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Identification and study of novel genes expressed in the heart precursor cells
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Authors
Advisor(s)
Abstract(s)
Understanding
heart
development
on
a
molecular
level
is
a
requirement
for
uncovering
the
causes
of
congenital
heart
diseases.
Several
genes
have
been
implicated
as
critical
for
heart
development.
However,
the
inducers
of
these
genes
as
well
as
their
targets
and
pathways,
remain
largely
unknown.
We
have
identified
a
promoter
element
of
chick
cCer
able
to
drive
EGFP
expression
in
a
population
of
cells
that
consistently
exit
from
the
anterior
primitive
streak
region,
from
as
early
as
stage
HH3+,
and
that
later
will
populate
the
heart.
Using
this
promoter
element
as
a
tool
allowed
us
to
identify
novel
genes
previously
not
known
to
potentially
play
a
role
in
heart
development.
In
order
to
identify
and
study
genes
expressed
and
involved
in
the
correct
development
and
differentiation
of
the
vertebrate
heart
precursor
cell
(HPC)
lineages,
a
differential
screening
using
Affymetrix
GeneChip®
system
technologies
was
performed.
Remarkably,
this
screening
led
to
the
identification
of
more
than
700
transcripts
differentially
expressed
in
the
heart
forming
regions
(HFR).
Bioinformatic
tools
allowed
us
to
filter
the
large
amount
of
data
generated
from
this
approach
and
to
select
a
few
transcripts
for
in
vivo
validation.
Five
genes
were
selected
for
further
characterization
by
whole
mount
in
situ
hybridization
leading
to
the
validation
of
their
expression
in
the
HPC.
From
those,
Adtk1
and
Ccbe1
were
selected
for
functional
analysis.
Regarding
to
ccbe1,
a
more
detailed
WISH
analysis
was
performed
and
showed
that
Ccbe1
is
expressed
specifically
on
the
cardiac
progenitors
regions
at
HH4,
more
specifically
in
primary
heart
field
and
at
later
stages
is
present
in
the
second
heart
field.
Further
functional
analyses
by
knockdown
and
overexpression
revealed
an
important
role
for
Ccbe1
in
early
heart
tube
formation.
Moreover,
the
results
presented
in
this
thesis
suggested
that
Ccbe1
is
a
key
gene
during
heart
development
and
might
be
limited
to
multipotent
and
highly
proliferative
progenitors
and
downregulated
upon
cellular
commitment
into
more
specific
cardiac
phenotypes.
Other
of
the
genes
identified,
Adtk1
was
also
subjected
to
further
functional
studies.
Knockdown
of
Adtk1
using
morpholino
oligonucleotides
suggested
that
it
might
be
necessary
for
the
migration
and
fusion
of
the
heart
tube
as
well
as
for
neural
tube
closure.
Description
Keywords
Ciências biomédicas Coração Rastreio Embrião