Barrere-Lemaire S, Kondo R, Norman LW, Gourdie RG, Rahme MM, Feld

GK, Clark RB, Giles WR, Chien KR. A novel genetic pathway for sudden cardiac death

via defects in the transition between ventricular and conduction system cell lineages.

Cell. 2000;102:671-682.

16. Kupershmidt S, Yang T, Anderson ME, Wessels A, Niswender KD, Magnuson MA,

Roden DM. Replacement by homologous recombination of the minK gene with lacZ

reveals restriction of minK expression to the mouse cardiac conduction system. Circ Res.

1999;84:146-152.

17. Logan C, Khoo WK, Cado D, Joyner AL. Two enhancer regions in the mouse En-2 locus

direct expression to the mid/hindbrain region and mandibular myoblasts. Development.

1993;117:905-916.

18. Stroud DM, Darrow BJ, Kim SD, Zhang J, Jongbloed MR, Rentschler S, Moskowitz IP,

Seidman J, Fishman GI. Complex genomic rearrangement in CCS-LacZ transgenic mice.

Genesis. 2007;45:76-82.

19. Rentschler S, Vaidya DM, Tamaddon H, Degenhardt K, Sassoon D, Morley GE, Jalife J,

Fishman GI. Visualization and functional characterization of the developing murine

cardiac conduction system. Development. 2001;128:1785-1792.

WoWoWoWoWoWoWollllllllllllllererererererert t t t t tt KCKCKCKCKCKCKC,,,, , BrBrBrBrBrBrBr

ourdddddddieieieieieieie R R R R RRRG,G,G,G,G,G,G, R R R R R R Rahahahahahaha m

B, Giles WR, Chien KR. A novel genetic pathway for sudde

n

0

t S, Yang T, Anderson ME, Wessels A, Niswender KD, M g

B, Giles WR, Chien KR. A novel genetic pathway for sudde

n the transition between ventricular and conduction system c

02:671-682.

t S, Yang T, Anderson ME, Wessels A, Niswender KD, Mag



17

20. Myers DC, Fishman GI. Toward an understanding of the genetics of murine cardiac

pacemaking and conduction system development. Anat Rec A Discov Mol Cell Evol Biol.

2004;280:1018-1021.

21. Coppen SR, Kaba RA, Halliday D, Dupont E, Skepper JN, Elneil S, Severs NJ.

Comparison of connexin expression patterns in the developing mouse heart and human

foetal heart. Mol Cell Biochem. 2003;242:121-127.

22. Miquerol L, Meysen S, Mangoni M, Bois P, van Rijen HV, Abran P, Jongsma H, Nargeot

J, Gros D. Architectural and functional asymmetry of the His-Purkinje system of the

murine heart. Cardiovasc Res. 2004;63:77-86.

23. Gu H, Smith FC, Taffet SM, Delmar M. High incidence of cardiac malformations in

connexin40-deficient mice. Circ Res. 2003;93:201-206.

24. Kirchhoff S, Nelles E, Hagendorff A, Kruger O, Traub O, Willecke K. Reduced cardiac

conduction velocity and predisposition to arrhythmias in connexin40-deficient mice. Curr

Biol. 1998;8:299-302.

25. Greenway SC, Pereira AC, Lin JC, DePalma SR, Israel SJ, Mesquita SM, Ergul E, Conta

JH, Korn JM, McCarroll SA, Gorham JM, Gabriel S, Altshuler DM, Quintanilla-Dieck

Mde L, Artunduaga MA, Eavey RD, Plenge RM, Shadick NA, Weinblatt ME, De Jager

PL, Hafler DA, Breitbart RE, Seidman JG, Seidman CE. De novo copy number variants

identify new genes and loci in isolated sporadic tetralogy of Fallot. Nat Genet.

2009;41:931-935.

26. Ismat FA, Zhang M, Kook H, Huang B, Zhou R, Ferrari VA, Epstein JA, Patel VV.

Homeobox protein Hop functions in the adult cardiac conduction system. Circ Res.

2005;96:898-903.

27. Poliak S, Salomon D, Elhanany H, Sabanay H, Kiernan B, Pevny L, Stewart CL, Xu X,

Chiu SY, Shrager P, Furley AJ, Peles E. Juxtaparanodal clustering of Shaker-like K+

channels in myelinated axons depends on Caspr2 and TAG-1. J Cell Biol.

2003;162:1149-1160.

28. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time

quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402-408.

,,,,,, W W W W W W Wilililililililleleleleleleleckckckckckckcke e e e e e e K.K.K.K.K.K.K. R R R R R R R

connnnnnnnexexexexexexexininininininn40404040404040-d-d-d-d-d-d-deeeeefefe

:

C M

M n

n t

:299-302.

C, Pereira AC, Lin JC, DePalma SR, Israel SJ, Mesquita SM

M, McCarroll SA, Gorham JM, Gabriel S, Altshuler DM, Quin

nduaga MA, Eavey RD, Plenge RM, Shadick NA, Weinblatt



18

29. Hirose M, Stuyvers BD, Dun W, Ter Keurs HE, Boyden PA. Function of Ca release

Channels in Purkinje cells that survive in the Infarcted Canine Heart; a Mechanism for

triggered Purkinje Ectopy. Circ Arrhythm Electrophysiol. 2008;1:387-395.

30. Boyden PA, Albala A, Dresdner KP, Jr. Electrophysiology and ultrastructure of canine

subendocardial Purkinje cells isolated from control and 24-hour infarcted hearts. Circ

Res. 1989;65:955-970.

31. Liu N, Colombi B, Memmi M, Zissimopoulos S, Rizzi N, Negri S, Imbriani M,

Napolitano C, Lai FA, Priori SG. Arrhythmogenesis in catecholaminergic polymorphic

ventricular tachycardia: insights from a RyR2 R4496C knock-in mouse model. Circ Res.

2006;99:292-298.

32. Chambers J, Zhao J, Terracciano C, Bezzina C, Zhang W, Kaba R, Navaratnarajah M,

Lotlikar A, Sehmi J, Kooner M, Siedlecka U, Wass M, Dekker L, de Jong J, Sternberg M,

McKenna W, Severs N, DeSilva R, Wilde A, Anand P, Yacoub M, Scott J, Elliot P,

Wood J, Kooner J. Genetic Variation in SCN10a is Associated With Cardiac Conduction,

Heart Block and Risk of Ventricular Fibrillation. Paper presented at: American Heart

Association Annual Meeting; November 14-18 2009; Orlando, Florida.

33. Oosthoek PW, Viragh S, Mayen AE, van Kempen MJ, Lamers WH, Moorman AF.

Immunohistochemical delineation of the conduction system. I: The sinoatrial node. Circ

Res. 1993;73:473-481.

34. Walsh FS, Doherty P. Neural cell adhesion molecules of the immunoglobulin

superfamily: role in axon growth and guidance. Annu Rev Cell Dev Biol. 1997;13:425-

456.

35. Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ,

Joyner A, Leblanc G, Hatten ME, Heintz N. A gene expression atlas of the central

nervous system based on bacterial artificial chromosomes. Nature. 2003;425:917-925.

36. Haissaguerre M, Shah DC, Jais P, Shoda M, Kautzner J, Arentz T, Kalushe D, Kadish A,

Griffith M, Gaita F, Yamane T, Garrigue S, Hocini M, Clementy J. Role of Purkinje

conducting system in triggering of idiopathic ventricular fibrillation. Lancet.

2002;359:677-678.

37. Wit AL, Bigger JT, Jr. Possible electrophysiological mechanisms for lethal arrhythmias

accompanying myocardial ischemia and infarction. Circulation. 1975;52:III96-115.

eeeeeeekkkkkkkkkkkkkkererererererer L L LL L LL,,,,,,, dedededededede J JJJJJJonononononononggggggg

Yacooububububububub M MM M MMM, , , , , , , ScScScScScScScototooooottt

o d

m

A

W, Viragh S, Mayen AE, van Kempen MJ, Lamers WH, M o

oner J. Genetic Variation in SCN10a is Associated With Card

and Risk of Ventricular Fibrillation. Paper presented at: Am

Annual Meeting; November 14-18 2009; Orlando, Florida.

W, Viragh S, Mayen AE, van Kempen MJ, Lamers WH, Moo



19

38. Deo M, Boyle P, Plank G, Vigmond E. Role of Purkinje system in cardiac arrhythmias.

Conf Proc IEEE Eng Med Biol Soc. 2008;2008:149-152.

39. Vigmond E, Vadakkumpadan F, Gurev V, Arevalo H, Deo M, Plank G, Trayanova N.

Towards predictive modelling of the electrophysiology of the heart. Exp Physiol.

2009;94:563-577.

40. Cohen NR, Taylor JS, Scott LB, Guillery RW, Soriano P, Furley AJ. Errors in

corticospinal axon guidance in mice lacking the neural cell adhesion molecule L1. Curr

Biol. 1998;8:26-33.

41. Rasband MN, Shrager P. Ion channel sequestration in central nervous system axons. J

Physiol. 2000;525 Pt 1:63-73.

42. Traka M, Goutebroze L, Denisenko N, Bessa M, Nifli A, Havaki S, Iwakura Y,

Fukamauchi F, Watanabe K, Soliven B, Girault JA, Karagogeos D. Association of TAG-

1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of

myelinated fibers. J Cell Biol. 2003;162:1161-1172.

43. Malhotra JD, Tsiotra P, Karagogeos D, Hortsch M. Cis-activation of L1-mediated

ankyrin recruitment by TAG-1 homophilic cell adhesion. J Biol Chem. 1998;273:33354-

33359.

44. Bhat MA. Molecular organization of axo-glial junctions. Curr Opin Neurobiol.

2003;13:552-559.

45. Hashemi SM, Hund TJ, Mohler PJ. Cardiac ankyrins in health and disease. J Mol Cell

Cardiol. 2009;47:203-209.

46. Mohler PJ, Rivolta I, Napolitano C, LeMaillet G, Lambert S, Priori SG, Bennett V.

Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and

expression of Nav1.5 on the surface of cardiomyocytes. Proc Natl Acad Sci U S A.

2004;10:17533-17538.

47. Mohler PJ, Splawski I, Napolitano C, Bottelli G, Sharpe L, Timothy K, Priori SG,

Keating MT, Bennett V. A cardiac arrhythmia syndrome caused by loss of ankyrin-B

function. Proc Natl Acad Sci U S A. 2004;101:9137-9142.

48. Dun W, Robinson R, Lowe J, Mohler P, Boyden P. Ankyrin-G participates in the

remodeling of INa in myocytes of the EBZ of the infarcted heart. Biophys J. 2008;94:1.

gogogogogogogogegegegegegegeososososososos D D D D D D D. . ..... AsAsAsAsAsAsAssososososososo

of juuuuuuuxtxtxtxtxtxtxtapapapapapapaparararararararanananananananooooododo

i

, m

u 9

ibers. J Cell Biol. 2003;162:1161-1172.

, Tsiotra P, Karagogeos D, Hortsch M. Cis-activation of L1-m

uitment by TAG-1 homophilic cell adhesion. J Biol Chem. 19



20

Table 1. Functional Properties of Cntn2-EGFP- and Cntn2-EGFP+ myocytes

Action Potential Characteristics Ca2+ Transient Kinetics Cell Type

RMP (mV)

APA (mV)

APD50 (ms)

APD90 (ms)

Time to Max (ms)

Peak Rate of Rise (F/Fo/s)

Tau of decay (ms)

EGFP(-) (n=10)

-71.3±1.1 119.2±3.2 5.5±0.9 54.7±9.7 29.5±3.6 103.0±20 110.4±13.4

EGFP(+) (n=10)

-71.5±1.3 128.3±3.7 24.7±4.3 139.3±17.8 49.4±4.8 49.4±5.9 136.7±23.2

P value .87 .08 .00035 .00057 < .000001 < .00001 0.02

Data are expressed as mean values ± SEM. N, number of cells; RMP, resting membrane

potential; APA, AP amplitude; APD50,90, AP duration at 50% and 90% of repolarization,

respectively. APDs are significantly longer for Cntn2-EGFP+ myocytes compared to Cntn2-

EGFP- myocytes. Ca2+ transient measurements demonstrated that Cntn2-EGFP+ myocytes have

significantly slower Ca2+-handling kinetics compared to Cntn2-EGFP-myocytes.

yoyyyyyy cyyyyyyytes compmpmpmpmpmpmpararaaaaa

at CnCnCCCCC tntntntttt 2-2-22222 EGEGEGEGEGEGEGFPFPFPFPFPFPFP+++

2+ .Ca2+-handling kinetics compared to Cntn2-EGFP-myocytes.



