Page 121 - 80_02

The
role
of
the
catecholaminergic…

was
further
confirmed
by
blocking
the
endogenous
synthesis
of
L-­-DOPA
and

dopamine,
which
led
to
a
decrease
in
expression
of
AMHC1
(13).

Table
1.
Sequence
of
the
primers
used
in
semiquantitative
PCR.

Gene
Primer
Forward
Primer
Reverse

Th
CACGAGTGAAGATGCCAAC
CACGAGTGAAGATGCCAAC

AAAGAAGGCAGCCAAGACA
GAAAGAGCTGGGGTTTTTCC

a -1b

a -1d
CTTCTTCGTCCTGCCTTTTG
AGGAGCTGTTCAGGGTGCTA

a -2c

AGAGCAGCACCTCAGAGAGC
GGCTACTGGAGTGCTTGGAG

ß -1

ß -2
CCAAGGAGCAGATCAGGAAG
GCCCAACCAGTTGAAGAAAA

CGAGTTCTGGACCTCTCTGG
GGGTCTTCAGAGCTTTGTGC

gapdh

GCAATGCATCGTGCACCACC
TGTGATGGCATGGACAGTGG

Table
2.
Sequence
of
the
primers
used
in
quantitative
PCR
with
Roche
probes.

Gene
Primer
Forward
Primer
Reverse
Roche

probe

D1
TGTCATTAGTGTGGACAGATACTG GGGTCATTTTCCTCTCGTACC

G
19

D3
GATAAGCTCTTTAGGAAGGATAG CCTAATACAGAGAGCCACTCCA
GC
C
12

D5
GTCTGCTGCACTGGAGGTC
GAATCGTTCGTCTCCGTAGG

gapdh
GTCCTCTCTGGCAAAGTCCA
ACCATGTAGTTCAGATCGATG 27

AA
49

Interestingly,
in
a
parallel
study,
carried
out
with
mouse
embryonic
stem

cells,
the
addition
of
L-­-DOPA
and
dopamine
enhanced
cardiac
myocyte

differentiation
(international
patent
presented
by
Hernández
Sánchez,
C.,
Bártulos

Encinas,
O.,
De
Pablo
Dávila,
F.,
Aránega
Jiménez,
A.
on
“The
use
of
catecholamine

for
the
differentiation
of
stem
cells
to
cardiomyocytes”.
PCT/ES09/070339).

Overexpression
and
knock-­-down
of
TH
disrupts
cardiac
markers
gene

expression
and
affects
chamber
formation

We
set
out
gain-­-of-­-function
experiments
using
constructs
that
led
to

overexpression
of
TH
together
with
GFP
(Figure
4),
or
just
GFP
as
a
control,
in

electroporated
chick
embryos.

One
of
the
first
features
of
anterior-­-posterior

patterning
is
the
restriction
of
the
ventricular
(VMHC-­-1)
and
atrial
(AMHC-­-1)

myosin
heavy
chain
to
the
anterior
and
posterior
pole,
respectively,
of
the

primitive
heart
tube
(18-­-20).

We
showed
that
TH
overexpression
increased
and
expanded
the
expression

of
the
posteriorly
restricted
genes
AMHC-­-1
and
Tbx5
toward
more
rostral
regions

(Figure
8).
These
results
suggested
that
TH
is
involved
in
the
heart
tube
patterning

by
specifying
the
atrial
region.
In
addition,
TH
also
had
major
functional

357