Int. J. Dev. Biol.

57: 907-916 (2013)

doi: 10.1387/ijdb.130317dr
www.intjdevbiol.com

Differential expression of angiogenic and anti-angiogenic

molecules in the chick embryo chorioallantoic membrane
and selected organs during embryonic development
CHRISTIAN MARINACCIO, BEATRICE NICO and DOMENICO RIBATTI*
Department of Basic Medical Sciences, Neurosciences, and Sensory Organs, University of Bari Medical School,
Bari, Italy

ABSTRACT In this study we have investigated by real time polymerase chain reaction (RT-PCR)
the expression of cytokines, which are well known angiogenic and anti-angionenic molecules,
during chick embryo development in four organs, namely the chorioallantoic membrane (CAM),
heart, liver and optic tectum at four different stages. We have studied the expression of vascular
endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), angiopoietin-1 (ANG-1),
hypoxia inducible factor-1 and -2 alpha (HIF-1a and HIF-2a), hepatocyte growth factor (HGF), and
of endostatin. All the pro-angiogenic cytokines examined showed a progressive increase in their
expression in brain, heart, and liver, through all the stages of development, and parallel endostatin
reduces its expression. In contrast, in the CAM, all the pro-angiogenic factors examined, with the
exception of ANG-1, showed a stably-decreased expression during development, whereas endostatin progressively increased its expression. The CAM as an extraembryonic membrane which
mediates gas and nutrient exchange until hatching, may be considered an outer organ and not an
intrinsic organ of the developing embryo in which the mechanisms regulating the development of
the vascular tree are different.

KEY WORDS: angiogenesis, chorioallantoic membrane, chick embryo, heart, liver, optic tectum

Introduction
There is an extensive literature of the genetic background, the
molecular and cellular mechanisms responsible for blood vessel
formation. During embryonic development, the vasculature is
formed by both vasculogenesis and angiogenesis (Risau, 1995,
Risau and Flamme, 1995). The nascent vasculature is stabilized
via the recruitment of mural cells, namely pericytes and smooth
muscle cells, and the generation of the extracellular matrix. Vasculogenesis seems to be restricted to early developmental periods,
whereas angiogenesis can occur during the entire life span but
also occurs in the early embryo. Although establishment of the
vasculature of most organs occurs by angiogenesis, development
of the vasculature of certain endodermal organs, including liver,
lung, pancreas, stomach/intestine, and spleen, occurs by vasculogenesis (Pardanaud and Dieterlen-Lievre, 1999).
Angiogenesis is controlled by the balance between molecules
that have positive and negative regulatory activity and this concept
had led to the notion of the angiogenic switch, which depends

on an increased production of one or more positive regulators of

angiogenesis (Ribatti et al., 2007). The control mechanisms involved
in the regulation of angiogenesis might be better investigated
during embryonic development, because the vascular pattern is
reproducible and forms in the absence of inflammatory processes.
Vascular endothelial growth factor (VEGF) is most likely the primary
factor involved as both a diffusible attractant for migrating endothelial cells and as substrate-bound vessel stabilization factor once
vascular tubes are formed. Fibroblast growth factor-2 (FGF-2) and
VEGF play a crucial role in the angiogenic expansion of the early
network. Angiopoietins (ANGs) play a critical role in endothelial
sprouting, vessel wall remodeling and mural cell recruitment. Disruption of ANG-1 is responsible for a failure of endothelial cells to
associate properly with pericytes during embryonic angiogenesis
(Suri et al., 1996). Moreover, during embryonic development of
Abbreviations used in this paper: ANG-1, angiopoietin-1; CAM, chorioallantoic membrane; FGF-2, fibroblast growth factor-2; HGF, hepatocyte growth factor; HIF-1a
hypoxia inducible factor-1 alpha; VEGF-A, vascular endothelial growth factor-A.

*Address correspondence to: Domenico Ribatti. Department of Basic Medical Sciences, Neurosciences, and Sensory Organs, University of Bari Medical School, and
National Cancer Institute Giovanni Paolo II, Piazza Giulio Cesare, 11, 70124 Bari, Italy.Tel: +39-080-547-8326. Fax: +39-080-547-8310. E-mail: domenico.ribatti@uniba.it
Accepted: 5 December 2013. Final, author-corrected PDF published online: 21 February 2014

ISSN: Online 1696-3547, Print 0214-6282

2014 UBC Press
Printed in Spain

908

C. Marinaccio et al.
stages (E7, E12, E14, and E18), and we have compared the expression of angiogenic and anti-angiogenic molecules between
heart, liver, optic tectum and CAM at different stages.