21

Figure Legends

Fig. 1. Identification of novel Purkinje fiber-specific transcripts by comparative

microarray analysis. (A) PF+ and PF- samples were micro dissected distal to the bundle

branches (Top, arrow) from the subendocardial and subepicardial regions (Bottom) of CCS-lacZ

hearts. (B) Enrichment of PF-specific transcripts in PF+ samples was confirmed by increased

expression of the PF markers CCS-lacZ and Cx40 compared to PF- samples, assessed by XGal

staining (Top) and/or RT-PCR (Bottom). (C) QRT-PCR showing up-regulation of Cntn2 and the

PF-specific markers CCS-lacZ ( Gal) and Cx40 in micro dissected PFs. Values are expressed as

Arbitrary Units (1AU= RV in PF- sample). PFs, Purkinje fibers; RW, right ventricular wall; LW,

left ventricular wall; RV, right ventricle; LV, left ventricle; e, endocardium; E, epicardium.

Black outline, subendocardial region; Red outline, subepicardial region; IS, interventricular

septum. Size bar: 200 M.

Fig. 2. Cntn2 is specifically expressed in Purkinje fibers. (A Left) Confocal image of mouse

heart sections confirming that Cntn2+ cells (green) express the cardiac marker sarcomeric actinin

(red, Left). (Inset) High power image showing an optical slice of a Cntn2+/sarcomeric actinin+

myocyte. (B Left) Epifluorescent images of CCS-lacZ hearts showing a Cntn2+/ Gal+

(green/red) cell (arrowhead) under a layer of Cntn2-/ Gal- endocardial cells (arrows). (B Right)

Images of CCS-lacZ heart serial sections stained for XGal (Upper) and Cntn2 (red, Lower)

showing co-localization of Cntn2 and the PF-specific marker CCS-lacZ. (C) Epifluorescent

image of E13.5 mouse brain used as positive control, showing Cntn2 expression (red) in the

intermediate and marginal zones (arrowheads) and in the preplate region (arrows) of the ventral

telenchephalon. LW, left ventricular wall; LV, left ventricle; RW, right ventricular wall; RV,

right ventricle. Size bars: 20 M. A Left, Inset: 35 M.

Fig. 3. Cntn2 expression delineates the CCS in the adult heart. (A) Whole mounts (Top) and

cryosections (Bottom) of adult Cntn2-lacZ (Left; XGal staining) and Cntn2-EGFP hearts (Right)

showing expression of Cntn2 throughout the CCS. (B) Epifluorescent images of Cntn2-EGFP

hearts confirming expression of Cntn2 (green) in the SAN (Top), AVN (Center) and PFs

(Bottom), identified by anatomical landmarks and expression of the nodal marker Hcn4 (red) or

rererererereregigigigigigigiononononononon; ; ; ;;;; ISISISISISISIS, , , , , , , inininininininteteteteteteterrrrrrr

c m

m a

H o

cifically expressed in Purkinje fibers. (A Left(( ) Confocal im

ming that Cntn2+ cells (green) express the cardiac marker sa

High power image showing an optical slice of a Cntn2+/sarco//



22

the PF marker Cx40 (red). (Top) Cntn2-EGFP+ cells (green) were found in the SAN, localized

at the junction of the VC with the right atrium and extending from the CT (0 M), posteriorly, to

the base (+40 M), and the walls (+190 M) of the VC, anteriorly. Cntn2 identifies a sub-

population of elongated Hcn4+ cells in the SAN. Cntn2-EGFP+/Hcn4+ cells were also found in

the AVN, 20-40 M posteriorly to the His Bundle (Center). (Bottom) Cntn2-EGFP+

(green)/Cx40+ (red) PFs were observed in both ventricles. (C) Cntn2-EGFP transgene

expression recapitulates Cntn2 protein expression as shown by co-localization of anti-Cntn2-TR

antibody (red) and EGFP (green) signals in the AVN (Top) and the PFs (Bottom). (Bottom

Right) Confocal image detail of PFs. His/Br, His bundle and bundle branches; AVN,

atrioventricular node; SAN, sinoatrial node; lPFs, left ventricle Purkinje fibers; rPFs, right

ventricle Purkinje Fibers; Ct, crista terminalis; VC, vena cava; A, aorta and pulmonary artery;

arrow, SAN (in B); CCS, cardiac conduction system. Blue fluorescence: DAPI nuclear counter

stain, in A (Bottom, Right,), B and C. Size bars: (A) 50 M, all except for Bottom, Left, 500 M;

(B) 20 M, all except for Top, Left, 200 M, and Bottom, Left, 50 M; (C), 100 M.

Fig. 4. Cntn2 is expressed in canine Purkinje fibers. (A) Epifluorescent images (Top) of

micro dissected canine Purkinje fiber (PF) bundles showing expression of Cntn2 (red, Left). In

the negative control (Right) the primary antibody was omitted. (B) Confocal images of the same

canine Purkinje Fiber bundles, showing expression of Cntn2 (red) on the membrane

(arrowheads) of Cx40+ (green) canine Purkinje cells (PCs), sectioned either longitudinally (Left)

or transversally (Right). Cx40+ intercalated disks (stars) are clearly visible at the junction of

Cntn2+ adjacent PCs. (Inset) High power en face image of an entire intercalated disk showing

Cntn2 signal at the periphery of Cx40+ domains with typical gap junction organization with

larger fluorescent patches at the periphery and smaller areas in the center. Cntn2 (arrowhead) is

also visible on the sarcolemma of two longitudinal PCs, separated by a Cx40+ intercalated disk

(star). (C) High power confocal image of cell surface optical slice of a single canine PC

confirming expression of Cntn2 on the sarcolemma, identified by expression of Cx40 (green) at

the intercalated disk junctions (arrowheads). Cntn2+ cells co-express the cardiac marker Nav1.5

as visualized at the cell surface (D), or in a subsurface slice (E). (F) Negative control: no

primary antibody. Size bars: 20 M (A, B); 10 M (C-F).

esesesesesesescececececececencncncncncncnce:e:e:e:e:e:e: D DDDDDDAPAPAPAPAPAPAPI II I I II nnnnnnn

xceppppttttttt fofofofofofoforrrrrrr BoBoBoBoBoBoBottttttttttttttomomoomomomo

t M

p g

2

a

t for Top, Left, 200 M, and Bottom, Left, 50 M; (C), 100CC M

pressed in canine Purkinje fibers. (A(( ) Epifluorescent imag

ne Purkinje fiber (PF) bundles showing expression of Cntn2

(Right( ) the primary antibody was omitted. (B( ) Confocal ima



23

Fig. 5. Analysis of electrophysiological features of Cntn2-EGFP+ cells. (A) Representative

phase contrast and corresponding epifluorescence micrographs of dissociated myocytes from

Cntn2-EGFP transgenic hearts. Cntn2-EGFP+ cells can be differentiated from surrounding

cardiac myocytes by their rod-shaped morphology (Left) and their Cx43- (Center), Cx40+ (Right)

phenotype. (B) Representative whole cell recordings of action potentials from Cntn2-EGFP- and

Cntn2-EGFP+ cells. (C) APs from Cntn2-EGFP+ myocytes demonstrated spontaneous electrical

oscillations in phase 2 which resulted in EADs (Top, arrows), at lower pacing rates (1Hz), and

DADs (Bottom, arrowheads), at higher pacing rates (5Hz). (D) Line-scan fluorescence images

and corresponding fluorescence profiles generated by Ca2+ transients in Cntn2-EGFP+ (Right)

and Cntn2-EGFP- (Left) myocytes. (E) Comparison of kinetic parameters corresponding to Ca2+

transients in Cntn2-EGFP+ and Cntn2-EGFP- myocytes. (F) Line plots (G) and graphs showing

that Cntn2-EGFP+ myocytes displayed a higher frequency of unstimulated Ca2+ events arising

from DADs compared to Cntn2-EGFP- myocytes. EAD, early afterdepolarization; DAD,

delayed afterdepolarization; AP, action potential. N=number of cells analyzed. VM, ventricular

myocyte; PF, Purkinje Fiber; APs, action potentials; EADs, early afterdepolarizations; DADs,

delayed afterdepolarizations.

ssstititititititimumumumumumumulalalalalalalateteteteteteted d dd d dd CaCaCaCaCaCaCa2+2+2+2+2+2+2+ ee

fterdrddddddepepepepepepepolololololololarararararararizizizizizzzatatatatatatatioio

r

n a

r

rization; AP, action potential. N=number of cells analyzed.

nje Fiber; APs, action potentials; EADs, early afterdepolariza

rizations.













Dun, Penelope A. Boyden and Glenn I. FishmanBenedetta A. Pallante, Steven Giovannone, Fang-Yu Liu, Jie Zhang, Nian Liu, Guoxin Kang, Wen

Contactin-2 Expression in the Cardiac Purkinje Fiber Network

Print ISSN: 1941-3149. Online ISSN: 1941-3084 Copyright © 2010 American Heart Association, Inc. All rights reserved.

Dallas, TX 75231is published by the American Heart Association, 7272 Greenville Avenue,Circulation: Arrhythmia and Electrophysiology

published online January 28, 2010;Circ Arrhythm Electrophysiol.

http://circep.ahajournals.org/content/early/2010/01/28/CIRCEP.109.928820World Wide Web at:

The online version of this article, along with updated information and services, is located on the

http://circep.ahajournals.org/content/suppl/2010/01/28/CIRCEP.109.928820.DC1.htmlData Supplement (unedited) at:

http://circep.ahajournals.org//subscriptions/

is online at: Circulation: Arrhythmia and Electrophysiology Information about subscribing to Subscriptions:

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

document. Permissions and Rights Question and Answerinformation about this process is available in the

requested is located, click Request Permissions in the middle column of the Web page under Services. FurtherCenter, not the Editorial Office. Once the online version of the published article for which permission is being

can be obtained via RightsLink, a service of the Copyright ClearanceCirculation: Arrhythmia and Electrophysiology Requests for permissions to reproduce figures, tables, or portions of articles originally published inPermissions:


http://circep.ahajournals.org/content/early/2010/01/28/CIRCEP.109.928820

http://circep.ahajournals.org/content/suppl/2010/01/28/CIRCEP.109.928820.DC1.html

http://www.ahajournals.org/site/rights/

http://www.lww.com/reprints

http://circep.ahajournals.org//subscriptions/


SUPPLEMENTAL MATERIAL Expanded Methods

Microarray Profiling. Purkinje fiber (PF) enriched samples were obtained by micro-

dissecting the subendocardial layer of 6-8 month old CCS-lacZ murine hearts and PF

depleted samples were micro-dissected from the sub-epicardial layer of the ventricular

walls, using a dissection microscope (Zeiss) and 0.125 mm tungsten micro-needles. The

tissue from three hearts was pooled together to generate one sample and three

independent pooled samples of each tissue type were analyzed in transcriptional profiling

performed on total RNA using the GeneChip 3’ IVT Express Labeling Kit (Affymetrix)

to generate biotin labeled probes hybridized to Mouse Genome 430 2.0 Array chips,

covering 39,000 mouse genes. Probe level was converted to expression values using both

the Robust Multi-array Average (RMA) procedure. 415 probe sets for genes/mRNAs

differentially regulated between PF-enriched versus PF-depleted samples were identified

using triplicate sets of Robust multichip average (RMA)-normalized Affymetrix CEL

files 1 followed by feature selection algorithm Pavlidis Template Matching (PTM,

p<0.05)2 and thresholding at 33% for reproducible fold change in expression level

between the two sample types.

