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Establishment and properties of a stable transfected epicardial cell line expressing a dominant negative retinoic acid receptor

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Establishment and properties of a stable transfected epicardial cell line expressing a dominant negative retinoic acid receptor

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Content ESTABLISHMENT AND PROPERTIES OF A STABLE TRANSFECTED
EPICARDIAL CELL LINE EXPRESSING A DOMINANT NEGATIVE RETINOIC
ACID RECEPTOR
by
Tsai-Ching Chang
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISRTY AND MOLECULAR BIOLOGY)
August 2002
Copyright 2002 Tsai-Ching Chang
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UMI Number: 1414871
UMI
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES, CALIFORNIA 90089-1695
This thesis, written by
approved by all its members, has been presented to and
accepted by the Director o f Graduate and Professional
Programs, in partial fulfillment o f the requirements fo r the
degree o f
cHxwe, ch/vn^
under the direction o f h thesis committee, and
Director
nrnmitfpp
Chair
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DEDICATION
This work is dedicated to my family, for their unconditional love, support and
encouragement through the years.
ii
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ACKNOWLEDGMENTS
I would like to thank my mentor, Dr. Henry M. Sucov, for welcome me into his lab
and for his patience, encouragement and guidance.
I also thank my committee members, Dr. Zoltan Tokes and Dr. Hide Tsukamoto, for
their time and effort in helping me prepare this thesis.
Everyone in Henry’s lab (Tim H.-P. Chen, Takako Makita, Bibha Choudhary, Ji-One
Kang, and Monica Flores) for their support. I couldn’t have done this without your
help.
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TABLE OF CONTENTS
DEDICATION............................................................................................... ii
ACKNOWLEDGMENTS.................................................................................................iii
LIST OF FIGURES........................................................................................................... vi
ABSTRACT......................................................................................................................vii
CHAPTER I: Retinoic Acid Receptor R X R a...................................................................1
1.1 Retinoic acid................................................................................................. 1
1.2 Retinoic Acid Receptor R X R a....................................................................1
1.3 Non cell autonomous...................................................................................2
1.3.1 RXRa is not required in the myocardium............................................3
1.3.2 RXRa is required in the epicardium...................................................4
1.4 Epicardial cells secrete a RA-inducible trophic factor............................ 4
CHAPTER II: Specific aim................................................................................................6
2.1 Purpose............................................................................................................ 6
2.2 Study approach................................................................................................6
CHAPTER III: Generation of the stably transfected clone............................................. 9
3.1 Dominant negative RAR (RAR-E)............................................................... 9
3.2 Establish a dominant negative RAR (RAR303E) construct....................... 9
3.2.1 Materials and method.......................................................................... 13
3.3 EMC subline: EMCdn...................................................................................13
3.3.1 Materials and method.......................................................................... 13
iv
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CHAPTER IV: Validation of the stably transfected clone.............................................15
4.1 PCR—DNA level........................................................................................... 15
4.1.1 Materials and method........................................................................ 15
4.1.2 Result...................................................................................................16
4.2 RT-PCR—RNA level.....................................................................................17
4.2.1 Materials and method........................................................................ 18
4.2.2 Result...................................................................................................19
4.3 Western blot analysis—protein level............................................................ 20
4.3.1 Result.................................................................................................. 21
CHAPTER V: Bioassay.................................................................................................... 22
5.1 Purpose............................................................................................................22
5.2 Conditioned media.........................................................................................22
5.2.1 Introduction........................................................................................ 22
5.2.2 Materials and method........................................................................ 24
5.2.3 Result...................................................................................................25
CHAPTER VI: Validation of the cell morphology.........................................................27
6.1 Development of the epicardium....................................................................27
6.2 Immunofluorescence cell staining................................................................ 28
6.2.1 Purpose................................................................................................28
6.2.2 Materials and method.........................................................................28
6.2.3 Result...................................................................................................29
CHAPTER VII: Discussion..............................................................................................31
REFFERENCES................................................................................................................34
V
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LIST OF FIGURES
Figure 2.1 Mechanism of action of retinoic acid........................................................... 7
Figure 3.1 Schematic organization of the dominant negative retinoic acid receptor
(RAR303E) gene.........................................................................................10
Figure 3.2 Comments for pcDNA3.1(+).......................................................................12
Figure 4.1 Electrophoretic fractionation of DNA extracted from constructed
dnEMC cells after PCR analysis................................................................ 17
Figure 4.2 RAR303E mRNA expression in the dominant negative subline
EMCdn......................................................................................................... 20
Figure 5.2 Proliferation of mouse embryonic myocytes..............................................26
Figure 6.1 Immunofluorescence staining demonstrates the epithelial....................... 30
vi
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ABSTRACT
Retinoic acid (RA), the active derivative of vitamin A, is an important signaling
molecule for the vertebrate embryo. In the mouse heart development, the absence of
RA receptor RXRa fail to initiate a proliferative expansion of cardiomyocytes that
leads to the hypoplastic ventricular chamber and the embryos die in utero, although
the embryos undergo the early stages of heart morphogenesis (i.e. looping of the
heart tube and chamber determination). In this study, we investigated the underlying
mechanistic basis of this phenotype. Based on the previous finding of that wildtype
primary epicardial cells, and an established epicardial cell line (EMC cells), secrete
trophic protein factors into conditioned media that stimulate thymidine incorporation
in primay fetal cardiomyocytes (Chen et al.), we further showed that an EMC subline
constitutively expressing a dominant negative receptor construct failed to secrete
activity into conditioned media. The epicardial-derived trophic signaling is induced
by retinoic acid treatment. Therefore we hypothesize that the fetal epicardium
secretes trophic factors to stimulate fetal cardiomyocyte proliferation and promote
ventricular chamber morphogenesis. This mechanism is through the RXRa mediated
retinoids action.