Results
Relative expression of pro- and anti-angiogenic factors in
the developing CAM
All the pro-angiogenic factors examined, with the exception of
ANG-1 and HIFs, showed a stably-decreased expression during
development (Fig. 2). On the contrary, endostatin progressively
increased its expression. VEGF-A, FGF-2 and HGF showed a
peak of expression at ED7 of CAM development comparable
to the reference expression at ED18, while ANG-1 and HIF-2a
display the peak of expression at ED12 after an initial lower
expression at ED7. HIF-1a showed a low expression at ED7

Fold Change

1.0

1.28

0.5

1.00

0.86

1.0

0.5

0.62

0.52

1.00

*
Fold Change

1.0
1.47

1.21

0.5

1.00

0.29

0.5

18
ED
C

0.98

1.00

0.75

18
ED

14
A
M
C

A
M

A
M

ED

ED

12

7
ED
A
M

ED
A
M
C

A
M

A
M

ED

ED

14

18

0.0

ED
A
M
C

1.0

0.47

12

HIF-1a

HIF-2a
1.5

Fold Change

1.5

1.0

0.64

0.62

0.5

1.00
0.45

1.0

0.5

1.00

0.99

0.72

0.55

1.0

0.5

1.00
0.56

0.72

0.55

18
ED

14
C

A
M

ED
C

A
M

ED
A
M
C

A
M

ED

12

0.0

18

14
A
M

ED

ED
C

A
M
C

A
M
C

ED

ED

18
ED
A
M
C

Endostatin

1.5

Fold Change

A
M
C

A
M

ED

ED

12

7
ED

12

0.0
14

0.0

A
M

Fold Change

HGF
1.5

1.5

0.0

A
M

A
M
C

ANG-1
2.0

14
ED

ED
A
M
C

12

7
ED
A
M

ED

14
A
M

A
M

ED

ED
A
M
C

7
ED
A
M
C

18

0.0

12

0.0

*
0.92

Fold Change

1.5

0.41

Fold Change

Fig. 2. Relative expression of VEGF-A, FGF-2, ANG-1,

HGF, HIF-1a, HIF-2a, and endostatin in the developing
chorioallantoic membrane at ED7, 12, 14 of incubation in comparison to expression at E18 assumed as
reference stage. All the pro-angiogenic factors except
ANG-1 display a decreased expression during development, while endostatin shows an increased expression.
A peak in expression can be seen at ED7 for VEGF-A,
FGF-2 and HGF and at ED12 for ANG-1 and HIF-2a. HIF1a shows a stable increase of expression at ED12 and
ED14, while endostatin increased its expression at ED14
after a stable expression at ED7 and ED12. * p<0.05 vs
CAM at ED7. List of abbreviations: CAM, chorioallantoic
membrane; VEGF-A, vascular endothelial growth factor-A;
FGF-2, fibroblast growth factor-2; ANG-1, angiopoietin-1;
HGF, hepatocyte growth factor; HIF-1a and HIF-2a, hypoxia
inducible factor 1 alpha and 2 alpha; ED, embryonic day.

FGF-2

1.5

A
M

the vascular system, hypoxia activates hypoxia

inducible factor-1 and 2-alpha (HIF-1a and HIF2a), which turn on the expression of angiogenic
genes, such as VEGF, inducing vessels to branch
towards the hypoxic tissue (Pugh and Ratcliffe,
2003). Among the anti-angiogenic molecules,
endostatin is a proteolytic fragment of collagen
XVIII (a component of basement membrane),
which is able to inhibit endothelial cell survival
and migration (Ribatti, 2009).
In this study we have investigated by Real time
polymerase chain reaction (RT-PCR) the expression of well known angiogenic cytokines, VEGF-A,
FGF-2, ANG-1, HIF-1a and HIF-2a, hepatocyte
growth factor (HGF) and of an endogenous antiangiogenic molecule, namely endostatin during
chick embryo development. We have chosen four
organs, namely chorioallantoic membrane (CAM),
a useful model to study angiogenesis in ovo (Fig.
1), heart, liver and optic tectum at four different

REAL TIME CAM

VEGFA

Fig. 1. A strong angiogenic response induced by a

tumor bioptic specimen implanted on the top of a
chick embryo chorioallantoic membrane at ED12.
Original magnification, x 50.

Angiogenic and anti-angiogenic molecules in the chick embryo 909

followed by a stable increase at ED12 and ED14. Endostatin
increased its expression at ED14 after an initial stable expression at ED7 and ED12.

12, VEGF-A showed a peak in its expression during the 14 day

stage and a subsequent decrease at day 18 of heart development. FGF-2 showed a stable expression through ED7, ED12 and
ED14 and a decrease at ED18. ANG-1 showed a decreasing trend
starting with a strong expression at ED7 followed by a reduction
at ED12, ED14 and ED18 of heart development. Conversely,
HGF showed a progressive increasing trend from ED7 through
ED12 and ED14, followed by a strong decrease in its expression at ED18 of heart development. HIF-1a showed a fluctuating
expression from ED7 through ED12, ED14 and ED18 of heart
development. After a stable expression in ED7 and ED12, HIF-2a
showed a peak in its expression during
the ED14 and a subsequent decrease at
ED18 of heart development. Endostatin
FGF-2
showed a fluctuating expression from
ED7 through ED12, ED14 and a reduction at ED18 of heart development. For
all pro-angiogenic and anti-angiogenic
1.31
factors examined, the lower expression
*
was reached at ED18.