Immunohistochemistry Mouse hearts or canine PFs were fixed in 4% PFA, then

equilibrated in 10% sucrose and embedded in OCT. Cryosections, 6-10µM thick, were

post-fixed for 10 min in 4% PFA (Cx40), permeabilized for 10 min in 0.5% Triton X-100

and blocked for 1 h at 37oC in 5% Normal Donkey Serum/0.1% Triton X-100/0.1% NP-

40. Slides were then incubated overnight at 4oC with the primary antibody followed by

incubation for 1 hr at 37oC with the secondary antibody and mounted with Vectashield

with or without the nuclear counter stain DAPI (VectorLabs). The permeabilization step

was omitted for Cx40, HCN4 and Cntn2 single staining. Staining for β-galactosidase

(βGal) and whole mount XGal staining were performed as previously described 3, 4. For

Cntn2/XGal serial staining, CCS-lacZ hearts were snap frozen in OCT, and sequential

cryosections were stained for Cntn2 or fixed and stained for XGal.

Epifluorescent images were acquired with an Axiovert 200M inverted microscope (Zeiss)

with 10x and 40x dry objectives, or a Semi SV 11 Apo dissection microscope, equipped

for fluorescence (Zeiss). With both microscopes images were acquired with an AxioCam

MRc camera (Zeiss) and AxioVision 4.6 software. Confocal images were acquired with

a SP5 confocal laser system (Leica), using 20x (water) and 40x (dry) objectives, and Ar

(488 nm line; green fluorescence) or Green HeNe (543 nM line; red fluorescence) lasers.

Immunocytochemistry of canine PCs. Freshly isolated PCs were seeded onto laminin–

coated glass coverslips. After attachment the cells were fixed for 15 min with 4% PFA,

pemeabilized for 20 min with 0.7% Triton X-100, incubated for 30 min in 5% Normal

Goat Serum blocking buffer. Cells were then incubated overnight at 4oC with the

primary antibodies, followed by incubation for 1.5 h with the appropriate combination of

secondary antibodies. Immunoreactivity was visualized by 488/568 excitation using a

Zeiss LSM 510 scope with 100x objective.

Antibodies. The following primary antibodies were used: rabbit anti-HCN4 (Millipore),

goat anti-Cntn2 (R&D Systems); mouse anti-sarcomeric actinin (Sigma); rabbit anti-

Cx40 (Alpha Diagnostics); rabbit anti-βGal (5’ -3’, Boulder, CO); rabbit anti-Nav1.5

rabbit (Alomone Laboratories, Israel). The following fluorophore-conjugated secondary

antibodies were used: biotinilated goat anti-rabbit (Sigma); FITC avidin D (Sigma);

Alexa Fluor 555 goat anti-rabbit (Invitrogen); FITC donkey anti-rabbit; TR-conjugated

donkey anti-rabbit; TR or FITC donkey anti-goat; Cy3 donkey anti-mouse; FITC goat

anti-mouse. Canine PCs were incubated with the following secondary antibody

combination: FITC donkey-anti-Rabbit (Jackson ImmunoResearch) and Cy3-conjugated

donkey anti-goat IgG. Antibodies were purchased from Jackson ImmunoResearch,

unless otherwise stated, and were diluted in blocking buffer at a concentration of 2-10

µg/ml.

Electrophysiological recordings in isolated ventricular myocytes

Ventricular myocytes were isolated using an established enzymatic digestion protocol5

yielding to 60-80% rod-shaped, calcium-tolerant myocytes. Within 6 hours from

isolation, laminin-coated dishes containing isolated ventricular myocytes were mounted

on the stage of an inverted microscope. The myocytes were bathed with the solution

containing (mmol/L): 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES and 5 glucose, pH

adjusted to 7.4 with NaOH at 35°C. Transmembrane potentials were recorded in whole

cell current mode as previously described. All signals were acquired at 5 kHz (Digidata

1322A, Axon Instruments) and analyzed with pCLAMP version 9.2 software (Axon

Instruments). Patch electrodes had a resistance of 2–3 M when filled with patch

electrode solutions containing (mmol/L): 120 potassium aspartate, 20 KCl, 1 MgCl2, 4

Na2ATP, 0.1 GTP, 10 HEPES, 10 glucose, pH adjusted to 7.2 with NaOH. Myocytes

were electrically stimulated by intracellular current injection through patch electrodes

using depolarizing pulses with duration of 3 ms and amplitude of 0.5-1 nA. Action

potential duration (APD) was measured at 90% and 50% of repolarization (APD90 and

APD50). DADs were defined as phase 4 positive (depolarizing) deflections of the

membrane potential. EADs were defined as positive (depolarizing) oscillations occurring

during phase 2 or 3 of action potential.

Ca2+ imaging. Isolated myocytes from Cntn2-EGFP transgenic mice were loaded with

the membrane-permeant Ca2+ indicator dye Rhod-2/AM. Cells were placed in solution

with 5 µM Rhod-2/AM (Invitrogen) at room temperature for 20 min. The loading

solution was removed, and cells was washed and equilibrated in fresh Tyrode’s solution

for 30 min to allow deesterification of the dye before recording. Epifluorescence light

was used to identify the EGFP+ and EGFP- cells. Ca2+ images were acquired using a 63x

water immersion objective (N.A.=1.2) with a Leica SP5 confocal microscope in line-scan

mode (400Hz). Rhod-2 was excited at 540-nm. Emission was collected at >560-nm.

During the experiments, cells were field stimulated with 1 Hz at least 1 min to reach a

steady-state of SR Ca2+ loading. In some experiments, the Ca2+ transients were measured

by microfluorimetry. Freshly isolated myocytes were incubated with 2µM Indo-1/AM

(Invitrogen) at room temperature for 15min. Then the loading solution was replaced with

fresh Tyrode’s solution for 30 min to allow deesterification of the dye before recording.

The Indo-1 loaded cells were illuminated by a Xenon lamp (excitation was 340nm) and

the dual emission light (405-nm and 495-nm) was collected by two separate PMT tubes.

The fluorescence ratio was calculated by Ionwizard (IonOptix, Milton, MA).

References

1. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of

Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003;31(4):e15.

2. Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M,

Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A,

Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J.

TM4: a free, open-source system for microarray data management and analysis.

Biotechniques. 2003;34(2):374-378.

3. Rentschler S, Vaidya DM, Tamaddon H, Degenhardt K, Sassoon D, Morley GE,

Jalife J, Fishman GI. Visualization and functional characterization of the

developing murine cardiac conduction system. Development. 2001;128(10):1785-

1792.

4. Visconti RP, Richardson CD, Sato TN. Orchestration of angiogenesis and

arteriovenous contribution by angiopoietins and vascular endothelial growth

factor (VEGF). Proc Natl Acad Sci U S A. 2002;99(12):8219-8224.

5. Liu N, Colombi B, Memmi M, Zissimopoulos S, Rizzi N, Negri S, Imbriani M,

Napolitano C, Lai FA, Priori SG. Arrhythmogenesis in catecholaminergic

polymorphic ventricular tachycardia: insights from a RyR2 R4496C knock-in

mouse model. Circ Res. 2006;99(3):292-298.

Table S1. Primers specific for mouse genes Gene Forward Reverse βactin 5’-CTGCCTGACGGCCAAGTCATCAC-3’; 5’-GTCAACGTCACACTTCATGATGG-3’ GAPDH 5’-TCTCCCTCACAATTTCCATCCCAG-3’ 5’-GGGTGCAGCGAACTTTATTTATTGATGG-3’ Cx40 5’-CCACCGCACTGGTGCCTA-3’ 5’-TTGAGTAGGGTGCCCTGGAG-3’ βGalactosidasea 5’-ATAACGAGCTCCTGCACTGG-3’ 5’-AAAAATCCATTTCGCTGGTG-3’ βGalactosidaseb 5’-ACTATCCCGACCGCCTTACT-3’ 5’-TAGCGGCTGATGTTGAACTG-3’ Cntn2/TAG1 5’-CATGTCTTCAGCCACTGACC-3’ 5’-TGGCCTTGTCCTGGGTTAT-3’ a conventional RT-PCR; b QRT-PCR