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CHAPTER I: Retinoic Acid Receptor RXRa
1.1 Retinoic acid
Retinoic acid (RA), the active metabolite of vitamin A, is an important
signaling molecule for the vertebrate embryo. Administration of excess RA has
striking teratogenic effects on early heart development. According to the species,
stage and mode of administration, excess RA can result in either lack of fusion of
the paired cardiac primordia, impaired or reversed heart looping, and/or truncation
of the posterior portion of the heart tube (prospective atrium) with abnormal
expansion of anterior (prospective ventricle) structures (Niederreither et al., 2001;
Osmond et al., 1991; Yutzey et al., 1994; Dickman and Smith, 1996; Drysdale et al.,
1997).
1.2 Retinoic Acid Receptor RXRa
Retinoids play an important role in development, differentiation, and
homeostasis. In the presence of RA, the retinoid receptors are able to up-regulate
transcription directly by binding to RA-responsive element on the promoters of
responsive genes. Because the retinoic acid receptors are transcriptional regulators,
perturbation of RA signal transduction pathway leads to several types of defects.
Both RA excess and RA deficiency could cause a spectrum of defects by the
induction of ectopic gene expression and by the interference of the endogenous
gene expression respectively (Shenefelt, 1972; Lammer et al., 1985). There are two
1
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families of retinoid receptors, retinoic acid receptor (RAR), the RAR isoforms
(alpha, beta, and gamma) and the retinoid X receptor (RXR), the RXR isoforms
(alpha, beta, and gamma)(Chambon, 1996). These retinoid receptors form
homodimers or heterodimers to regulate transcription after binding to ligand. For
example, RA binds to RARs homodimers and activates RAR-RXR heterodimers
and then these complexes up-regulate transcription by binding to particular cis
elements, retinoic acid response elements (Love and Gudas, 1994).
1.3 Non cell autonomous
In the developing heart, at least four independent primary cell
types-myocardium, epicardium, endocardium and endothelium, and neural crest are
involved and contribute to the morphogenesis. Morphogenesis of the ventricular
chamber involves proliferation and accumulation of cardiomyocytes in the
ventricular chamber wall, resulting in a thickened muscular compact zone. A
numerous different mutations in mice give rise to an underdeveloped hypoplastic
ventricular camber that leads to embryonic lethality. Mouse embryos homozygous
for a mutation in the RXRa gene have the hypoplastic development of the
ventricular chambers of the heart and die in utero between E13.5 to E 15.5. The
extremely thinned ventricular wall accompany with defects in ventricular septation.
The phenotype of the RXRa is also concurrent with a subset of the effects of
embryonic vitamin A deficiency. Therefore, RXRa plays an important role in
cardiac morphogenesis by being a genetic component of the vitamin A signaling
2
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pathway. The cardiomyocyte phenotype in RXR a-/- embryos is a
non-cell-autonomous phenotype (Tran and Sucov, 1998). A possible model of this
phenotype is that another tissue responds to retinoid signaling through RXRa, and
then influences the proliferation of cardiomyocytes and chamber maturation.
Candidate tissues could be epicardium, endocardium and neural crest for the site of
RXRa action (Chen et al.). RXRa is ubiquitously expressed during early and
midgestation (Dolle et al., 1994; Mangelsdorf et al., 1992). Another lineage of the
heart could respond to RA signaling through RXRa and then in a secondary
process inductively direct cardiomyocyte proliferation and accumulation.
1.3.1 RXRa is not required in the myocardium
The evidence for the involvement of retinoic acid in the formation of compact
zone comes from the studies on rat embryos deficient in vitamin A (Wilson and
Warkany, 1949), and mouse embryos lacking RXRa (Sucov et al., 1994), where
both of them fail to undergo compact zone expansion. This results in a profound
cardiac defect that is manifested as underdeveloped, thin-walled and hypoplastic
ventricular chamber, which leads to midgestational lethality. Although RXRa is
ubiquitously expressed in the heart, in chimeric embryos made with embryonic
stem cells lacking RXRa, cardiomyocytes result in a normal proliferation and
contribute to the ventricular chamber wall in a normal manner (Tran and Sucov,
1998). Tissue-specific knockout of the RXRa gene in the myocardium leads a
normally formed heart (Chen et al., 1998), and transgenic expression of RXRa
3
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specifically in the myocardium doesn’t rescue the defects of RXRa A hearts
(Subbarayan et al., 2000). From these observations, it is evident that myocardium
does not utilize RXRa to respond to retinoic acid signaling.
1.3.2 RXRa is required in the epicardium
Since we already know that hypoplastic ventricular chamber phenotype seen
in RXRa' ' embryo is not because of the absence of function in the myocardium,
the further investigation showed that RXRa finction is required in the epicardium
(Chen et al.). The human keratin 18 clone, K18, (Chen et al.; Theory et al., 1993)
was used for transgenic studies. The endogenous ATG site of K18 clone was altered
by RAR303E, the dominant-negative form of RARa, sequence containing a
consensus translation initiation sequence. The defect in retinoid receptor function
within the domain of expression of the K18 transgenic promoter, the K18
promoter/enhancer construct, which is expressed in the epicardium of transgenic
mouse embryos, displays the induction of the ventricular hypoplastic phenotype.
That suggested that the site of action of retinoid receptors during heart development
is in the epicardium (Chen et al.).
1.4 Epicardial cells secrete a RA-inducible trophic factor
The previous studies showed that myocardium does not utilize RXRa to
respond to retinoic acid (Tran and Sucov, 1998; Chen et al., 1998; Subbarayan et
4
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al., 2000). On the other hand, RXRa is required in the epicardium. Also, epicardial
cells secrete RA-induced trophic protein factors into conditioned media that
stimulate fetal cardiomyocyte proliferation and promote ventricular chamber
morphogenesis (Chen et al.). Consequently, the processes that are initiated by RA
to promote cardiomyocyte proliferation and formation of the ventricular chamber
should be indirect.