Relative expression of pro- and anti-angiogenic factors in

the developing heart
All the factors analyzed showed a higher expression through
the ED7, ED12, ED14 stage of heart development considered
when compared to the ED18 CAM, with the exception of HIF-2a,
which relative expression was substantially lower or comparable
to the ED18 CAM (Fig. 3). After a stable expression in day 7 and
REAL TIME PCR HEART-CAM
VEGFA
1.6

1.4
1.2

Fold Change

10.0
13.83

*
6.59
4.53

30

2.2

18
ED

14

12
C

A
M

A
M

ED

ED
A
M

A
M

ED

18

14

12

ED

HIF-2a
1.2

1.0

18

A
M

ED

12

ED

ED

A
M

18

T
R
H

EA

R
EA
H

ED

14

12

ED
T

T
EA

R
EA
H

ED

ED

18

A
M
C

A
M

ED

14

12

ED

Percentage change of expression

of pro- and anti-angiogenic factors
between CAM and heart at different
stages of development
All the factors considered, with the
exception of HIF-2a, showed a positive
percentage change of expression when
compared to the CAM in all the stages
of development, except for ED18, when
FGF-2, ANG-1, HGF, HIF-2a and endostatin showed a negative percentage
change (Fig. 4). HIF-2a showed an initial
decrease at ED7 and ED12 followed by
an increase at ED14. HGF showed the
highest percentage increases at ED12,
VEGF and FGF-2 at ED14, while ANG-1
and endostatin showed a progressive
decrease after an initial high positive
percentage change at ED7.
Relative expression of pro- and antiangiogenic factors in the developing
liver
VEGF-A, FGF-2, HIF-1a and HIF-2a
showed a peak in their relative expression at ED14 and a significant reduction
at ED18 of liver development (Fig. 5).

1.8

1.6

1.2

1.0

1.64

0.8

1.35

0.6

0.96

0.4

0.46

14
ED

A
M

A
M
C

12
ED

A
M

18

A
M

ED
C

EA

ED

ED
T
R

EA
H

EA
H

14

12

7
ED
T
R
EA

0.62

0.16

0.0

1.00

0.86

0.2

Fold Change

1.4

ED

ED

ENDOSTATIN

14

0.12

0.93

0.67

0.55

0.41

0.35

0.0

A
M

18

A
M
C

ED
T

0.2

1.00

EA

R
EA
H

ED

14

12
T

ED
R

EA
H

ED

7
ED

T
R

0.88

0.64

0.49

0.0

0.5

0.4

1.00

1.00

ED

2.54

1.0

0.93

1.5

0.6

A
M

3.61

18

2.0

0.8

4.03

ED

2.5

Fold Change

3.0

A
M

3.5

4.0

Fold Change

EA

HIF-1a

EA

T
R

T
R
H

EA

R
EA

18
ED

14

12

ED

7
T

T
R
EA

ED

ED

18
ED

A
M

A
M
C

0.27

0.0

4.5

1.00

0.87
0.56

A
M

1.00

14

12

ED

ED

R
EA

1.21

1.07

0.2

EA

A
M

18

A
M

ED
T
R

R
EA
H

ED

14

12

7
ED

ED
T

T
R
EA
H

1.94

0.45

ED

3.64

1.03

0.4

*
0.15

ED

1.64

0.8
0.6

10.52

A
M
C

EA
H
2.17

1.0

1.2

1.4

ED

31.67

1.6

A
M

21

Fold Change

Fold Change

1.8

24

0.64

2.0

27

12

18

14
R

ED

ED

12
EA
H

EA

EA

ED

ED

14
ED

A
M

A
M

12

R
EA

2.4

15

0.65

HGF

ANG-1
33

18

1.00

0.93

0.63

0.0

1.41

ED

18

A
M

ED
T

T
R
EA

R
EA
H

ED

14

12

ED

ED

ED

T
R

1.28

0.2

1.00

0.57
1.37

0.0

EA

1.21

0.4

2.5

0.6

18

6.77

0.8

ED

5.0

1.0

ED

7.5

A
M

Fold Change

12.5

A
M

15.0

Fig. 3. Relative expression of VEGF-A, FGF-2, ANG-1, HGF, HIF-1a,

HIF-2a, and endostatin in the developing heart at ED7, 12,14 and 18
in comparison to CAM expression. All the factors except HIF-2a display
an higher expression during heart development than the expression during
CAM development. A peak of expression was detected during ED14 of
heart development in all the factors except for HIF-1a, ANG-1, Endostatin
in which the peak is at ED7.* p<0.05 vs heart at ED7. List of abbreviations:
CAM, chorioallantoic membrane; VEGF-A, vascular endothelial growth
factor-A; FGF-2, fibroblast growth factor-2; ANG-1, angiopoietin-1; HGF,
hepatocyte growth factor; HIF-1a and HIF-2a, hypoxia inducible factor 1
alpha and 2 alpha; ED, embryonic day.