PTM 0-05 DOWN or PTM 0-05 UP

Sample Number ()*Systematic AVG FC p-value Common Genbank Gene Symbol_AffymetrixGene Title_Affymetrix Gene Symbol1435165_at 5.872759433 0.029247865 Cntn2 AV339091 --- Mus musculus transcribed sequence with moderate similarity to protein pdb:1LBG (E. coli) B Chain B, Lactose Operon Repressor Bound To 21-Base Pair Symmetric Operator Dna, Alpha Carbons OnlyCntn21421072_at 4.175814167 0.003160417 Irx5 NM_018826 Irx5 Iroquois related homeobox 5 (Drosophila)Irx51432420_a_at 3.082203933 0.064473846 2310003M01Rik AK009097 9430066I12Rik RIKEN cDNA 9430066I12 gene2310002L09Rik1426298_at 2.886283067 0.001470412 Irx2 AF295369 Irx2 Iroquois related homeobox 2 (Drosophila)Irx2 /// LOC1000456121440788_at 2.804869233 0.003811745 BB316368 --- Mus musculus transcribed sequencesGas81430068_at 2.6503976 0.011630521 C030011L09Rik BB356045 --- Mus musculus adult male corpus striatum cDNA, RIKEN full-length enriched library, clone:C030011L09 product:unknown EST, full insert sequenceC030011L09Rik1430635_at 2.602525433 0.012603521 Mlana BB774399 Mlana melan-A Mlana1443821_at 2.5550902 0.051939433 AW122758 --- Mus musculus transcribed sequence with weak similarity to protein pir:A36298 (H.sapiens) A36298 proline-rich protein PRB3M (null) - human (fragment)Lect21452270_s_at 2.486160467 0.032094027 Cubn AF197159 Cubn cubilin (intrinsic factor-cobalamin receptor)Cubn1449015_at 2.4667787 0.039646164 Retnla NM_020509 Retnla resistin like alpha Retnla1423286_at 2.4645071 0.050849899 Cbln1 AA016422 Cbln1 cerebellin 1 precursor proteinCbln1 /// LOC1000463141434240_at 2.457186067 0.003646211 4632434I11Rik BB463610 4632434I11Rik RIKEN cDNA 4632434I11 gene4632434I11Rik1452642_at 2.4130641 0.001295682 BE372017 --- Mus musculus adult male pituitary gland cDNA, RIKEN full-length enriched library, clone:5330434I02 product:unknown EST, full insert sequence---1419414_at 2.3831449 0.02847821 Gng13 AB030194 Gng13 guanine nucleotide binding protein 13, gammaGng131447986_at 2.356622133 0.045589351 D17892 D17892 --- Mus musculus transcribed sequencesD178921460372_at 2.318730333 0.019879261 BC019755 BC019755 BC019755 cDNA sequence BC019755Duoxa11431751_a_at 2.307472033 0.050148872 2700082O15Rik AK012553 --- Mus musculus similar to Fetal brain protein 239 (239FB) (LOC383738), mRNAMpped21449031_at 2.288619667 0.021115906 Cited1 U65091 Cited1 Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1Cited11449588_at 2.257555233 0.063903913 Abca4 NM_007378 Abca4 ATP-binding cassette, sub-family A (ABC1), member 4Abca41436617_at 2.235074767 0.005057972 Cetn4 AV211098 LOC207175 centrin 4 Cetn41448321_at 2.191838967 0.037612386 Smoc1 NM_022316 Smoc1 SPARC related modular calcium binding 1Smoc11450266_at 2.145880067 0.002831577 Scn10a NM_009134 Scn10a sodium channel, voltage-gated, type X, alpha polypeptideScn10a1436376_s_at 2.129053867 0.010465475 AI461933 C85414 AI461933 expressed sequence AI461933Srbd11425178_s_at 2.1113407 0.000555249 Shmt1 AF237702 Shmt1 serine hydroxymethyl transferase 1 (soluble)Shmt11424107_at 2.107242533 0.003856724 Kif18a BC016095 Kif18a kinesin family member 18AKif18a1452651_a_at 2.0986456 0.036770258 Myl1 AK003182 Myl1 myosin, light polypeptide 1, alkali; atrial, embryonicMyl11447966_a_at 2.061129933 0.011057409 A630048M13 AW123020 A630048M13 hypothetical protein A630048M13Tmem691417654_at 2.034422133 0.006613708 Sdc4 BC005679 Sdc4 syndecan 4 Sdc41437669_x_at 1.992355033 0.00211137 Ccrl1 BB543291 Ccrl1 chemokine (C-C) receptor-like 1Ccrl11460555_at 1.9746743 0.027309394 6330500D04Rik BM242294 6330500D04Rik RIKEN cDNA 6330500D04 gene6330500D04Rik1427428_at 1.956474467 0.021168378 4930572L20Rik BC025069 4930572L20Rik RIKEN cDNA 4930572L20 geneClec4g1418151_at 1.955439233 0.053068159 Mtmr4 BQ032797 Mtmr4 myotubularin related protein 4Mtmr41456971_at 1.9531528 0.001899647 Slc1a7 BB280296 A930031E15Rik RIKEN cDNA A930031E15 geneSlc1a71456983_at 1.949645467 0.058659942 BB360644 --- Mus musculus adult male corpus striatum cDNA, RIKEN full-length enriched library, clone:C030048M04 product:unknown EST, full insert sequence---1418835_at 1.945700933 0.000846799 Phlda1 NM_009344 Phlda1 pleckstrin homology-like domain, family A, member 1Phlda11457756_at 1.9297795 0.000464687 Zfp192 BB483373 --- Mus musculus 13 days embryo lung cDNA, RIKEN full-length enriched library, clone:D430019P06 product:unknown EST, full insert sequenceZfp1921437149_at 1.918663967 0.011875297 Slc6a6 AI648846 Slc6a6 solute carrier family 6 (neurotransmitter transporter, taurine), member 6Slc6a61434073_at 1.91563 0.017166091 5330440H13Rik BB028100 3110031O14Rik RIKEN cDNA 3110031O14 geneGprasp21419080_at 1.893283867 0.040458973 Gdnf NM_010275 Gdnf glial cell line derived neurotrophic factorGdnf1444399_at 1.872906367 0.039875131 5830411E10Rik AI506429 5830411E10Rik RIKEN cDNA 5830411E10 geneObfc2a1420849_at 1.8456137 0.075778781 Crnkl1 AV143435 Crnkl1 Crn, crooked neck-like 1 (Drosophila)Crnkl11442611_at 1.824272267 0.059956137 4930592C13Rik BB019852 --- Mus musculus adult male testis cDNA, RIKEN full-length enriched library, clone:4930592C13 product:unclassifiable, full insert sequence4930592C13Rik1444122_at 1.8105262 0.009022712 4930518F03Rik AW556894 4930518F03Rik RIKEN cDNA 4930518F03 geneSycp21458014_at 1.8086192 0.018463882 Fmr2 BB245809 Fmr2 fragile X mental retardation 2 homologAff21459311_at 1.777227 0.0301045 9630011N22Rik BB255824 9630011N22Rik RIKEN cDNA 9630011N22 genePde4d1418953_at 1.775498267 0.017316266 Fbxo16 NM_015795 Fbxo16 F-box only protein 16 Fbxo161449098_a_at 1.774916333 0.066754312 Poli NM_011972 Poli polymerase (DNA directed), iotaPoli1428217_at 1.7677801 0.039827603 1600012H06Rik AK017276 1600012H06Rik RIKEN cDNA 1600012H06 gene1600012H06Rik1455616_at 1.766615767 0.048419245 AI586002 BB667191 Aebp2 AE binding protein 2 AI5860021420349_at 1.765067833 0.004577787 Ptgfr NM_008966 Ptgfr prostaglandin F receptorPtgfr1435166_at 1.741166167 0.022470971 Cntn2 AV339091 --- Mus musculus transcribed sequence with moderate similarity to protein pdb:1LBG (E. coli) B Chain B, Lactose Operon Repressor Bound To 21-Base Pair Symmetric Operator Dna, Alpha Carbons OnlyCntn21424974_at 1.739579933 0.06433412 A230102I05Rik BC024761 A230102I05Rik RIKEN cDNA A230102I05 geneZfp4181456956_at 1.733587 0.054457188 Zfpn1a2 BB291816 Znfn1a2 zinc finger protein, subfamily 1A, 2 (Helios)Ikzf21446934_at 1.726845 0.067207482 BB548287 --- --- ---1427582_at 1.7238651 0.004711807 Fgf6 M92416 Fgf6 fibroblast growth factor 6Fgf61432108_at 1.7206513 0.058449878 Rnf134 AK016709 Rnf134 ring finger protein 134Pcgf61456517_at 1.7168144 0.026342758 9330161C17Rik BB035891 9330161C17Rik RIKEN cDNA 9330161C17 geneTmem441447807_s_at 1.701295433 0.007039448 Plekhh1 AV336001 Plekhh1 pleckstrin homology domain containing, family H (with MyTH4 domain) member 1Plekhh11445550_at 1.6972071 0.034708678 BB252788 --- Mus musculus transcribed sequencesKlhl321422533_at 1.680870933 0.02385404 Cyp51 NM_020010 Akap9 A kinase (PRKA) anchor protein (yotiao) 9Cyp511429225_at 1.6786911 0.033977285 6330417K15Rik AI847460 --- Mus musculus adult male hippocampus cDNA, RIKEN full-length enriched library, clone:C630005J10 product:unknown EST, full insert sequenceSlc24a21440450_at 1.675953233 0.051587325 Hspa1l AW611466 Hspa1l heat shock protein 1-likeLsm21440845_at 1.669638433 0.026563013 E030031F02Rik BB323696 --- Mus musculus 0 day neonate lung cDNA, RIKEN full-length enriched library, clone:E030031F02 product:unknown EST, full insert sequence4930420K17Rik1429939_at 1.660952533 0.033221306 D19Ertd703e AK015925 D19Ertd703e DNA segment, Chr 19, ERATO Doi 703, expressedSaps31432700_at 1.660788267 0.070889235 2810047J09Rik AV138957 --- Mus musculus 10, 11 days embryo whole body cDNA, RIKEN full-length enriched library, clone:2810047J09 product:unclassifiable, full insert sequence2810047J09Rik1431300_at 1.660725833 0.024159546 3110007P09Rik AK014022 3110007P09Rik RIKEN cDNA 3110007P09 geneSgip11435229_at 1.658439433 0.038250135 A930008A22Rik BI729743 A930008A22Rik RIKEN cDNA A930008A22 geneGramd1b1437469_at 1.646847433 0.048394798 A030007D23Rik BM230508 A030007D23Rik RIKEN cDNA A030007D23 geneZfp7501449034_at 1.642419833 0.010208957 Klkb1 BC026555 Klkb1 kallikrein B, plasma 1 Klkb11418090_at 1.641480833 0.052083807 Plvap NM_032398 Plvap plasmalemma vesicle associated proteinPlvap1437015_x_at 1.6308531 0.004229261 Pla2g1b AV060866 Pla2g1b phospholipase A2, group IB, pancreasPla2g1b1437668_at 1.628822967 0.029870867 Ccrl1 BB543291 Ccrl1 chemokine (C-C) receptor-like 1Ccrl11453959_at 1.611580967 0.011786659 1700065O13Rik AK006897 1700065O13Rik RIKEN cDNA 1700065O13 gene1700065O13Rik1455279_at 1.600071433 0.025953695 BG070552 --- Mus musculus similar to CG10958-like (LOC381738), mRNAGm10601442167_at 1.5990051 0.063428741 Cyfip2 BG068238 Cyfip2 cytoplasmic FMR1 interacting protein 2Cyfip21442404_at 1.590610667 0.04548012 BB624790 --- Mus musculus adult male hypothalamus cDNA, RIKEN full-length enriched library, clone:A230061K20 product:unknown EST, full insert sequenceNcl1438053_at 1.582804033 0.016141339 Tfg BB428499 Tfg Trk-fused gene Tfg1431795_a_at 1.5694533 0.035690552 Sema3b AK014333 Sema3b sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3BSema3b1452732_at 1.5634657 0.002791972 2300003P22Rik AK004007 2300003P22Rik RIKEN cDNA 2300003P22 geneAsprv11451186_at 1.558746733 0.040475695 2700083B06Rik BI440638 2700083B06Rik RIKEN cDNA 2700083B06 geneIsg20l11454190_at 1.553051967 0.02825443 9630013K17Rik AK020677 --- Mus musculus 16 days neonate cerebellum cDNA, RIKEN full-length enriched library, clone:9630013K17 product:unclassifiable, full insert sequence9630013K17Rik1439823_at 