The roleof the catecholaminergic…
357
was further confirmed by blocking the endogenous synthesis of L-‐DOPA and
dopamine,which ledtoadecrease inexpressionofAMHC1(13).
Table1.
Sequenceof theprimersused insemiquantitativePCR.
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 presentedbyHernández Sánchez, C., Bártulos
Encinas, O., De PabloDávila, F., Aránega Jiménez, A. on “The use of catecholamine
for thedifferentiationof stemcells tocardiomyocytes”. PCT/ES09/070339).
Overexpression and knock-‐down of TH disrupts cardiac markers gene
expressionandaffects 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
primitiveheart tube (18-‐20).
We showed that THoverexpression increasedandexpanded the expression
of the posteriorly restricted genes AMHC-‐1 andTbx5 towardmore rostral regions
(Figure8). These results suggested that THis involved in theheart tubepatterning
by specifying the atrial region. In addition, TH also had major functional
Gene
PrimerForward
PrimerReverse
Th
CACGAGTGAAGATGCCAAC
CACGAGTGAAGATGCCAAC
α
-1b
AAAGAAGGCAGCCAAGACA
GAAAGAGCTGGGGTTTTTCC
α
-1d
CTTCTTCGTCCTGCCTTTTG
AGGAGCTGTTCAGGGTGCTA
α
-2c
AGAGCAGCACCTCAGAGAGC
GGCTACTGGAGTGCTTGGAG
β
-1
CCAAGGAGCAGATCAGGAAG
GCCCAACCAGTTGAAGAAAA
β
-2
CGAGTTCTGGACCTCTCTGG
GGGTCTTCAGAGCTTTGTGC
gapdh
GCAATGCATCGTGCACCACC
TGTGATGGCATGGACAGTGG
Gene
PrimerForward
PrimerReverse
Roche
probe
D1
TGTCATTAGTGTGGACAGATACTG
G
GGGTCATTTTCCTCTCGTACC 19
D3
GATAAGCTCTTTAGGAAGGATAG
GC
CCTAATACAGAGAGCCACTCCA
C
12
D5
GTCTGCTGCACTGGAGGTC
GAATCGTTCGTCTCCGTAGG
27
gapdh
GTCCTCTCTGGCAAAGTCCA
ACCATGTAGTTCAGATCGATG
AA
49
Table2.
Sequenceof theprimersused inquantitativePCRwithRocheprobes.