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CHAPTER II: Specific aim
2.1 Purpose
RXRa expresses ubiquitously in mouse development during early and
midgestation (Dolle et al., 1994; Mangelsdorf et al., 1992). It also be used in
epicardium. Based on this, by observation in epicardial cells, the further step is to
evaluate the role of RA in fetal cardiomyocyte proliferation and compact zone
morphogenesis through the action of RXRa in epicardium.
2.2 Study approach
RARs and RXRs both are members of the superfamily of ligand-inducible
transcriptional regulators that act as RAR-RXR heterodimers (Green and Chambon,
1988; Kastner et al. 1997; Evans, 1988). RARs are activated by both all-trans and
9-cis RA, whereas RXRs are only activated by 9-cis RA with high affinity
(Chambon, 1996). These heterodimers modulate the frequency of transcription
initiation of target genes after directly binding to RA response elements (RAREs)
in their promoters or indirectly interacting with other transcription factors (figure
2.1).
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Cytoplasm
Nucleus
RARE
mRNA
Figure 2.1 Mechanism of action of retinoic acid
1. RA passes into the cell and diffuses into the nucleus.
2. RAR binds to RA and undergoes a conformational change.
3. The liganded protein dimerizes (RAR-RXR) and binds to specific
DNA sequences RARE (RA response elements).
4. In conjunction with a series of co-proteins, RNA polymerase binds
to the receptor and transcribes the target gene located downstream of
the RARE.
7
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Previous studies showed that RXRa null mutant mice displayed ocular and
cardiac malformations, liver developmental delay, and die from cardiac failure
around embryonic day (E) 14.5 pc (Sucov et al., 1994; Ruiz-Lozano et al., 1998;
Kastner et al., 1994; Dyson, et al., 1995). Previous studies have also documented
that the dominant-negative form of the RARa is able to block the RXR-RAR
heterodimer pathway (Saitou et al., 1995). In this regard, our approach is to
establish a stable dominant-negative RARa expressing epicardial cell line.
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CHAPTER III: Generation of the stably transfected clone
3.1 Dominant negative RAR (RAR-E)
In order to investigate the physiological functions of RA, some investigators
have disrupted the RAR genes (Li et al., 1993; Lohnes et al., 1993) or constructed
the targeted expression of a dominant-negative RAR (RAR-E) (Saitou et al., 1995).
The dominant-negative RAR has a point mutation in the ligand binding domain that
mimics a dominant-negative thyroid hormone receptor. This mutated RAR
inhibited the endogenous activities of RARs, and then could display the functions
of RA by blocking RA signaling in the target organ such as in skin development
(Saitou et al., 1995). The dominant-negative form of the RARa (RAR303E) is able
to block the RXR-RAR heterodimer pathway. Thus, by using the same strategy, we
can address the function of RA during heart development through a
dominant-negative RAR construct.
3.2 Establish a dominant negative RAR (RAR303E) construct
The dominant negative RAR (RAR303E) construct used in studies contains a
single amino-acid substitution (G303E) that is homologous to a point mutation in
the thyroid hormone receptor that cause generalized thyroid resistance (figure3.1).
9
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HA epitope
SV40
Small t Intron
88 153 198 G303-*E 462
C'MV Promoter
AF1 H A F2 |
poly A
Figure 3.1 Schematic organization of the dominant negative
retinoic acid receptor (RAR303E) gene
CMV: cytomegalovirus
HA: hemagglutinin
AF: transcriptional activation domain
DBD: DNA binding domain
H: hinge region
LBD: ligand binding domain
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The RAR303E construct described above was cloned into the plasmid
pcDNA3.1(+) (Invitrogen), where its expression is driven by the CMV
(cytomegalovirus) promoter (figure 3.2).
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Figure 3.2 Comments for pcDNA3.1(+)
CMV promoter: bases 232-819
T7 promoter/priming site: bases 863-882
Multiple cloning site: bases 895-1010
pcDNA3.1/BGH reverse priming site: bases 1022-1039
BGH polyadenylation sequence: bases 1028-1252
f1 origin: bases 1298-1726
SV40 early promoter and origin: bases 1731-2074
Neomycin resistance gene (ORF). bases 2136-2930
SV40 early polyadenylation signal: bases 3104-3234
pUC origin: bases 3617-4287 (complementary strand)
Ampicillin resistance gene (b/a): bases 4432-5428 (complementary strand)
ORF: bases 4432-5292 (complementary strand)
Ribosome binding site: bases 5300-5304 (complementary strand)
b/a promoter (P3): bases 5327-5333 (complementary strand)
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3.2.1 Materials and method
RAR303E construct was made by insertion of a 1449 bp Hind III-BamH I
fragment of the human RARa gene, which contains a single amino-acid
substitution (G303E), into the pcDNA3.1(+) vector (Invitrogen).
3.3 EMC subline: EMCdn
We derived a stable subline of EMC cells, in which the RARE303 dominant
negative receptor described above was stably and constitutively expressed. The
RAR303E-pcDNA3.1(+) plasmid, and a control lacking the insert, were stably
transfected into EMC, a stable rat epicardial cell line that has previously been
derived (31), by calcium phosphate.
3.3.1 Materials and method
Stable transfection of EMC cells was performed by a
calcium-phosphate-mediated transfection method (Chen and Okayama, 1987). 5x
105 EMC cells were cultured in DMEM with 20% FBS and plated on 10-cm tissue
culture dishes the day prior to transfection. 10 pg of plasmid DNA, RAR303E
construct or pcDNA3.1(+) vector only, were mixed with 500 pi of 0.25 M CaCh.
To that 500 pi of 2x BBS has added then mixed well, and incubated 20 min at room
temperature. The calcium phosphate-DNA solution was then dropwise added onto
13
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the medium-containing plate while swirling plates. Cells were incubated in a 37°C,
3.5% CO2 incubator for 16 hours, and then the transfection medium was replaced
with fresh medium. After 24 hours of incubation at 37°C, 5% CO2, cells were split
into 1:10 dilution, and were incubated for another 24 hours. Transfected cells were
replaced with medium containing 250 pg/ml G418 (Life Technologies, Inc.) and
selected for a period of 2 weeks prior to isolation of colonies. Stable colonies were
individually picked and expanded. The dominant negative subline EMCdn was
chosen for use on the basis of expression of the full-length RAR303E sequence.