1.552689967 0.062492098 BG797330 --- Mus musculus transcribed sequences---1435510_at 1.5490099 0.005301688 A430075L18Rik BB283101 A430075L18Rik RIKEN cDNA A430075L18 genePpm1h1431870_at 1.5483554 0.021732473 4930463O16Rik AK015500 4930463O16Rik RIKEN cDNA 4930463O16 gene4930463O16Rik1417069_a_at 1.5464078 0.013701031 Gmfb BG228815 Gmfb glia maturation factor, betaGmfb1429084_at 1.546337667 0.062825472 Vezf1 AV308858 Vezf1 vascular endothelial zinc finger 1Vezf11418996_a_at 1.537936433 0.025525281 4930469P12Rik BC021522 4930469P12Rik RIKEN cDNA 4930469P12 geneLyrm51435751_at 1.533610967 0.035110291 Abcc9 BG791642 Abcc9 ATP-binding cassette, sub-family C (CFTR/MRP), member 9Abcc91438692_at 1.529229933 0.001690782 Gtf3c4 BG069995 AI426938 expressed sequence AI426938Gtf3c41442008_at 1.527865933 0.009737656 BB181330 --- Mus musculus adult male hypothalamus cDNA, RIKEN full-length enriched library, clone:A230090L04 product:unknown EST, full insert sequence---1431394_a_at 1.526478333 0.013993255 4921513O20Rik AK014938 4921513O20Rik RIKEN cDNA 4921513O20 geneLrrk21459851_x_at 1.524062533 0.008460153 Riok1 AV348356 5430416A05Rik RIKEN cDNA 5430416A05 geneRiok11425823_at 1.523956433 0.059768351 Cfh BC026782 Cfh complement component factor hBC0267821455033_at 1.5225902 0.004547972 B430201A12Rik BB325849 B430201A12 hypothetical protein B430201A12B430201A12Rik1455512_at 1.5213474 0.019024793 AI852151 --- Mus musculus transcribed sequence with strong similarity to protein sp:P00722 (E. coli) BGAL_ECOLI Beta-galactosidase (Lactase)Gm8791450754_at 1.521134567 0.031941987 Cacna2d2 AF247139 Cacna2d2 calcium channel, voltage-dependent, alpha 2/delta subunit 2Cacna2d2 /// LOC1000462591420641_a_at 1.513318067 0.071465452 Sqrdl AF174535 Sqrdl sulfide quinone reductase-like (yeast)Sqrdl1427344_s_at 1.509712933 0.037326867 Rasd2 BC026377 4930526B11Rik RIKEN cDNA 4930526B11 geneRasd21450468_at 1.506817967 0.032508595 Myoc AW125804 Myoc myocilin Myoc1430346_at 1.504389167 0.036958631 5730507A09Rik BB550527 5730507A09Rik RIKEN cDNA 5730507A09 gene5730507A09Rik1425627_x_at 1.503127967 0.024129374 Gstm1 J03952 Gstm1 glutathione S-transferase, mu 1Gstm1 /// LOC100043965 /// LOC433943 /// LOC436518 /// LOC6740861458918_at 1.502905167 0.002276123 BB552741 --- Mus musculus transcribed sequence with moderate similarity to protein pir:S12207 (M.musculus) S12207 hypothetical protein (B2 element) - mouseSlc12a81417768_at 1.500294333 0.03644326 1200006O19Rik BC019364 1200006O19Rik RIKEN cDNA 1200006O19 genePnpla81459418_at 1.4980453 0.020766664 BG076300 --- --- ---1450823_at 1.4952512 0.044313331 Og9x NM_008759 Og9x OG9 homeobox gene Sebox1458515_at 1.494538533 0.016836933 Zfp128 BB502692 Zfp128 zinc finger protein 128Zfp1281428023_at 1.4936301 0.010058946 3110009E18Rik AA879840 3110009E18Rik RIKEN cDNA 3110009E18 gene3110009E18Rik1429190_at 1.493552733 0.045273774 1110007C02Rik BI440651 --- Mus musculus similar to Arylsulfatase B (ASB) (N-acetylgalactosamine-4-sulfatase) (G4S) (LOC218452), mRNAArsb1439798_at 1.491406433 0.039369876 Hoxc10 BB779859 Hoxc10 homeo box C10 Hoxc101448536_at 1.4828737 0.022016313 Lsm3 NM_026309 Lsm3 LSM3 homolog, U6 small nuclear RNA associated (S. cerevisiae)Lsm31415894_at 1.4819761 0.063099152 Enpp2 BC003264 Enpp2 ectonucleotide pyrophosphatase/phosphodiesterase 2Enpp21454002_at 1.475856233 0.03366323 2700071L08Rik AK012512 --- Mus musculus 16 days embryo head cDNA, RIKEN full-length enriched library, clone:C130043J02 product:unknown EST, full insert sequence---1417867_at 1.4755646 0.008390207 Adn NM_013459 Adn adipsin Cfd1436433_at 1.474765667 0.037206304 BC049762 AV264828 4930503F14 hypothetical protein 4930503F14BC0497621440911_at 1.469189233 0.055143177 2810458L13Rik AI429655 Col23a1 procollagen, type XXIII, alpha 1Col23a11427155_at 1.468465567 0.041154207 Fchsd1 AK014510 4631403P03Rik RIKEN cDNA 4631403P03 geneFchsd11436229_at 1.4684648 0.103100483 BC049806 BB256501 --- Mus musculus 12 days embryo spinal cord cDNA, RIKEN full-length enriched library, clone:C530033E13 product:unknown EST, full insert sequenceBC0498061438588_at 1.466968667 0.007051284 Plagl1 BB540903 Plagl1 pleiomorphic adenoma gene-like 1Plagl11456116_at 1.4629025 0.018101582 Catnd2 BB431091 Catnd2 catenin delta 2 Ctnnd2 /// LOC1000459791457377_at 1.461455467 0.034266679 BB253657 --- Mus musculus transcribed sequences---1427571_at 1.461199033 0.031502758 Shh X76290 Shh sonic hedgehog Shh1458425_at 1.460135733 0.072345185 BM945176 --- Mus musculus 0 day neonate thymus cDNA, RIKEN full-length enriched library, clone:A430046D13 product:unclassifiable, full insert sequenceA430046D13Rik1444505_at 1.459583067 0.028890248 BC065120 BB729667 4931406H21Rik RIKEN cDNA 4931406H21 geneZmiz11446773_at 1.459290267 0.021502941 E230025N22 BB069871 E230025N22 hypothetical protein E230025N22E230025N22Rik1436417_at 1.455243267 0.027632994 Slc19a3 BG070700 Slc19a3 solute carrier family 19 (sodium/hydrogen exchanger), member 3LOC6652461458277_at 1.455083133 0.071980525 Ccl25 BB640315 Ccl25 chemokine (C-C motif) ligand 25Ccl251438883_at 1.449604333 0.025706492 Fgf5 AV240088 Fgf5 fibroblast growth factor 5Fgf51449113_at 1.445590567 0.014203188 5330440M15Rik NM_030247 5330440M15Rik RIKEN cDNA 5330440M15 geneGpbp1l11436406_at 1.445580633 0.038662272 BB327942 --- Mus musculus 4 days neonate male adipose cDNA, RIKEN full-length enriched library, clone:B430304M13 product:unknown EST, full insert sequence---1456963_at 1.439867267 0.034324466 1110039B18Rik BM508503 1110039B18Rik RIKEN cDNA 1110039B18 gene1110039B18Rik1442176_at 1.439518433 0.032592475 Arid5b BB006508 Desrt developmentally and sexually retarded with transient immune abnormalitiesArid5b1434989_at 1.438669133 0.083130543 BQ176485 --- Mus musculus 6 days neonate skin cDNA, RIKEN full-length enriched library, clone:A030001O10 product:unknown EST, full insert sequenceBC0427201430182_a_at 1.436083 0.017007927 1700006J14Rik AK011105 1700006J14Rik RIKEN cDNA 1700006J14 gene1700006J14Rik1437177_at 1.434941667 0.025339512 DXErtd793 BI964325 D330037H05Rik RIKEN cDNA D330037H05 geneLarp4 /// LOC627412 /// LOC6326841443050_at 1.432759567 0.042299758 BC032265 BB072270 BC032265 cDNA sequence BC032265---1448832_a_at 1.4322935 0.033083104 Cplx1 BC014803 Cplx1 complexin 1 Cplx11426506_at 1.431665833 0.01109184 Evi2b AI122415 Evi2a ecotropic viral integration site 2aEvi2a /// Evi2b1425767_a_at 1.431623033 0.068943587 Six4 D50416 Six4 sine oculis-related homeobox 4 homolog (Drosophila)Six41458108_at 1.429959133 0.014795575 BB322169 --- Mus musculus adult male adrenal gland cDNA, RIKEN full-length enriched library, clone:B330013C06 product:unclassifiable, full insert sequence---1455737_at 1.429907533 0.072634707 C030002B11Rik AU040848 --- Mus musculus 2 days neonate thymus thymic cells cDNA, RIKEN full-length enriched library, clone:E430020E24 product:unknown EST, full insert sequencePpm1h1420755_a_at 1.4198147 0.005768645 Park2 AF250293 Park2 parkin Park21439058_at 1.4191984 0.075897211 5730453G22Rik BG061796 Sfpq splicing factor proline/glutamine rich (polypyrimidine tract binding protein associated)Sfpq1424707_at 1.4178778 0.038102743 1110014C03Rik BI409239 1110014C03Rik RIKEN cDNA 1110014C03 geneTmed101419886_at 1.4158133 0.027832224 DXErtd223e BG079602 --- Mus musculus transcribed sequencesDXErtd223e1425360_at 1.413360733 0.009699784 AI315037 AY050217 Mllt6 myeloid/lymphoid or mixed lineage-leukemia translocation to 6 homolog (Drosophila)Mllt61441765_at 1.408534767 0.082293011 BB822342 --- Mus musculus transcribed sequences---1418313_at 1.4056525 0.06365691 Zfp276 BB667131 Zfp276 zinc finger protein (C2H2 type) 276Zfp2761457997_at 1.4049338 0.040848577 5730589L02Rik BB186022 Tmc4 transmembrane channel-like gene family 4Leng41421752_a_at 1.403573333 0.038942109 Serpinb5 NM_009257 Serpinb5 serine (or cysteine) proteinase inhibitor, clade B, member 5Serpinb51440026_at 1.396455433 0.027455133 BM120634 --- Mus musculus 12 days embryo spinal ganglion cDNA, RIKEN full-length enriched library, clone:D130046C24 product:unknown EST, full insert sequence---1450676_at 1.3894312 0.063364992 Tceb3 BI965936 Tceb3 transcription elongation factor B (SIII), polypeptide 3Tceb31446122_at 1.389156967 0.052221753 4732416N19Rik AV236932 1200009B18Rik RIKEN cDNA 1200009B18 gene4732416N19Rik1435252_at 1.3862108 0.027655787 B3galt6 AV328064 B3galt6 UDP-Gal:betaGal beta 1,3-galactosyltransferase, polypeptide 6B3galt61440932_at 1.381405167 0.037710948 Hist3h2a BB428585 Trim17 tripartite motif protein 17---1429276_at 1.377461733 0.049109212 C030048J01Rik AK021160 --- Mus musculus similar to mKIAA0342 protein (LOC235639), mRNALba11423344_at 1.3733098 0.035733342 Epor AK010968 Epor erythropoietin receptorEpor1457442_at 1.3732432 0.053229232 AW125324 AW125324 --- Mus musculus transcribed sequencesAW1253241430317_at 1.368468633 0.028484652 Ube2j2 BB279437 Ube2j2 ubiquitin-conjugating enzyme E2, J2 homolog (yeast)Ube2j21452598_at 1.3658368 0.04922318 2810418N01Rik AK013116 2810418N01Rik RIKEN cDNA 2810418N01 geneGins11417375_at 1.3561107 0.053191178 Tuba4 AW491660 Stk16 serine/threonine kinase 16Tuba4a1426960_a_at 1.347902367 0.051314866 Fa2h BM118638 Fa2h fatty acid 2-hydroxylaseFa2h1422520_at 1.342046333 0.052424892 Nef3 NM_008691 Nef3 neurofilament 3, mediumNefm1443418_at 1.3290223 0.05269736 BB236368 --- Mus musculus 3 days neonate thymus cDNA, RIKEN full-length enriched library, clone:A630058O09 product:unknown EST, full insert sequence---1443241_at 0.773418267 0.032363534 AW544264 --- Mus musculus 13 days embryo stomach cDNA, RIKEN full-length enriched library, clone:D530023N15 product:unclassifiable, full insert sequence---