910

C. Marinaccio et al.

HGF, a specific growth factor for the liver, showed a peak in its
expression at the beginning of the chick embryo development
suggesting an early role for this factor in the liver development.
ANG-1 also showed a peak of its relative expression at ED7 of
liver development. HIF-1a and HIF-2a showed a comparable
trend through all the stages of development. Endostatin showed
a significant reduction at the end of development. Percentage
change of expression of pro- and anti-angiogenic factors between
CAM and liver at different stages of development (Fig. 6)
VEGF-A

FGF-2

2500

150

2250

125
100

1750

Variation (%)

1500
1250
1000
750

75
50
25

ea
rt
ED

14

ANG-1

HGF

7000

200

6000

175
150

5000

125
100

Variation (%)

4000
3000
2000
1000

75
50
25
0
-25

-50
-75

-1000

25

ED
18
H
ea
rt
18
ED
M

C
A

18
H
18
ED
18
ea
rt
ED

14

ea
rt
ED

18
ED
M
A
C

M
A
C

ED
14
ED
M

C
A

C
A

ED

12

14

H
ea
rt

H
ea
rt

ED
12

ED
7
H
ea
rt
7

14

12
ED
M
A

M
A
C

Variation (%)

ED
M
C
A

H
7
ED
M
A

18
ED

ED
M
A

ea
rt
ED

18
H

H
14

H
12
ED
M
A

ea
rt
ED

ea
rt
ED

12
ea
rt
ED

ea
rt
ED
H

Endostatin
200
150
100
50
0
-50
-100

ED

-100

ea
rt
ED

-75

0
14

100

-50

12

200

-25

300

ED

Variation (%)

400

ED
M
A
C

500

Variation (%)

600

Percentage change of expression of pro- and

anti-angiogenic factors between CAM and optic tectum at different stages of development
VEGF-A, FGF-2 and HGF showed a progressive positive percentage change during ED7,
ED12 and ED14 of the chick embryo development
and a reduction at ED18 (Fig. 8). ANG-1 reached
a positive percentage change peak towards the
end of chick embryo development. HIF-1a and
HIF-2a showed a positive and, respectively, a
negative variation during all the stages of the
chick embryo development. Endostatin showed a
progressive decrease of percentage change from
positive at ED7 to negative in the other stages
of chick embryo development.

Discussion

HIF-2a
50

A
C

ea
rt
ED

14
ea
rt
ED
H
14

12
ED
A

ED

18

ED

ea
rt
ED

ea
rt
ED

ea
rt
ED

18

14
ea
rt
ED
H
14
ED
M

ED

ED

12

ea
rt
ED

ea
rt
ED

12

12

-100

HIF-1a

Relative expression of pro-and anti-angiogenic factors in the developing optic tectum

VEGF-A, ANG-1, HGF, HIF-1a, HIF-2a
showed a progressive increase of relative expression through all the stages of the optic tectum
development with a peak at ED18 (Fig. 7). FGF-2
showed a peak of relative expression at ED12 of
optic tectum development. Endostatin reduced
its expression at ED12 and slightly increased at
ED14 and ED18.

H
18

ED

ED

14

12
ED
A

18

ED

ea
rt
ED

ea
rt
ED

18
ea
rt
ED

14
ED
M
A
C

ED

ED

12

14

ea
rt
ED

ea
rt
ED

12

7
ea
rt
ED
H
7
ED
M
A
C

-50

ea
rt
ED

-25

250

18

500

12

Variation (%)

2000

Variation (%)

VEGF-A was the only factor showing positive percentage

changes during all stages of liver development with a peak at ED14.
FGF-2, HIF-2a and endostatin showed a negative percentage
change of expression during all the stages of liver development.
HIF-1a showed the same trend with the exception of ED14 where
the percentage change was positive, while HGF had its only
negative percentage change at ED18. ANG-1 showed a peak of
positive percentage change at ED7 and a negative percentage
change from ED12 to ED18.

The intricate patterning process that establish

the complex architecture of the vascular system
depend on a combination of genetic and epigenetic factors (Ribatti, 2006). It currently appears
that blood vessels have the ability to provide
instructive regulatory signals to surrounding non
vascular target cells (Crivellato et al., 2007). On
one hand, organ instructions to endothelial cells
provides cues for vascular development and acquisition of local specialties; on the other hand,
endothelial signals back to organ cells provide

Fig. 4. Percentage change of expression of VEGF-A, FGF-2, ANG-1,

HGF, HIF-1a, HIF-2a, and endostatin between CAM and heart at
ED7, 12, 14 and 18. A positive percentage change can be seen in all the
factors for ED7, ED12 and ED14 except HIF-2a. At ED18 FGF-2, ANG-1,
HGF, HIF-2a and endostatin display a negative percentage change. List
of abbreviations: CAM, chorioallantoic membrane; VEGF-A, vascular
endothelial growth factor-A; FGF-2, fibroblast growth factor-2; ANG-1,
angiopoietin-1; HGF, hepatocyte growth factor; HIF-1a and HIF-2a, hypoxia
inducible factor 1 alpha and 2 alpha; ED, embryonic day.