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seconds for 30 cycles. Primers had the following sequences: T7 promoter
(pcDNA3.1(+) T7 promoter primer binding site)
(5 '-TT AAT ACGACTC ACTAT AGGG-3'); hRARoc reverse
(5 '-GC AGTTCTT GT CCCGGTG AC A-3'); N eoF
(5'-CGCCCCATGGCTGACTAATT-3'); NeoR
(5'-AACACGGCGGCATCAGAG-3'). Amplified PCR products were
electrophoresed on 2% agarose gel stained with ethidium bromide. DNA fragments
were then visualized by UV transilluminator.
4.1.2 Result
A 500 b.p. fragment amplified using T7 primer and hRARa reverse primer
was seen only in the EMCdn cell line (land 5) and not in the control EMCc (line 6).
Plasmid DNA containing the insert (RAR303E-pcDNA3.1(+)) was used as positive
control (land 3) and only vector used as negative control (land 4). Land 2 showed a
control has no DNA put in. Land 1 showed DNA molecular mass markers. Neo F
(neomycin forward) and Neo R (neomycin reverse) primers were used as internal
controls to show that DNA was present in the positive and negative controls by
showing a 290-b.p. band, (figure 4.1).
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1 2 3 4 5 6
517 -
396 _
344 -
298 “
220 -
no DNA positive negative EMCdn EMCc
control control
Figure 4.1 Electrophoretic fractionation of DNA extracted from constructed
dnEMC cells after PCR analysis.
Human RARa primer (hRARa reverse) and a primer sequence
located in pcDNA3.1(+) vector (T7 promoter) gave a 500-b.p band to
identify clone. Neo F and Neo R (neomycin resistance gene primers) locating in
pcDNA3.1(+) vector showed a 290-b.p. band.
4.2 RT-PCR—RNA level
The expression of dominant negative subline EMCdn and control subline
EMCc were determined using RT-PCR.
17
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4.2.1 Materials and method
Total RNA was extracted from EMCdn and EMCc by the single-step
guanidinium-isothiocyanate-phenol-chloroform technique (Chomczynski and
Sacchi, 1987). A total of lpg of total RNA was reverse transcribed in a 20pl
reaction containing 2pl (100-500ng) of Random Decamer Primer (Ambion), lOmM
dNTPs, and 200 units of M-MuLV Reverse Transcriptase (GIBCO BRL) in First
Stand Synthesis buffer (50Mm Tris-HCl at pH 8.3, 75 mM KC1, 3mM MgCls, and
5mM DTT). The RT reaction was diluted with water to 50pl, and 2pi of RT
reaction was used for PCR. PCR was performed in a 30 pi reaction containing lpg
of each primer, 2.5 units of Taq DNA Polymerase (GIBCO) in PCR buffer (20%
DMSO, 134 mM Tris at pH 8.8, 33 mM (N H ^S O ^O mM P-mercaptoethanol, 6
mM MgCE, and 1 mM dNTPs) with the following parameters: 40 cycles each for 1
minute at 95°C, 1 minute at 68°C, and 30 seconds at 72°C. The PCR primers for
amplification are hRARa reverse (5'-GCAGTTCTTGTCCCGGTGACA-3'),
hRARaHA (5’ -GTACCCCTACGACGTGCCCGA-3'), and rRARa
(5'- CTGCCCCGGGTCCCTACTCCA-3'). Amplified PCR products were
separated on 2% agarose gel stained with ethidium bromide. As an internal positive
control for the efficiency of gene amplification, rat specific RARa primer sequence
located in DNA binding domain of rat RARa (rRARa) and human RARa primer
(hRARa reverse) were designed to give a 280-b.p band. Oligonucleotide primers,
hRARa reverse and hRARa HA (HA epitope sequence involved in RAR303E
construct), gave a 430-b.p band to identify clone.
18
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4.2.2 Result
Total RNA was isolated from EMCdn cells and control construct cells and
then reverse-transcribed into cDNA. Equal amounts of the cDNA were used as
template for PCR with primer pairs specific for the nuclear receptors RARa. The
430 b.p. fragment was seen in the EMCdn (land 5) and not in the vector control
(EMCc) cells (land 7). Positive control RAR303E-pcDNA3.1(+) plasmid showed
the expected 430 b.p. fragment (land 3). Rat RARa was expressed in both EMCdn
and EMCc cell lines which amplified as 280 b.p. fragment (land 5 and land 7). -RT
was used as control to showed that the RNA did not have DNA contamination, and
in absence of reverse transcriptase, the RNA was not transcribed to cDNA and do
not amplified (land 4 and land 6). Land 1 showed DNA molecular mass markers.
Land 2 showed a control has no DNA put in. (figure 4.2).
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1 2 3 4 5 6 7
5 1 7 -
396 -
344 -
298 “
220 -
no DNA positive — + - + R T
control ^ ^ ^ ------------1
EMCdn EMCc
Figure 4.2 RAR303E mRNA expression in the dominant negative subline
EMCdn.
Rat specific RARa primer (rRARa) and human RARa primer (hRARa reverse)
were designed to give a 280-b.p band for internal control. hRARa reverse and
hRARa HA (HA epitope sequence involved in RAR303E construct), gave a
430-b.p band to identify clone.
4.3 Western blot analysis—protein level
RAR303E construct contains a HA (hemagglutinin) epitope sequence for HA
antibody recognition. Western analysis was performed with antibodies specific for
RARa and HA-tag protein.
20
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4.3.1 Result
Validation of this stably transfected clone was done by Western blot analysis.
HA antibody was used to detect the transgenic expression of dnRARa, and RARa
antibody was used to detect both the transgenic expression and endogenous RARa.