1425035_s_at 0.7712053 0.026400272 Dnmt3l AF220524 Dnmt3l DNA (cytosine-5-)-methyltransferase 3-likeDnmt3l1448839_at 0.76172948 0.03017889 D17Ertd288e NM_030697 0610013D04Rik RIKEN cDNA 0610013D04 geneAnkrd471426748_s_at 0.755697033 0.069575595 Abcf3 AI552141 Abcf3 ATP-binding cassette, sub-family F (GCN20), member 3Abcf31446094_at 0.75163398 0.0173615 BG076353 --- Mus musculus transcribed sequences---1420743_a_at 0.7465365 0.048480979 Ppp3cc NM_008915 Ppp3cc protein phosphatase 3, catalytic subunit, gamma isoformPpp3cc1421673_s_at 0.74539486 0.057381261 Stx1b2 NM_024414 Stx1bl syntaxin 1 b-like Stx1b1 /// Stx1b21433880_at 0.742279333 0.056164339 E030019A03Rik BB531733 E030019A03Rik RIKEN cDNA E030019A03 geneDnajc111437182_at 0.740279567 0.024323954 Dido1 BM224349 Dido1 death inducer-obliterator 1Dido11444972_at 0.740011243 0.018994385 C77626 BG065987 --- --- C776261447301_at 0.739465867 0.043216765 BG228875 --- Mus musculus similar to A-kinase anchor protein 5; RII-B-binding protein (LOC238276), mRNAAkap51430021_a_at 0.7393637 0.001185022 Uble1a AK011772 Uble1a ubiquitin-like 1 (sentrin) activating enzyme E1ASae11418125_at 0.738324133 0.018083785 4632409L19Rik BM238259 4632409L19Rik RIKEN cDNA 4632409L19 geneInoc1 /// LOC1000484761441123_at 0.73802759 0.004849593 ORF34 BB023801 ORF34 open reading frame 34ORF341441158_at 0.7375835 0.038774524 BB707145 --- Mus musculus adult male aorta and vein cDNA, RIKEN full-length enriched library, clone:A530046H22 product:unknown EST, full insert sequence---1450594_at 0.735007233 0.009064867 Magea4 NM_020280 Magea4 melanoma antigen, family A, 4Magea41455547_at 0.734205723 0.036541861 Scrg3 BM125518 Scrg3 scrapie responsive gene 3Zc3h7b1422341_s_at 0.73282398 0.019446052 Lypla3 NM_133792 Lypla3 lysophospholipase 3 Lypla31452171_at 0.732759533 0.063299397 Grwd1 BM200151 --- Mus musculus transcribed sequence with moderate similarity to protein sp:Q9BQ67 (H.sapiens) GRWD_HUMAN Glutamate-rich WD repeat proteinGrwd11416274_at 0.731424187 0.019478862 Ctns NM_031251 Ctns cystinosis, nephropathicCtns1426334_a_at 0.730825517 0.039299473 Bcl2l11 AF032460 Bcl2l11 BCL2-like 11 (apoptosis facilitator)Bcl2l111433116_at 0.729115593 0.046627339 5730552O08Rik AK017837 --- --- 5730552O08Rik1455745_at 0.72809771 0.054796368 BM249363 --- Mus musculus transcribed sequencesCln81424718_at 0.727628453 0.027933287 Mapt M18775 Mapt microtubule-associated protein tauMapt1423478_at 0.727118233 0.025626938 Prkcb BF660388 Prkcb protein kinase C, beta Prkcb11435108_at 0.72457435 0.001340729 Arhgap22 BG295243 B230341L19Rik RIKEN cDNA B230341L19 geneArhgap221426869_at 0.723745767 0.023020737 Boc BB005556 Boc biregional cell adhesion molecule-related/down-regulated by oncogenes (Cdon) binding proteinBoc1419584_at 0.722864707 0.020464949 AI428795 AW492543 AI428795 expressed sequence AI428795Ttc281457910_at 0.721580733 0.020410601 AW552893 --- Mus musculus transcribed sequences---1446946_at 0.721510767 0.008812469 BM217698 --- Mus musculus transcribed sequences---1440562_at 0.7213552 0.00781423 BB308531 --- Mus musculus adult male corpora quadrigemina cDNA, RIKEN full-length enriched library, clone:B230304I02 product:hypothetical protein, full insert sequence---1458803_at 0.7207291 0.011344145 9830137M10Rik BB837976 9830137M10Rik RIKEN cDNA 9830137M10 geneSlfn91457384_at 0.719557397 0.066112367 AV252220 --- Mus musculus 0 day neonate head cDNA, RIKEN full-length enriched library, clone:4833438M18 product:unknown EST, full insert sequence---1457922_at 0.716056993 0.02089127 BB038696 --- Mus musculus transcribed sequencesUng1441630_at 0.71597627 0.024058788 Ep400 AW541237 Ep400 E1A binding protein p400Ep4001421217_a_at 0.715687533 0.004458069 Lgals9 NM_010708 Lgals9 lectin, galactose binding, soluble 9Lgals91455022_at 0.715265437 0.02385327 Strn BG070684 --- Mus musculus 12 days embryo male wolffian duct includes surrounding region cDNA, RIKEN full-length enriched library, clone:6720407O05 product:unknown EST, full insert sequenceStrn1429007_at 0.7141988 0.03105821 Slc35b1 AK003377 Slc35b1 solute carrier family 35, member B1Slc35b21447913_x_at 0.712922307 0.074639927 Akap9 BB109183 Akap9 A kinase (PRKA) anchor protein (yotiao) 9Akap91441544_at 0.712385793 0.027644202 BM114572 --- Mus musculus adult male medulla oblongata cDNA, RIKEN full-length enriched library, clone:6330578K09 product:unclassifiable, full insert sequence---1444628_at 0.712195407 0.018450208 Adam33 AW986061 Adam33 a disintegrin and metalloprotease domain 33Adam331454095_at 0.711703673 0.056850135 4930579H20Rik AK016323 --- Mus musculus adult male testis cDNA, RIKEN full-length enriched library, clone:4930579H20 product:unclassifiable, full insert sequence4930579H20Rik1443709_at 0.709487983 0.033402187 BE979452 --- Mus musculus transcribed sequences---1450976_at 0.70677633 0.035409397 Ndrg1 AI987929 Ndr1 N-myc downstream regulated 1Ndrg11431943_at 0.706177967 0.01126024 2210011K15Rik AK008708 --- Mus musculus adult male stomach cDNA, RIKEN full-length enriched library, clone:2210011K15 product:unclassifiable, full insert sequence2210011K15Rik1427747_a_at 0.70609417 0.021920901 Lcn2 X14607 Lcn2 lipocalin 2 Lcn21436184_at 0.705330113 0.022322988 mKIAA1196 BM117370 mKIAA1196 mKIAA1196 protein Znf512b1443571_at 0.7043409 0.023226941 AI788586 --- Mus musculus transcribed sequences---1434480_at 0.703093013 0.024953988 4930402E16Rik BB667201 4930402E16Rik RIKEN cDNA 4930402E16 gene4930402E16Rik1424312_at 0.702633937 0.028889709 2810031L11Rik BC014875 2810031L11Rik RIKEN cDNA 2810031L11 geneAdipor11429897_a_at 0.701948447 0.005953743 D16Ertd472e AK009258 D16Ertd472e DNA segment, Chr 16, ERATO Doi 472, expressedD16Ertd472e1460556_at 0.701154347 0.011349094 D15Mit260 AF096898 D15Mit260 DNA Segment, Chr 15 Massachusetts Institute of Technology 260---1424367_a_at 0.701088367 0.030686229 Homer2 AB017136 Homer2 homer homolog 2 (Drosophila)Homer21421834_at 0.700880633 0.019214078 Pip5k1a NM_008846 Pip5k1a phosphatidylinositol-4-phosphate 5-kinase, type 1 alphaPip5k1b1416762_at 0.700264303 0.022404173 S100a10 NM_009112 S100a10 S100 calcium binding protein A10 (calpactin)S100a101421462_a_at 0.699454247 0.010448877 Lepre1 NM_019783 Lepre1 leprecan 1 Lepre11456733_x_at 0.698303283 0.04298588 Serpinh1 BB329489 Serpinh1 serine (or cysteine) proteinase inhibitor, clade H, member 1Serpinh11425090_s_at 0.697392583 0.03745836 Kcnc4 BC024837 Kcnc4 potassium voltage gated channel, Shaw-related subfamily, member 4Kcnc41455435_s_at 0.696732617 0.009002844 Chdh BG072100 D630034H06Rik RIKEN cDNA D630034H06 geneChdh1440363_at 0.696061267 0.01091535 BB560961 --- Mus musculus transcribed sequences---1442845_at 0.69492398 0.045888927 C130075A20Rik AV320517 --- Mus musculus 16 days neonate heart cDNA, RIKEN full-length enriched library, clone:D830015N20 product:unknown EST, full insert sequenceC130075A20Rik1417173_at 0.693355553 0.019089627 Crebl1 BC013534 Crebl1 cAMP responsive element binding protein-like 1Crebl11422572_at 0.693030287 0.027600935 Rhog NM_019566 Arhg ras homolog gene family, member GRhog1417885_at 0.69283492 0.050519362 Mapt BC014748 Mapt microtubule-associated protein tauMapt1452151_at 0.69248673 0.011185599 BC021523 BC021523 BC021523 cDNA sequence BC021523BC0215231419160_at 0.690342767 0.012364516 Golga3 D78270 Golga3 golgi autoantigen, golgin subfamily a, 3Golga31422079_at 0.69016589 0.012972316 Prkch NM_008856 Prkch protein kinase C, eta Prkch1424870_at 0.689252813 0.027806206 Osbpl10 BC027256 C820004B04Rik RIKEN cDNA C820004B04 geneLOC100047530 /// Osbpl101457789_at 0.688351977 0.050145865 Cln3 AV378786 Cln3 ceroid lipofuscinosis, neuronal 3, juvenile (Batten, Spielmeyer-Vogt disease)Cln31446245_at 0.68771392 0.009050515 BB042558 --- Mus musculus 13 days embryo male testis cDNA, RIKEN full-length enriched library, clone:6030463N01 product:unknown EST, full insert sequence---1417868_a_at 0.68701763 0.031651545 Ctsz NM_022325 Ctsz cathepsin Z Ctsz1420749_a_at 0.686650157 0.074943521 Pou6f1 NM_010127 Pou6f1 POU domain, class 6, transcription factor 1Pou6f11429207_at 0.686096087 0.021013916 5730408K05Rik BB257344 5730408K05Rik RIKEN cDNA 5730408K05 gene5730408K05Rik1424797_a_at 0.685713247 0.08833289 Pitx2 U80011 Pitx2 paired-like homeodomain transcription factor 2Pitx21442012_at 0.684758443 0.025303285 AU015791 AV381755 --- Mus musculus transcribed sequencesAU0157911421964_at 0.68322083 0.031152272 Notch3 NM_008716 Notch3 Notch gene homolog 3 (Drosophila)Notch31416398_at 0.682335967 0.007433071 Mesdc1 BB474887 Mesdc1 mesoderm development candidate 1Mesdc11437231_at 0.681271057 0.030983684 Slitrk6 AV246497 Slitrk6 SLIT and NTRK-like family, member 6Slitrk61460741_x_at 0.679826477 0.00868005 D17Wsu92e BG069802 D17Wsu92e DNA segment, Chr 17, Wayne State University 92, expressedD17Wsu92e1446063_at 0.6793176 0.047143549 C730034F03Rik BB667700 C730034F03Rik RIKEN cDNA C730034F03 geneC730034F03Rik1424770_at 0.678951733 0.037948636 Cald1 BI248947 Cald1 caldesmon 1 Cald11441732_at 0.676066863 0.051465717 AU042873 --- Mus musculus transcribed sequences---1448965_at 0.675911567 0.015606474 4632409L19Rik BM238259 4632409L19Rik RIKEN cDNA 4632409L19 geneInoc1 /// LOC1000484761439984_at 0.675408033 0.047746335 Ptdss2 BB474782 Ptdss2 phosphatidylserine synthase 2Ptdss21457668_x_at 0.675237733 0.031107847 Ebna1bp2 BB249696 Ebna1bp2 EBNA1 