Angiogenic and anti-angiogenic molecules in the chick embryo 911

ED

0.6
1.00

0.4

0.73

18

A
M
C

A
M
C

ED

14

12

ED

7
C

A
M

ED

ED

18

LI
VE
R

ED

14

12
LI
VE
R

ED

ED

LI
VE
R

ED

0.6

0.03

0.1

0.0

0.62

0.27

A
M

0.36

0.2

18

A
M

A
M

ED

A
M

ED

12

14

18

A
M

ED

ED

A
M

ED

14

12

ED

ED

A
M

A
M

ED

18

14

18

ED

LI
VE
R

LI
VE
R

ED
0.8

LI
VE
R

A
M

A
M
C
1.0

Fold Change

18

14

12

ED
A
M

18

ED

A
M
C

ED

LI
VE
R

LI
VE
R

ED

12

ED

LI
VE
R

ED
LI
VE
R

ED

ENDOSTATIN
1.2

ED

0.33

0.0

14

0.0

14

0.2

0.31

0.21

0.4

ED

0.70

A
M

0.80

0.66

12

0.65

0.2

0.6

ED

0.4

1.00

A
M

0.86

0.6

0.8

1.0

0.8

Fold Change

Fold Change

1.0

ED

1.2

A
M

HIF-1a
1.2

LI
VE
R

12

7
ED

ED

LI
VE
R

LI
VE
R

ED

A
M

A
M
C

A
M
C

18

14
ED

12

ED

18

ED

A
M

ED

14
LI
VE
R

LI
VE
R

ED

ED

LI
VE
R

LI
VE
R

ED

12

0.0

0.5

0.0

18

1.0

0.48

A
M

1.5

ED

0.4

1.00

A
M

0.36

1.72

4.64

2.0

18

1.55

0.5

2.5

LI
VE
R

2.0

14

1.0

3.0

ED

2.17

3.5

LI
VE
R

1.5

12

Fold Change

Fold Change

4.0

ED

4.5
2.0

5.0

14

ANG-1
2.5

ED

7
LI
VE
R

LI
VE
R

ED

ED
LI
VE
R

12

18

14

ED

ED
A
M

0.41

0.0

A
M

12

A
M
C

0.5

1.00

0.41

1.21

ED

18
C

A
M

ED

14

ED

ED

LI
VE
R

LI
VE
R

ED

LI
VE
R

ED
LI
VE
R

1.51

2.9

1.0

ED

8.51

6.00

Fold Change

15.14

ED

1.5

LI
VE
R

16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0

12

Fold Change

REAL TIME PCR LIVER-CAM

VEGFA

Few literature data are available concerning the time-course

in the expression of angiogenic and anti-angiogenic molecules
during the development of different organs in the chick embryo.
As concerns the CAM, primitive vessels proliferate and differentiate into an arteriovenous system until day 8, thus originating a
network of capillaries that migrate to occupy an area beneath the
chorion and mediate gas exchanges with the outer environment.
Rapid capillary proliferation goes until ED11; thereafter, their mitotic
index declines just as rapidly, and the vascular system attains its
final arrangement on ED18, just
before hatching (Ausprunk et al.,
1974). A morphometric investigation by DeFouw et al., (1989)
FGF-2
has shown rapid extension of
the CAM surface between ED6
and ED14. During this period, the
number of feed vessels increased
predominantly due to growth and
remodeling after ED10. Dusseau
1.33
1.00
and Hutchins (1989) studied the
0.76
* 0.5
0.69
effects of hypoxia on the number
0.32
0.17
of pre and post-capillary vessels
in the CAM and demonstrated
that the low oxygen regimen
stimulated a preferential increase
HGF
in the number of arterioles. Ribatti
et al., (1995) demonstrated that
endogenous FGF-2 is intrinsically
*
involved in CAM vascularization
on the basis of the evidence that
FGF-2 is present in elevated
* 3.31
amounts in the CAM from ED6
* 1.2
to ED18, maximal concentrations
1.11
1.00
0.67
0.56
0.48
being observed between ED10
and ED14. We have confirmed
the highest amount of FGF-2 beHIF-2a
tween ED7 and ED12. Moreover,
neutralizing antibodies to FGF-2
inhibit vessel growth, confirming a
role of FGF-2 in vascularization of
the CAM. Parson-Wingerter et al.,
*
(1998) demonstrated that FGF-2
1.00
0.96
increased the rate of angiogen0.57
0.64
0.57
esis in the CAM by a maximum
*
*
of 72%, whereas angiostatin
0.18
0.16
decreases the rate of vascular
growth by a maximum of 68%.
As concerns the central nerLI
VE
R

morphogenetic and patterning instructions during organ formation

(Cleaver and Melton, 2003).
In this study, we have demonstrated that all the pro-angiogenic
cytokines examined show a progressive increase in their expression
in brain, heart, and liver, through all the stages development, and
parallel endostatin reduces its expression. On the contrary in the
CAM, all the pro-angiogenic factors examined, with the exception
of ANG-1, showed a stably decreased expression during development, whereas endostatin progressively increased its expression.