We failed to detect the transgenic expression as well as the endogenous expression
despite the in vitro translated dnRAR and wildtype RAR were detected cleanly.
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CHAPTER V: Bioassav
5.1 Purpose
To examine whether epicardium secretes trophic factors which stimulate fetal
cardiomyocyte proliferation and compact zone morphogenesis, rat epicardial cell
lines were used in these studies.
5.2 Conditioned media
5.2.1 Introduction
Previous data showed that proliferation of chick embryonic myocytes is
induced by epicardial conditioned media (Chen et al.) (figure 5.1). By preparing
conditioned media, we can investigate the effect of RXRa on epicardial cells that
secrete RA-induced trophic protein factors into conditioned media.
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Thymidine Incorporation (CPM
2000
1500
1000
500
0% 10%
FBS FBS
JL
DMSO RA DMSO RA
Conditioned Media Conditioned Media
of Epicardial cells of Hela Cells
Figure 5.1 Retinoic acid treated epicardial conditioned media induces
embryonic chick cardiomyocyte proliferation.
23
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5.2.2 Materials and method
Conditioned media: To prepare conditioned media, cells were grown in the
presence of serum to near confluency and then switched to DMEM alone for one
day. The media was then replaced with fresh DMEM, retinoids (all-trans retinoic
acid at 10'6 M) or solvent (DMSO) added, and the cells cultured two more days
before the conditioned media was collected.
Cardiomyocyte proliferation assay: Ventricular tissue was isolated from E l3.5
mouse embryos. Following serial enzymatic digestion with collagenase (0.5 mg/ml)
and pancreatin (lmg/ml), the cell suspension was diluted with lx ADS buffer (116
mM NaCl, 20 mM HEPES pH 7.3, 1 mM NaH2P 04, 5.36 mM KC1, 0.83 mM
MgS04, 0.1% dextrose), and layered on top of a Percoll step gradient. The bottom
layer of this gradient contained a 4:1 ratio of Percoll stock (90% Percoll
(Amersham-Pharmacia) plus 10% lOx ADS buffer): lx ADS buffer; the top layer
contained a 9:11 ratio. The step gradient was spun at 3000 rpm for 30 min, and
cardiomyocytes collected at the interface above the bottom layer. We can routinely
obtain cardiomyocytes with >98% purity (assessed by MF20 immunostaining)
through this method. Cardiomyocytes were then plated on gelatin-coated 48-well
dishes in plating media (4:1 DMEM/M199, 5% FBS, 10% horse serum). After 24
hr, the media was replaced with serum-free DMEM, and the cells cultured another
24 hr. Thereafter the media was replaced with serum-free conditioned media, and
0.5 p Ci 3 H-thymidine was added per well. After 48 hr of incorporation, cells was
washed three times with ice-cold PBS, fixed with ice-cold 10% TCA, and lysed in
24
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IN NaOH. Cell lysates were pH neutralized with IN acetic acid and transferred
into scintillation vials to count radioactivity. A background subtraction (generally
50-100 CPM) was calculated for each experiment, measured by adding
3 H-thymidine to cells cultured in DMEM alone for 5 min before extraction.
5.2.3 Result
Mouse cardiomyocytes cultured without any additional factors (serum free) in
the media have a basal level of thymidine incorporation, whereas treatment of these
cells with serum induced a substantial increase in incorporation of the label.
However, the conditioned media from the epicardial cells treated with DMSO had
higher basal level compared to the serum free media alone while high basal level
probably reflects endogenous production of retinoic acid, since these cells express
the RA synthetic enzyme RALDH2 (as do epicardial cells in vivo) (Moss et al.,
1998). This conditioned media from the RA treated epicardial cells showed two
times were incorporation of the thymidine compared to the DMSO treated control.
The DMSO treated conditioned media from the EMCdn cells reduced the basal
thymidine incorporation compared to the DMSO treated control epicardial cells
(EMCc). Treatment with RA conditioned media did not enhance the incorporation
of the thymidine and hence proliferation of the cardiomyocytes compared to the
DMSO treated conditioned media from EMCdn cells (figure 5.2). Based on this,
the forced expression of the dominant negative RA receptor blocked the basal
25
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production of secreted trophic activity, and prevented the induction of activity in
response to RA treatment.
1500
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H H
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1000
500
SF DMSO RA DMSO RA
Conditioned Media Conditioned Media
of EMC control cells of EMCdn cells
Figure 5.2 Proliferation of mouse embryonic myocytes.
E l3.5 mouse ventricular myocytes were cultured in serum free media to which
conditioned media from solvent (DMSO) treated or RA treated EMCdn cells or
EMC control cells were added.
26
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CHAPTER VI: Validation of the cell morphology
6.1 Development of the epicardium
In vertebrate embryos, myocardium eventually is bordered by endocardium
beneath and epicardium above and is separated from each layer by extensive
extracellular matrix (Morabito et al., 2001). The epicardium is derived from the
proepicardial organ, proepicardium, which deposits epicardial cells onto the heart
(Sucov, 1998; Viragh and Challice, 1981). These cells then migrate over the
developing heart. Epicardial cells are the developmental precursors to coronary
vascular smooth muscle cells and are derived from the epicardium through
epithelial-mesenchymal transformation (EMT) (Dettman et al., 1998; Landerholm
et al., 1999). The model of epicardial EMT suggests that after epicardial cells
migrate over the developing heart, the stimulated epicardial cells, which are
stimulated by myocardially secreted FGFs, separate from the epicardium and then
break through the basement membrane, invade the subepithelial matrix, and
become mesenchymal cells. These mesenchymal cells then differentiate into
coronary vascular smooth muscle cells, perivascular fibroblasts, or intermyocardial
fibroblasts. The coronary vascular smooth muscle cells and perivascular fibroblasts
form a nascent coronary artery within the subepicardial matrix by assembling
around an endothelial cell (Morabito et al., 2001).