binding protein 2LOC100041290 /// LOC1000449721428484_at 0.674170943 0.003350951 Osbpl3 AK004768 Osbpl3 oxysterol binding protein-like 3Osbpl31440554_at 0.674137667 0.072456925 BB667363 --- Mus musculus transcribed sequences---1437958_at 0.67336165 0.019607688 Xpr1 BG066856 Xpr1 xenotropic and polytropic retrovirus receptor 1Xpr11434201_at 0.672901433 0.003573812 Chrdl1 AV144145 Nrln1 neuralin 1 Chrdl11418226_at 0.671921513 0.014958997 Orc2l BB830976 Orc2l origin recognition complex, subunit 2-like (S. cerevisiae)Orc2l1429617_at 0.671871683 0.06803419 Cyld BM119209 Cyld cylindromatosis (turban tumor syndrome)Cyld1440625_at 0.6713363 0.056031827 D930015E06Rik BB547420 D930015E06Rik RIKEN cDNA D930015E06 geneD930015E06Rik1448462_at 0.671043853 0.019885776 Tdg NM_011561 Tdg thymine DNA glycosylaseEG545124 /// LOC434200 /// LOC624784 /// Tdg1447540_at 0.67069522 0.059618044 MGC67569 AV167924 MGC67569 Unknown (protein for MGC:67569)Tigd31427673_a_at 0.670405133 0.068712051 Sema3e Z93948 Sema3e sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3ELOC100044162 /// Sema3e1453762_at 0.669888723 0.006335749 1810012I05Rik AK010160 1810012I05Rik RIKEN cDNA 1810012I05 geneVps26b1455717_s_at 0.667221677 0.010325175 Daam2 BM206030 Daam2 dishevelled associated activator of morphogenesis 2Daam21452365_at 0.66573055 0.016998716 4732435N03Rik AV371987 4732435N03Rik RIKEN cDNA 4732435N03 gene4732435N03Rik1445328_at 0.665162403 0.026946415 E130010M05Rik BB530633 E130010M05Rik RIKEN cDNA E130010M05 geneCol4a41445255_at 0.664925877 0.034848639 Rapgef5 BB536074 Mrgef Mras regulated guanine nucleotide exchange factorRapgef51442188_at 0.664859457 0.023180082 BE979116 --- Mus musculus 0 day neonate cerebellum cDNA, RIKEN full-length enriched library, clone:C230031C13 product:unknown EST, full insert sequence---1437513_a_at 0.664562387 0.034680961 Tde2 BM239422 Tde2 tumor differentially expressed 2Serinc11447979_at 0.664295457 0.024857509 Rutbc2 AA408781 D5Bwg1524e DNA segment, Chr 5, Brigham & Women's Genetics 1524 expressedSgsm11442949_at 0.664165053 0.004268871 BG068088 --- Mus musculus transcribed sequences---1432101_a_at 0.66219592 0.016710726 4933424F08Rik AK006144 4933424F08Rik RIKEN cDNA 4933424F08 geneTssk41451027_at 0.66207899 0.049651338 Baiap2 AA796998 Baiap2 brain-specific angiogenesis inhibitor 1-associated protein 2Baiap21448428_at 0.661658593 0.018232025 Nbl1 NM_008675 Nbl1 neuroblastoma, suppression of tumorigenicity 1Nbl11438235_at 0.65887775 0.055610772 BG094104 --- Mus musculus transcribed sequences---1458920_at 0.658667017 0.052591229 AW555412 --- Mus musculus transcribed sequences---1417870_x_at 0.655202153 0.027547109 Ctsz NM_022325 Ctsz cathepsin Z Ctsz1447515_at 0.653850383 0.006780775 BE954996 --- Mus musculus transcribed sequences---1438404_at 0.65300983 0.006352572 Rnf144 BB125272 Rnf144 ring finger protein 144Rnf144a1449198_a_at 0.652985187 0.033211873 Siat9 BB829192 Siat9 sialyltransferase 9 (CMP-NeuAc:lactosylceramide alpha-2,3-sialyltransferase)St3gal51418962_at 0.64843245 0.032990362 1110005F07Rik AK009714 1110005F07Rik RIKEN cDNA 1110005F07 geneNecap21445031_at 0.64812042 0.010793711 BG075092 --- Mus musculus transcribed sequences---1418846_at 0.647418923 0.007198277 Ap4m1 NM_021392 Ap4m1 adaptor-related protein complex AP-4, mu 1Ap4m11422639_at 0.647334447 0.008435361 Calcb NM_054084 Calcb calcitonin-related polypeptide, betaCalcb1444982_at 0.645971167 0.045723212 C230007H23Rik BB380551 --- Mus musculus 16 days neonate heart cDNA, RIKEN full-length enriched library, clone:D830046F22 product:unclassifiable, full insert sequence---1445223_at 0.644867213 0.017274501 4933430H15Rik BB534764 4933430H15Rik RIKEN cDNA 4933430H15 gene4933430H15Rik1420731_a_at 0.6441529 0.025661855 Csrp2 NM_007792 Csrp2 cysteine and glycine-rich protein 2Csrp21460451_at 0.64396398 0.010567663 2310043I08Rik AK009779 2310043I08Rik RIKEN cDNA 2310043I08 geneTmem521453316_at 0.643834533 0.017388348 4933401F05Rik AK016601 4933401F05Rik RIKEN cDNA 4933401F05 gene4933401F05Rik1436350_at 0.64359455 0.099805851 D430039N05Rik BM117463 D430039N05Rik RIKEN cDNA D430039N05 geneD430039N05Rik1428131_a_at 0.641781437 0.034840275 Spec1 AK008084 1300002M12Rik RIKEN cDNA 1300002M12 geneCdc42se11422051_a_at 0.640314047 0.01304955 Gabbr1 NM_019439 Gabbr1 gamma-aminobutyric acid (GABA-B) receptor, 1Gabbr11448855_at 0.638968533 0.004752064 Rassf1 NM_019713 Rassf1 Ras association (RalGDS/AF-6) domain family 1Rassf11439968_x_at 0.63850104 0.044415208 BE949296 --- Mus musculus adult male corpora quadrigemina cDNA, RIKEN full-length enriched library, clone:B230215D24 product:unknown EST, full insert sequenceDbndd21449863_a_at 0.635210823 0.014981196 Dlx5 NM_010056 Dlx5 distal-less homeobox 5Dlx51452803_at 0.633876883 0.034495942 Glipr2 BM208214 Glipr2 GLI pathogenesis-related 2Glipr21458317_at 0.633813473 0.012941985 AI448971 --- Mus musculus transcribed sequences---1449554_at 0.633718657 0.064797057 Tle3 NM_009389 Tle3 transducin-like enhancer of split 3, homolog of Drosophila E(spl)LOC100048342 /// Tle31433147_at 0.63363228 0.029951737 Cald1 AK014755 Cald1 caldesmon 1 Cald11444517_at 0.62909819 0.030511414 AI551889 --- Mus musculus LOC380653 (LOC380653), mRNA---1426654_at 0.628657167 0.052281257 Nipa BI687848 1110054L24Rik RIKEN cDNA 1110054L24 geneZc3hc11436381_at 0.62831819 0.043342689 BC058433 BQ175774 DAP-3 disks large-associated protein 3Dlgap31430460_at 0.626871367 0.003349474 5830410F13Rik AW554864 2310047O13Rik RIKEN cDNA 2310047O13 gene2310047O13Rik1425214_at 0.625909423 0.012483025 P2ry6 BC027331 P2ry6 pyrimidinergic receptor P2Y, G-protein coupled, 6P2ry61440560_at 0.624791487 0.006202882 AU067691 --- Mus musculus 6 days neonate skin cDNA, RIKEN full-length enriched library, clone:A030001L21 product:hypothetical protein, full insert sequence---1418028_at 0.624406797 0.037961539 Dct NM_010024 Dct dopachrome tautomeraseDct1438878_at 0.623577713 0.002078579 6430537K16Rik AU067682 --- Mus musculus adult male olfactory brain cDNA, RIKEN full-length enriched library, clone:6430537K16 product:unknown EST, full insert sequence6430537K16Rik1439117_at 0.62227456 0.081398022 9330188N17Rik AU067755 Clmn calmin Clmn1453044_at 0.6209491 0.000888744 C030014I23Rik AK021081 C030014I23Rik RIKEN cDNA C030014I23 geneC030014I23Rik1416324_s_at 0.619187467 0.004176771 2410004N11Rik AV281062 2410004N11Rik RIKEN cDNA 2410004N11 geneKctd201423893_x_at 0.6187649 0.06864292 Apbb1 AF206720 Apbb1 amyloid beta (A4) precursor protein-binding, family B, member 1Apbb11436526_at 0.618234737 0.01640552 AU067744 BI965034 Mdcp1 M6PR domain containing protein 1Gnptg1422183_a_at 0.61668573 0.005643379 Adra1b NM_007416 Adra1b adrenergic receptor, alpha 1bAdra1b1427905_at 0.6141079 0.030241898 1810063B07Rik BI414448 1810063B07Rik RIKEN cDNA 1810063B07 gene1810063B07Rik1441230_at 0.61363548 0.020204875 BB470469 --- Mus musculus transcribed sequences---1427822_a_at 0.61340568 0.037127521 Copg2as2 U20265 --- --- Copg2as21422708_at 0.612867443 0.009071164 Pik3cg BB205102 Pik3cg phosphoinositide-3-kinase, catalytic, gamma polypeptidePik3cg1442651_at 0.611448087 0.028850449 BB797985 --- Mus musculus transcribed sequences---1417037_at 0.6112864 0.017728393 Orc6l NM_019716 Orc6l origin recognition complex, subunit 6-like (S. cerevisiae)Orc6l1442598_at 0.611001177 0.018754134 Prkrip1 AV324577 Prkrip1 Prkr interacting protein 1 (IL11 inducible)Prkrip11416845_at 0.61050948 0.001049066 Hspa5bp1 NM_133804 6720481D13Rik RIKEN cDNA 6720481D13 geneTmem132a1424895_at 0.609234513 0.036476691 Gpsm2 BC021308 6230410J09Rik RIKEN cDNA 6230410J09 geneGpsm21438451_at 0.608031693 0.036418215 Grit BI408524 Grit Rho GTPase-activating proteinGrit1417600_at 0.607974147 0.012368583 Slc15a2 NM_021301 Slc15a2 solute carrier family 15 (H+/peptide transporter), member 2Slc15a21437371_at 0.60620316 0.014018826 9930021J17Rik BB008528 9930021J17Rik RIKEN cDNA 9930021J17 gene9930021J17Rik1451571_s_at 0.604960323 0.036245993 8030466O12Rik BC010442 E430018J23Rik RIKEN cDNA E430018J23 gene6430604K15Rik /// 9130019O22Rik /// E430018J23Rik /// Zfp7641431826_a_at 0.602646067 0.06915989 4833424K13Rik AK014760 --- Mus musculus 0 day neonate head cDNA, RIKEN full-length enriched library, clone:4833424K13 product:unknown EST, full insert sequenceBrsk21455785_at 0.601451307 0.029652731 Kcna1 BQ175978 Kcna1 potassium voltage-gated channel, shaker-related subfamily, member 1Kcna11451679_at 0.599748267 0.003935349 6530401D17Rik BC016270 6530401D17Rik RIKEN cDNA 6530401D17 gene6530401D17Rik1434025_at 0.59930635 0.040474798 Klf5 BG069607 Klf5 Kruppel-like factor 5 ---1431398_at 0.599283263 0.061961556 5730446D14Rik AK012902 --- Mus musculus 10, 11 days embryo whole body cDNA, RIKEN full-length enriched library, clone:2810043O09 product:inferred: RIKEN cDNA 5730446D14 gene, full insert sequence5730446D14Rik1425854_x_at 0.597164213 0.010889899 Tcrb-V13 U07661 Tcrb-V13 T-cell receptor beta, variable 13Tcrb-V8.21453298_at 0.594756543 0.101640458 Ptpn21 AK013777 Ptpn21 protein tyrosine phosphatase, non-receptor type 21Ptpn211426955_at 0.589247647 0.014815399 Col18a1 D17546 Col18a1 procollagen, type XVIII, alpha 1Col18a11460181_at 0.58785988 0.054694964 Stmn3 NM_009133 Stmn3 stathmin-like 3 Stmn31417777_at 0.585473867 0.000128808 Ltb4dh BC014865 Ltb4dh leukotriene B4 12-hydroxydehydrogenaseLtb4dh