Fig. 5. Relative expression of VEGF-A, FGF-2, ANG-1, HGF, HIF-1a,

HIF-2a, and endostatin in the developing liver at ED7, 12, 14 and 18
in comparison to CAM expression. A peak of expression of VEGF-A,
FGF-2, HIF-1a and HIF-2a can be noted at ED14 followed by a significant
reduction at ED18 of liver development. ANG-1 and HGF showed a
peak at ED7 of liver development while endostatin showed a significant reduction at ED18. * p<0.05 vs liver at ED7. List of abbreviations:
CAM, chorioallantoic membrane; VEGF-A, vascular endothelial growth
factor-A; FGF-2, fibroblast growth factor-2; ANG-1, angiopoietin-1; HGF,
hepatocyte growth factor; HIF-1a and HIF-2a, hypoxia inducible factor
1 alpha and 2 alpha; ED, embryonic day.

912

C. Marinaccio et al.
VEGF-A

FGF-2

4000

20

3500

0
-20

Variation (%)

2500
2000
1500
1000

-40
-60

ED
18
ED
18
M

C
A

600

400

500

350
300

400

Variation (%)

250
200
150
100
50

300
200
100

18
D
18

Li
ve
rE

ED

14

A
C

C
A

ED

ED
M

A
C

Li
ve
rE

D
Li
ve
rE
12

7
ED
M

ED
18

14

7
ED
Li
ve
r

ED
18
Li
ve
r

Li
ve
r
ED
14
M
C
A

C
A

C
A

ED
12

ED
7

Li
ve
r

Li
ve
rE
D
7

ED
12

ED
14

-100

12

-50
-100

HIF-1a

HIF-2a

40

20

20

Variation (%)

Variation (%)

Li
ve
r

Li
ve
r
ED
14
M
C
A

C
A

C
A

ED
12

ED
7

Li
ve
r

18
D
Li
ve
rE
ED

A
C

ED

ED
M

M
A
C

18

12

Li
ve
rE

Li
ve
rE

14

12

7
ED
Li
ve
r
7
ED

14

HGF

ANG-1

Variation (%)

ED
12

-100

ED
14

-80

500

Li
ve
rE
D
7

Variation (%)

3000

-20
-40
-60

-20
-40
-60

-80

ED
18
ED
18
M

C
A

ED
14
M
C
A

Li
ve
r

Li
ve
r

ED
12
C
A

C
A

ED
12

ED
7

Li
ve
r

Li
ED
18
A
M
C

A
M
C

Li
ve
rE
D
7

ve

-100

ED
14

D
18
rE

D
14
rE
ve
Li
ED
14

ED
12
A
M
C

A
M

ED
7

Li

Li
v

ve

er

rE

ED

D
12

-80

-100

ED
18

M
A
C

ED

ED

14

18

Li
ve
r

Li
ve
r

ED
14

ED
12

M
A
C

ED

ED

12

Li
ve
r

Li
ve
rE
D

Variation (%)

Endostatin
20
0
-20
-40
-60
-80
-100

Fig. 6. Percentage change of expression of VEGF-A, FGF-2, ANG-1, HGF, HIF-1a, HIF-2a, and endostatin
between CAM and liver at ED7, 12, 14 and 18. Only VEGF-A displays a positive percentage change during all liver development, while FGF-2, HIF-2a and endostatin display an opposite trend. ANG-1, HGF and
HIF-1a showed a peak of positive percentage change at ED7 and ED14 respectively. List of abbreviations:
CAM, chorioallantoic membrane; VEGF-A, vascular endothelial growth factor-A; FGF-2, fibroblast growth
factor-2; ANG-1, angiopoietin-1; HGF, hepatocyte growth factor; HIF-1a and HIF-2a, hypoxia inducible factor
1 alpha and 2 alpha; ED, embryonic day.