27
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6.2 Immunofluorescence cell staining
6.2.1 Purpose
Since we already designated EMCdn subline, such cells can be expanded for
several passages in culture, the next step is to address that whether these epicardial
cells retain their epithelial morphology or become other cell types. The strategy to
distinguish the epithelial cells and myocardial cells is using the
immunofluorescence cell staining.
6.2.2 Materials and method
EMCdn cells were plated on 8-well camber slide (Nalge Nunc International,
IL) and fixed with 4% formaldehyde for 30min at 4°C. Cells were treated with 10
mM sodium citrate in water bath for 20 min at 95°C. After washing three times
with phosphate-buffered saline (PBS), non-specific binding sites were saturated for
45 min with blocking solution (5% dry milk, 0.05% Tween-20 in PBS). The slide
was then incubated overnight at 4°C in the primary antibody 1:100 dilution with
blocking solution. The control was incubated in blocking solution only.
After incubation, the slide was washed three times in PBST (0.05% tween 20
in PBS), incubated in the dark for 1 hour at room temperature in
fluorescence-conjugated goat anti-mouse or donkey anti-goat IgG (Santa Cruz
Biotechnology) diluted 1:200 in blocking solution, washed three times in PBST and
28
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washed again in H2O. After adding 1-2 drops of aqueous mounting medium
(Biomeda), covered with a glass coverslip, and then observed slide under
fluorescence microscope (Braun et al., 2000).
6.2.3 Result
Immunofluorescence staining was used for identification of the EMCdn cells
morphology. Monolayers grown in vitro were examined with immunofluorescence
for pan-cytokeratin, PECAM and MF20 expression. The cells demonstrated a
normal epicardial phenotype in pan-cytokeratin expression. PECAM-1, platelet
endothelial cell adhesion molecule, (Santa Cruz Biotechnology) is an endothelial
cell marker. And for these analyses, FITC-conjugated antibody to myosin heavy
chain (MF20) serves as a cell-specific marker for cardiac myocytes. The
observation of these assays showed most of epicardial cells retain their epithelial
morphology by immunofluorescence staining for pan-cytokeratin (fig 6.1).
29
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Pan-cytokeratin
PECAM-1
Figure 6.1 Immunofluorescence staining demonstrates the epithelial
Morphology.
The panels on tne rignt are corresponding light images. The control panel is a
monolayer stained with secondary antibodies alone. MF20 is a marker for
cardiomyocytes. Pan-cytokeratin is present in epicardial cells. The PECAM-1, an
endothelial cell marker, is not stained in epicardial cells. Thus, these data showed
that these EMC cells still maintain their morphology.
30
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CHAPTER VII: Discussion
After the stably transfected clones were generated, we next asked whether a
luciferase reporter gene utilizing the RARa gene driven by the CMV promoter was
RA responsive in a transfection assay. However, the normalized luciferase activity
(luciferase activity was normalized to P-galactosidase activity) was as low as
background. This may because of the extremely low transfect efficiency (<0.1% of
cells were stained by” In Situ Staining for Betagalactosidase”) no matter stably
transfected by calcium phosphate or Lipofectin (GIBCO BRL).
Based on other results described in this study, we propose that the mouse
embryo epicardium secretes one or more protein factors to stimulate embryo
cardiomyocyte proliferation. The retinoic acid-induced factors are probably
produced in an autocrine or paracrine manner by epicardial expression of
RALHD2, and this induction requires the involvement of RXRa.
A possible explanation of this hypothesis is that ablation of the epicardium
should result in a hypoplastic ventricular chamber wall. The epicardium, the
outmost layer of the heart, contributes to the population of cells. This population
migrates into the ventricular chamber wall to form the vessels of the coronary
arteries. In the absence of the cell adhesion molecules, such as VCAM-1 (Gurtner
et al., 1995; Kwee et al., 1995) and possibly a4 integrin (Yang et al., 1995), the
mutant mice failed to adhere to the myocardium, and consequently the
disintegration of the epicardium. VCAM-1 is expressed by the myocardium,
31
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whereas a4 integrin is expressed in the epicardium. The physical interaction
between VCAM-1 and a4 integrin results in an adherence of the epicardium to the
myocardium. Similarly, in chick embryos, the insertion of a piece of egg shell
membrane in front of the proepicardial organ (the precursor of the epicardium) can
partially disrupt the formation of the epicardium, thereby results in patches of
absent epicardium, with the myocardium underneath becoming hypoplastic
(Gittenberger-de Groot et al., 2000).
A number of interactions occur between adjacent layers of the developing
heart, mediated by secreted factors. Signals from the myocardium induce an
epithelial-mesenchymal transformation in the endocardium that allows
endocardial-derived cells to populate the atrioventricular and conotruncal cushions
(Brown et al., 1999; Runyan et al., 1992). The myocardium secretes factors,
including TGFp3, induce the epithelial-mesenchymal transformation in the
endocardium which secrets the TGFp3 receptors (Brown et al., 1996). Similarly,
the myocardium produces other factors, including FGFs, which induce an
epithelial-mesenchymal transformation in the epicardium. This process creates the
vascular endothelium of the coronary arteries, the smooth muscle cells that
surrounds these arteries, and the cardiac fibroblast lineages (Morabito et al., 2001).
In return, the endocardium secretes neuregulin, which induces cardiomyocyte
proliferation and trabecular ingrowth at the inner surface of the myocardium
(Gassmann et al., 1995; Lee et al., 1995; Theory et al., 1993). Based on this study,
we propose another possible axis of signaling for epicardium. The epicardium
32
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secretes factors to induce cardiomyocyte proliferation and compact zone formation
on the outer surface of the myocardium.
To identify the factors responsible for the cardiomyocyte trophic response, the
receptors of these factors, and the target genes that are immediately RA regulated,
the further investigations could utilize biochemical and molecular strategies. Also,
the identification of the receptor of the epicardial trophic signal should enable to
define the trophic stimulated signaling pathways in fetal cardiomyocytes. A
long-term goal of these studies is to facilitate the understanding of the roles of these
genes in these processes and then to assemble a pathway of genetic control of
cardiomyocyte proliferation and compact zone morphogenesis.