1460027_at 0.58508789 0.027378291 BC061194 BB013876 --- Mus musculus mRNA similar to sperm associated antigen 6 (cDNA clone MGC:58385 IMAGE:6774548), complete cdsBC0611941420374_at 0.582929063 0.037979067 Foxj2 BI249549 Foxj2 forkhead box J2 Foxj21446610_at 0.581723657 0.007227402 Elmo1 BB396016 Elmo1 engulfment and cell motility 1, ced-12 homolog (C. elegans)Elmo11460314_s_at 0.580674443 0.027620831 Hist2h3c2 NM_019469 Hist2h2be histone 2, H2be Hist1h3a /// Hist1h3b /// Hist1h3c /// Hist1h3d /// Hist1h3e /// Hist1h3f /// Hist1h3g /// Hist1h3h /// Hist1h3i /// Hist2h2aa2 /// Hist2h3b /// Hist2h3c1 /// Hist2h3c21416758_at 0.58003198 0.00891752 2310069I04Rik NM_133715 2310069I04Rik RIKEN cDNA 2310069I04 geneArhgap271458476_at 0.579293973 0.047993204 Eif2c3 AW455885 Eif2c3 eukaryotic translation initiation factor 2C, 3Eif2c31441442_at 0.579237137 0.023017867 BM232203 --- Mus musculus transcribed sequences---1443719_x_at 0.578500463 0.04054544 AW763628 --- Mus musculus transcribed sequencesDdx421444526_at 0.57398955 0.066712155 BF463841 --- Mus musculus 2 days neonate thymus thymic cells cDNA, RIKEN full-length enriched library, clone:E430005K05 product:TRANSLOCON-ASSOCIATED PROTEIN, ALPHA SUBUNIT PRECURSOR (TRAP-ALPHA) (SIGNAL SEQUENCE RECEPTOR ALPHA SUBUNIT) (SSR-ALPHA) homolog [Homo ...Ssr11429387_at 0.57286025 0.029998985 Grap AK018457 Grap GRB2-related adaptor proteinGrap1445708_x_at 0.572194597 0.043971402 3110021A11Rik AV158652 3110021A11Rik RIKEN cDNA 3110021A11 gene3110021A11Rik1458105_at 0.569102733 0.020190743 Syn2 BB183466 Syn2 synapsin II Syn21432869_at 0.5669739 0.060642894 4932432N04Rik AK016541 --- --- ---1422565_s_at 0.566637533 0.033288395 Nfic NM_008688 1110019L22Rik RIKEN cDNA 1110019L22 geneNfic1452484_at 0.565138087 0.002221348 Car7 AF291660 Car7 carbonic anhydrase 7 Car71415903_at 0.564733277 0.037304227 Slc38a1 NM_134086 Slc38a1 solute carrier family 38, member 1Slc38a11426772_x_at 0.564130013 0.019225821 Tcrb-V13 M11456 Tcrb-V13 T-cell receptor beta, variable 13Tcrb-J1449581_at 0.56379199 0.012380925 Emid1 NM_080595 Emu1 Emu1 gene Emid11452174_at 0.56348705 0.038559368 Srebf2 BM123132 Srebf2 sterol regulatory element binding factor 2Srebf21439352_at 0.562035487 0.001791032 Trim7 BB825733 Trim7 tripartite motif protein 7Trim71448902_at 0.561554247 0.038585149 1600012K10Rik NM_025905 1600012K10Rik RIKEN cDNA 1600012K10 geneTtc231444165_at 0.560764433 0.107324697 E030012M19Rik BB530994 LOC244864 similar to layilin Layn1428319_at 0.5605922 0.005962234 Pdlim7 AK010339 1110003B01Rik RIKEN cDNA 1110003B01 genePdlim71425822_a_at 0.55910945 0.006029854 Dtx1 AB015422 Dtx1 deltex 1 homolog (Drosophila)Dtx11454700_at 0.555206457 0.040560177 Lrfn4 BB093349 BC023156 cDNA sequence BC023156Lrfn41418706_at 0.554818797 0.009312473 Slc38a3 NM_023805 Slc38a3 solute carrier family 38, member 3Slc38a31456852_at 0.552716433 0.064679475 BF469258 --- Mus musculus transcribed sequencesPaip11448891_at 0.551452663 0.057542242 Msr2 BC016551 Msr2 macrophage scavenger receptor 2Msr21438829_at 0.548925367 0.009396725 BB801060 --- Mus musculus similar to ring finger protein 111; Arkadia (LOC225743), mRNARnf1651441092_at 0.545551233 0.003331008 9330159M07Rik BB214614 --- Mus musculus adult male diencephalon cDNA, RIKEN full-length enriched library, clone:9330159M07 product:unknown EST, full insert sequence9330159M07Rik1434606_at 0.54398972 0.044908087 Erbb3 BF140685 Erbb3 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian)Erbb31424499_s_at 0.543162137 0.013182781 5730596K20Rik BC017138 5730596K20Rik RIKEN cDNA 5730596K20 gene5730596K20Rik1449455_at 0.542494843 0.02196378 Hck NM_010407 Hck hemopoietic cell kinaseHck1440018_at 0.5411948 0.026210498 A330043J11Rik AU067735 --- Mus musculus adult male spinal cord cDNA, RIKEN full-length enriched library, clone:A330065M03 product:unknown EST, full insert sequenceA330043J11Rik1420643_at 0.540906393 0.058847965 Lfng NM_008494 Lfng lunatic fringe gene homolog (Drosophila)Lfng1432149_at 0.537001053 0.065591029 Dph2l1 AK015645 Dph2l1 diptheria toxin resistance protein required for diphthamide biosynthesis (Saccharomyces)-like 1Dph11429604_at 0.536430963 0.008582672 1810011H11Rik BB784910 1810011H11Rik RIKEN cDNA 1810011H11 gene1810011H11Rik1416730_at 0.532025653 0.009231597 Rcl1 BC004574 Rcl1 RNA terminal phosphate cyclase-like 1Rcl11459353_at 0.53115553 0.054405074 Kctd3 BB537940 Kctd3 potassium channel tetramerisation domain containing 3Kctd31439989_at 0.53043498 0.060392706 Tsc1 BB668097 Tsc1 tuberous sclerosis 1 Tsc11431178_at 0.528683857 0.000475473 A430108B07Rik BF016392 A430108B07Rik RIKEN cDNA A430108B07 geneUlbp11453101_at 0.528114443 0.022257553 2810402K13Rik AK012967 2810402K13Rik RIKEN cDNA 2810402K13 geneKlhl251436397_at 0.52489907 0.023955584 BC027057 BB040051 BC027057 cDNA sequence BC027057BC0270571422210_at 0.518235913 0.067726045 Foxd3 U41047 Foxd3 forkhead box D3 Foxd31436962_at 0.517138523 0.001185834 AV303905 --- Mus musculus similar to PR-domain zinc finger protein 6 (LOC225518), mRNAPrdm61430855_at 0.515846147 0.008859898 1700051I12Rik AK006759 1700051I12Rik RIKEN cDNA 1700051I12 geneCol20a11447411_at 0.502425727 0.030471877 AI506883 --- Mus musculus transcribed sequences---1456523_at 0.499449333 0.01166932 BB767153 --- Mus musculus transcribed sequencesC777131417514_at 0.494411453 0.03705633 Ssx2ip NM_138744 Ssx2ip synovial sarcoma, X breakpoint 2 interacting proteinSsx2ip1439593_s_at 0.494003387 0.022916982 1700007P14Rik BE945978 1700030G11Rik RIKEN cDNA 1700030G11 geneC030010B13Rik1438783_at 0.49362452 0.017850324 BB325257 --- Mus musculus transcribed sequencesAW7425601457093_at 0.491244253 0.016998849 4432404J10Rik AV370079 4432404J10Rik RIKEN cDNA 4432404J10 geneGapvd11420178_at 0.487794327 0.046886365 AI593797 --- Mus musculus transcribed sequences---1420704_at 0.487262957 0.060612103 Csf2ra BM941868 --- Mus musculus transcribed sequencesCsf2ra /// LOC1000452921457492_at 0.48686118 0.015310427 AW553130 --- Mus musculus transcribed sequencesTrio1444543_at 0.486188467 0.01882341 BI901592 --- Mus musculus transcribed sequences---1433452_at 0.480375453 0.026325026 B630019K06Rik BB179847 B630019K06Rik RIKEN cDNA B630019K06 geneB630019K06Rik1416822_at 0.48023899 0.005114508 Es2el BC013711 Es2el expressed sequence 2 embryonic lethalEs2el1424762_at 0.477688127 0.037871392 C1qtnf5 BC023068 C1qtnf5 C1q and tumor necrosis factor related protein 5C1qtnf5 /// Mfrp1443684_at 0.475883267 0.05174003 Pspc1 BE956557 Pspc1 paraspeckle protein 1 ENSMUSG000000580451435114_at 0.462970943 0.004381577 D630024B06Rik C77437 D630024B06Rik RIKEN cDNA D630024B06 geneWdhd11427496_at 0.460875873 0.023965884 AI851464 BC026366 AI851464 expressed sequence AI851464Cep1521434502_x_at 0.458789953 0.025519922 Slc4a1 BB448377 Slc4a1 solute carrier family 4 (anion exchanger), member 1Slc4a11417416_at 0.457742237 0.062419075 Kcna1 NM_010595 Kcna1 potassium voltage-gated channel, shaker-related subfamily, member 1Kcna11449465_at 0.452341203 0.006191262 Reln NM_011261 Reln reelin Reln1444902_at 0.45215938 0.023314517 Fbxl10 BM118394 Fbxl10 F-box and leucine-rich repeat protein 10---1433673_at 0.449944803 0.029090246 BG069691 --- Mus musculus transcribed sequencesE130309D14Rik1455699_at 0.44243309 0.01200292 BE943820 --- Mus musculus transcribed sequences---1456954_at 0.44172305 0.051987553 Kcna6 BM119753 Kcna6 potassium voltage-gated channel, shaker-related, subfamily, member 6Kcna61460484_at 0.438129273 0.035597476 Ttl AK010374 2410003M22Rik RIKEN cDNA 2410003M22 geneTtl1427121_at 0.437709697 0.015273473 Fbxo4 BF455337 Fbxo4 F-box only protein 4 Fbxo41418252_at 0.429175213 0.009241751 Padi2 NM_008812 Padi2 peptidyl arginine deiminase, type IILOC638935 /// Padi21442304_at 0.41175279 0.00538254 BB520468 --- Mus musculus 16 days neonate heart cDNA, RIKEN full-length enriched library, clone:D830044D09 product:unknown EST, full insert sequenceLOC100042588 /// LOC1000480391431808_a_at 0.403023067 0.003445504 Itih4 AK004893 Itih4 inter alpha-trypsin inhibitor, heavy chain 4Itih41444429_at 0.39920355 0.004429579 A930016D02Rik BB518226 A930016D02Rik RIKEN cDNA A930016D02 geneLrtm11420227_at 0.39858837 0.059121258 Hoxa4 AV206827 Hoxa4 homeo box A4 ---1416451_s_at 0.397230447 0.00420834 Tbn NM_022015 Tbn taube nuss Taf81422340_a_at 0.39356132 0.00991696 Actg2 NM_009610 Actg2 actin, gamma 2, smooth muscle, entericActg21418093_a_at 0.363376477 0.037458073 Egf NM_010113 Egf epidermal growth factorEgf1434121_at 0.330601933 0.065008871 Lgi4 BB756793 Lgi4 leucine-rich repeat LGI family, member 4Lgi41427433_s_at 0.27227232 0.084127639 5730446D14Rik BB496114 5730446D14Rik RIKEN cDNA 5730446D14 geneHoxa31419426_s_at 0.269957867 0.011857493 Ccl21b NM_011335 Ccl21a chemokine (C-C motif) ligand 21a (serine)Ccl21a /// Ccl21b /// Ccl21c /// LOC100038965 /// LOC100039131 /// LOC100041504 /// LOC100041593 /// LOC100042493 /// LOC1000425441434945_at 0.263139143 0.017636041 A330042H22 AW743924 A330042H22 hypothetical protein A330042H22Lpcat2

Documents

Contactin-2 Expression in the Cardiac Purkinje Fiber Network