vous system, angioblasts invade the

head and form a perineural vascular
plexus that covers the neural tube.
Vascular sprouts originating from this
plexus radially invade the neuroectoderm (Bar, 1980). Subsequently,
branching occurs in the subependymal
layer. Vascular endothelial cells are
among the first cells encountered by
neuroblasts during early development.
Neuroblasts release endothelial cell
mitogens, which promote angiogenesis by the invading vasculature
and endothelial cells release factors
that support neuronal development
(Bautch and James, 2009).
In normally developing chick
embryo optic tectum, the blood vessels enter the nervous wall as radial
vessels at ED 5-6, and from ED8, the
number of these main vessels and
of their collateral increases owing to
progressively more numerous branching vessels, parallel to the main tectum
histogenetic processes (Roncali et
al., 1985). In hypoxic condition, the
number of vessels was increased; a
transitory increment in vessel diameter
was recognized and the value of the
distance between contiguous vessels
was much lower than under normal
condition (Ribatti et al., 1989). Risau
isolated from embryonic chick brain
an endothelial cell growth factor and
found to be identical to acidic and basic
fibroblast growth factor (Risau, 1986,
Risau et al., 1988). The mitogenic
activity was found to increase during
embryonic chick brain development
and reached a plateau around ED14.
In this study, we have confirmed the
highest amount of FGF-2 between
ED12 and ED18. Breier et al., (1992)
demonstrated that the temporal and
spatial expression of VEGF in the
developing brain correlates with brain
vascularization and endothelial cell
growth. They suggested that VEGF
is releases by cell of the ventricular
layer and promotes angiogenesis by
initiating an angiogenic response of
endothelial cells from the perineural
vascular plexus. In this study, we have
demonstrated an high expression of
VEGF-A between ED12 and ED18.
The heart develops form the precardic lateral folds to form primitive
heart tube, consisting of an inner
endothelium and an outer myocardial

Angiogenic and anti-angiogenic molecules in the chick embryo 913

tube. Emergence of cardiac endothelium and cardiomyocytes occurs
almost concomitantly and, at first, they develop rather independently
from one another (Sugi and Markwald, 1996).
Experimental data in mouse and zebrafish support the view that
endocardial cells play a critical role in myocardial cell maturation.
A study concerning immunolocalization of FGF-2 during chicken
cardiac development demonstrated a progressive decrease in
its expression in the highly trabeculated region of the ventricular
myocardium with increasing developmental stage (Consigli and
Joseph-Silverstein, 1991).
Tomanek et al., (1998) tested the hypothesis that early vascularization of the embryonic heart prior to myocardial tube formation is enhanced after bolus injection of VEGF and FGF-2 into the
REAL TIME PCR OPTIC TECTUM-CAM
VEGFA
2.0