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REFFERENCES
Akmal, K. M., Dufour, J. M., and Kim, K. H. (1997). Retinoic acid receptor alpha
gene expression in the rat testis: potential role during the prophase of meiosis and in
the transition from round to elongating spermatids. Biol. Reprod. 56, 549-556.
Braun, K. W., Tribley, W. A., Griswold, M. D., and Kim, K. H. (2000).
Follicle-stimulating hormone inhibits all-/ram-retinoic acid-induced retinoic acid
receptor a nuclear localization and transcriptional activation in mouse sertoli cell
lines. The Journal o f Biological Chemistry. 275,4145-4151.
Brown, C. B., Boyer, A. S., Runyan, R. B., and Barnett, J. V. (1996). Antibodies to
the Type II TGFP receptor block cells activation and migration during
atrioventricular cushion transformation in the heart. Dev. Biol. 174,248-57.
Brown, C. B., Boyer, A. S., Runyan, R. B., and Barnett, J. V. (1999). Requirement
of type III TGF-beta receptor for endocardial cell transformation in the heart.
Science. 283,2080-2.
Chambon, R (1996); A decade of molecular biology of retinoic acid receptors.
FASEBJ. 10, 940-954.
Chen, C. and Okayama, H. (1987). High efficiency transformation of mammalian
cells by plasimd DNA. Mol. Cell. Biol. 7, 2745-52.
Chen, H.R, Chang, T. C., Kang, J. O., Choudhary, B., Makita, T., Tran, C. M., Eid,
H., Sucov, H. M. (submitted). Epicardial induction of fetal cardiomyocyte
proliferation via a retinoic acid-inducible trophic factor.
Chen, J., Kubalak, S. W., and Chien, K. R. (1998). Ventricular muscle-restricted
targeting of the RXRa gene reveals a non-cell-autonomous requirement in cardiac
chamber morphogenesis. Development 125, 1943-9.
34
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by
acid guanadinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,
156-159.
Dettman, R. W., Denetclaw, W., Jr., Ordahl, C. P., and Bristow, J. (1998). Common
epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and
intermyocardial fibroblasts in the avian heart. Dev. Biol. 193,169-181.
Dickman, E. D. and Smith, S. M. (1996). Selective regulation of cardiomyocyte gene
expression and cardiac morphogenesis by retinoic acid.Dev. Dyn. 206, 39-48.
Dolle, P., Fraulob, V., Kastner, P., and Chambon, P. (1994). Developmental
expression of murine retinoid X receptor (RXR) genes. Mech. Dev. 45, 91-104.
Drysdale, T. A., Patterson, K. D., Saha, M. and Krieg, P. A. (1997). Retinoic acid
can block differentiation of the myocardium after heart specification. Dev. Biol. 88,
205-215.
Dyson, E., Sucov, H. M., Kubalak, S. W., Schmid-Schonbein, G., Delano, F. A.,
Evans, R. M., Ross, J., Jr. and Chien, K. R. (1995). Atrial-phenotype is associated
with embryonic ventricular failure in retinoid X receptor a-/- mice. Proc. Natl. Acad.
Sci. USA 92, 7386-7390.
Eid, H., Larson, D. M., Springhom, J. P., Attawia, M. A., Nayak, R. C., Smith, T.
W., and Kelly, R. A. (1992). Role of epicardial mesothelial cells in the modification
of phenotype and function o f adult rat ventricular myocytes in primary coculture.
Circ. Res. 71,40-50.
Evans, R. M. (1988). The steroid and thyroid hormone receptor superfamily.
Science. 240,889-895.
Gassmann, M., Gasagranda, F., Orioli, D., Simon, H., Lai, C., Klein, R., and Lemke,
G. (1995). Aberrant neural and cardiac development in mice lacking the ErbB4
neuregulin receptor. Nature. 378, 390-4.
35
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Gittenberger-de Groot, A. C., Vrancken, Peeters, M. P., Bergwerff, M., Mentink, M.
M., and Poelmann, R. E. (2000). Epicardial outgrowth inhibition leads to
compensatory mesothelial outflow tract collar and abnormal cardiac septation and
coronary formation. Circ. Res. 87,969-71.
Green, S. and Chambon, P. (1988). Nuclear receptors enhance our understanding of
transcription regulation. Trends Genet. 4,309-314.
Gurtner, G. C., Davis, V., Li, H., McCoy, M. J., Sharpe, A. and Cybulsky, M. I.
(1995). Targeted disruption of the murine VCAM1 genes: essential role of VCAM-1
in chorioallantoic fusion and placentation. Genes Dev. 9 , 1-14.
Jiang, G., Nepomuceno, L., Hopkins, K., and Sladek, F. M. (1995); Exclusive
homodimerization of the orphan receptor hepatocyte nuclear factor 4 defines a new
subclass of nuclear receptors. Mol. Cell. Biol. 15, 5131-5143.
Kastner, P., Grondona, J., Mark, M., Gansmuller, A., LeMeur, M., Decimo, D.,
Vonesch, J. L., Dolle, P. and Chambon, P. (1994). Genetic analysis of
RXRa developmental function: convergence of RXR and RAR signaling pathways
in heart and eye morphogenesis. Cell 78,987-1003.
Kastner, P., Mark, M., Ghyselinck, N., Krezel, W., Dupe, V., Grondona, J. M. and
Chambon, P. (1997).Genetic evidence that the retinoid signal is transduced by
heterodimeric RXR/RAR functional units during mouse development. Development
124,313-326.
Kwee, L., Baldwin, H. S., Shen, H. M., Stewart, C. L., Buck, C. A., and Labow, M
A. (1995). Defective development of the embryonic and extraembryonic circulatory
systems in vascular cell adhesion molecule (VCAM-1) deficient mice. Development.
121,489-503.
Lammer, E. J., Chen, D. T., Hoar, R. M., Agnish, N. D., Benke, P. J. (1985).
Retinoic acid embryopathy. N. Engl. J. Med. 313, 837-41.
36
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Landerholm, T. E., Dong, X. R., Lun. J., Belaguli, N. S., Schwartz, R. J., and
Majesky, M. W. (1999). A role for serum response factor in coronary smooth muscle
differentiation from proepicardial cells. Development. 126,2053-2062.
Lee, K. F., Simon, H., Chen, H., Bates, B., Hung, M. C., and Hauser, C. (1995).
Requirement for neuregulin receptor erbB2 in neural and cardiac development.
Nature. 378, 394-8.
Li, E., Sucov, H. M., Lee, K., Evans, R. M., and Jaenisch, R. (1993). Normal
development and growth of mice carrying a targeted disruption of the a -1 retinoic
acid receptor gene Proc. Natl. Acad. Sci. U.S.A. 90,1590-1594.
Lohnes, D., Kastner, R, Dierich, A., Mark, M., LeMeur, M., and Chambon , P.
(1993). Function of retinoic acid receptor in the mouse. Cell 73,43-658.
Love, J. M., and Gudas, L.J. (1994). Vitamin A, differentiation and cancer. Curr.
Opin. Cell Biol. 6, 825-31.
Mangelsdorf, D.J., Borgmeyer, U., Heyman, R. A., Zhou, J. Y., Ong, E. S., Oro, A.
E., Kakizuka, A., and Evans, R. M. (1992). Characterization of three RXR genes that
mediate the action of 9-cis retinoic acid. Gene Dev. 6, 329-44.
Morabito, C. J., Dettman, R. W., Kattan, J., Collier, J. M., and Bristow, J. (2001).
Positive and negative regulation of epicardial-mesenchymal transformation during
avian heart development. Dev. Biol. 234, 204-15.
Moss, J. B., Xavier-Neto, J., Shapiro, M. D., Nayeem, S. M., McCaffery, P., Drager,
U. C., and Rosenthal, N. (1998). Dynamic patterns of retinoic acid synthesis and
response in the developing mammalian heart. Dev. Biol. 199, 55-71.
Niederreither, K., Vermot, J., Messaddeq, N., Schuhbaur, B., Chambon, P., and
Dolle, P. Embryonic retinoic acid synthesis is essential for heart morphogenesis in
the mouse. (2001) Development. 128, 1019-1031.
37
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Osmond, M. K., Butler, A. J., Voon, F. C. T. and Bellairs, R. (1991). The effects of
retinoic acid on heart formation in the early chick embryo. Development 113,
1405-1417.
Ruiz-Lozano, P., Smith, S. M., Perkins, G., Kubalak, S. W., Bossl, G. R., Sucov, H.
M., Evans, R. M., and Chien, K. R. (1998). Energy deprivation and a deficiency in
downstream metabolic target genes during the onset of embryonic heart failure in
RXRa-/- embryos Development 125, 533-544.
Runyan, R. B., Potts, J. D., and Weeks, D. L. (1992). TGF(33-mediated tissue
interaction during embryonic heart development. Mol. Reprod. Dev. 32, 152-9.
Saitou, M., Sugai, S., Tanaka, T., Shimouchi, K., Fuchs, E., Narumiya, S., and
Kakizuka, A. (1995); Inhibition of skin development by targeted expression of a
dominant-negative retinoic acid receptor. Nature 374, 159-62.
Shenefelt, R. E., (1972). Morphogenesis of malformation in hamsters caused by
retinoic acid: relation to dose and stage at treatment. Teratology 5 , 103-18.
Subbarayan, V., Mark, M., Messadeq, N., Rustin, P., Chambon, P., and Kastner, P.
(2000). RXRa overexpression in cardiomyocytes causes dilated cardiomyopathy but
fail to rescue myocardial hypoplasia in RXRa-null fetuses. J. Clin. Invest. 105,
387-94.
Sucov, H. M. (1998). Molecular insights into cardiac development. Annu. Rev.
Physiol. 60,287-308.
Sucov, H. M., Dyson, E., Gumeringer, C.L., Price, J., Chien, K. R., and Evans,
R.M.(1994). RXR alpha mutant mice establish a genetic basis for vitamin A
signaling in heart morphogenesis. Genes Dev. 8, 1007-18.
Theory, I. S., Meneses, J. J., Neznanov, N., Kulesh, D. A., Pedersen, R. A., and
Oshima, R. G. (1993). Embryonic expression of human keratin 18 and
38
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
K18-P-galactosidase fusion genes in transgenic mice. Dev. Biol. 160, 519-34.
Tran, C.M., and Sucov, H.M. (1998). The RXRalpha gene functions in a
non-cell-autonomous manner during mouse cardiac morphogenesis.
Development. 125,1951-6.
Viragh, S., and Challice, C. E. (1981). The origin of the epicardium and the
embryonic myocardial circulation of the mouse. Anat. Rec. 201,157-168.
Wilson, J. G., and Warkany, J. (1949). Aortic arch and cardiac anomalies in the
offspring of vitamin A deficient rats. Am. J. Anat. 85, 113-155.
Yang, J. T., Rayburn, H., and Hynes, R. O. (1995). Cell adhesion events mediated by
a4 integrins are essential in placental and cardiac development. Development. 121,
549-60.
Yutzey, K. E., Rhee, J. T. and Bader, D. (1994). Expression of the atrialspecific
myosin heavy chain AMHC1 and the establishment of anteroposterior polarity in the
developing chicken heart. Development 120,3638-3645.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Asset Metadata

Creator Chang, Tsai-Ching (author)

Core Title Establishment and properties of a stable transfected epicardial cell line expressing a dominant negative retinoic acid receptor

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Sucov, Henry (committee chair), Tokes, Zoltan A. (committee member), Tsukamoto, Hidekazu (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-300735

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