1.0

18

A
M

A
M

ED

ED

ED

A
M

14

12

18

A
M

18

A
M

A
M

ED

ED

14

12

ED

A
M

ED

18

18

14

ED

A
M

A
M

ED

ED

ED

A
M

14
ED
T

12

0.35

A
M

ED

ED

12

0.0

18

12

ED
A
M

0.39

0.28

0.29

ENDOSTATIN
1.2

0.8
0.6
0.4

0.65

0.2

0.31

0.31

1.00
0.72
0.56

0.2

0.55

18

14

ED

ED

A
M

A
M
C

12
ED

A
M
C

18

ED

A
M
C

14

ED

ED

ED
T

T
O

ED

12

0.0

Fold Change

1.0

ED
A
M

18

ED

A
M
C

ED

14
ED
T

ED
T
O

ED

12

0.0

0.72

0.55

0.2

1.00

0.62

14

0.64

0.45

0.5

1.00

0.99

0.4

1.0

0.6

1.5

4.01

3.63

ED

3.99
3.15

2.0

0.8

18

2.5

A
M

Fold Change

3.0

ED
T
O

1.2

3.5

Fold Change

A
M

14

12

7
ED

ED
T

T
O

14

ED
A
M

A
M
C

A
M
C

18

12

ED

ED

18

ED

A
M

ED
T

T
O

ED

14

ED

ED
T
4.0

1.00

0.75

0.47

0.24

HIF-1a

4.5

0.98

1.00

1.21

0.0

1.19

0.5
1.47

0.29

ED

14
ED

1.78

1.31

2.27

1.21

O
1.0

*
1.55

Fold Change

6.02

ED

ED

12

18

A
M
C

1.5

12

Fold Change

2.0

1.00

0.62

0.52

ANG-1
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0

1.39

0.92

0.0

ED

ED

1.00

14

12

0.86

ED

18

A
M
C

ED

14

ED

ED
T

T
O

12

1.28

ED

ED
T
O

1.31

0.61

0.41

A
M

1.64

A
M

1.66

0.5

6.7

7.59

1.0

ED

ED

10.13

ED

Fold Change

1.5

Fold Change

10

11

vitelline vein before the onset of myocardial vascularization and

demonstrated increased vascular growth. Guadix et al., (2006)
demonstrated that chick epicardium displays a high vasculogenic
potential which is modulated by VEGF and FGF-2. Tomanek et
al., (2002) have documented a role for multiple members of the
VEGF family and their receptors in the formation of the coronary
vascular bed in quail heart. Sugishita et al., (2000) suggested that
VEGF produced by chick embryonic heart myocytes may play an
important role in embryonic cardiovascular development by acting on surrounding endothelial cells and on ventricular myocytes
themselves. Tomanek et al., (2001) demonstrated that addition
to heart explants of anti-VEGF antibodies inhibited vascular tube
formation by about 25%, while anti-FGF-2 antibodies affected a
60% inhibition; when a combination of
two inhibitors was added, tubulogenesis
was inhibited by 80-90%. In this study,
FGF-2
we have confirmed an important role
played by VEGF-A and FGF-2 during
heart development.
* *
Song et al., (1999) using an immunohistochemical approach detected a
localization of HGF to the chick myocardial
cells and demonstrated that isolated endocardial cells responded to the addition of
HGF with increased motility, proliferation
and urokinase production. Wikenheiser
et al., (2006) identified areas of the emHGF
bryonic chicken heart that were intensely
*
positive for the hypoxia indicator (EF5) and
these areas were also positive for HIF*
1a. In this study, we have demonstrated
in heart samples a strong expression of
both HGF and HIF-1a at ED14.
The liver anlagen develops from the
ventral foregut endothelium (Wells and
Melton, 1999). Endodermal epithelial cells
of the primitive intestine receive stimuli
from the cardiac mesoderm which induces
changes in endodermal gene profile with
HIF-2a
expression of liver specific genes (Jung et
al., 1999). As a consequence, endodermal
cells differentiate and proliferate within the
foregut epithelium, and begin to delaminate into the surrounding mesenchyme
* *
of the septum transversum. Finally, the
condensed tissue mass becomes vascularized (Matsumoto et al., 2001).
HGF is a cytokine that stimulates pro-

Fig. 7. Relative expression of VEGF-A, FGF-2, ANG-1, HGF, HIF-1a, HIF-2a,

and endostatin in the developing optic tecum at ED7, 12, 14 and 18 in
comparison to CAM expression. A progressive increase of expression in
optic tectum development can be seen for VEGF-A, ANG-1, HGF, HIF-1a and
HIF-2a with a peak at ED18, while a progressive reduction of expression of
endostatin is present, with the lowest expression at ED12. FGF-2 displays a
peak of expression at ED12 of optic tectum development. * p<0.05 vs optic
tectum at ED7. List of abbreviations: CAM, chorioallantoic membrane; VEGF-A,
vascular endothelial growth factor-A; FGF-2, fibroblast growth factor-2; ANG-1,
angiopoietin-1; HGF, hepatocyte growth factor; HIF-1a and HIF-2a, hypoxia
inducible factor 1 alpha and 2 alpha; ED, embryonic day.

914

C. Marinaccio et al.
VEGFA

FGF-2

1600

180
160
140

500

120

Variation (%)

400
350
300
250
200
150

80
40
0

100
50

-40

18
ED

14
ED
M
C
A

C
A

ED

ED

12

14

18

O
T

O
T

ED

12
O
T

O
T

C
A

C
A

ED

ED

18

O
T

O
T
14
ED
M

ED

ED

18

-80

ED

ED

12
C
A

C
A

C
A

ED

ED

12

O
T

O
T

ED

ED

-100

14

-50

C
A

HIF-1a

HIF-2a

650
600
550
500
450
400
350
300
250
200
150
100
50
0

20

Variation (%)

0
-20
-40

18
ED
18

O
T
C
A

ED
M

ED

14

12
ED
M
C
A

C
A

O
T

ED

ED
O
T

O
T
7
ED
M
C
A

14

7
ED

18
ED
O
T
18
ED

ED
M
C
A

C
A

ED

12

14

O
T

O
T

ED

ED

12

7
ED
O
T
7
ED

14

-80

12

-60

C
A

Variation (%)

18

160

450

Variation (%)

ED

HGF
200

550

O
T

ED
M
C
A

ANG-1
600

C
A

18

14

O
T
ED
M
C
A

C
A

C
A

ED

18

ED

12

O
T

O
T

ED

ED

18
ED

14
ED
O
T
14
ED
M

C
A

C
A

C
A

ED

ED

12

O
T

O
T

ED

ED

12

20
0
-20
-40

ED

200

14

400

60
40

600

ED

800

120
100
80

C
A

1000

O
T

Variation (%)

Variation (%)

1200

12

1400

Variation (%)

Endostatin
20
0
-20
-40
-60
-80

M
CA

T
7O
ED

7
ED

M
CA

1
ED

T
2O

12
ED
M
CA

1
ED

T
4O

14
ED
M
CA

1
ED

T
8O

liferation and morphogenesis of epithelia

and is a hepatrophic factor for liver repair
(Nakamura et al., 1989). Moreover, HGF
is a potent angiogenic molecule through
direct actions on endothelial cells, including stimulation of cell motility, proliferation, protease production, invasion, and
organization into capillary-like tubes (Bussolino et al., 1992). Endothelial cells are
essential for early and later stages of liver
development. Accordingly, sinusoid endothelial cells supply HGF, which promotes
hepatocyte growth and expansion of the
liver mass (LeCouter et al., 2003). We
have confirmed an important role played
by HGF during chick liver organogenesis,
due to its highest expression in the first
stage of development examined at ED7.
Overall, in this study we have compared
for the first time the expression of well
known angiogenic cytokines and of one
endogenous inhibitor of angiogenesis in
four different organs of chick embryo in
different developmental stages. We have
demonstrated a common pathway of expression in liver, heart and optic tectum
samples, showing a progressive increase
in the expression of angiogenic cytokines
and a parallel decrease in the expression
of endostatin, whereas in the CAM this
pathway is opposite. Probably, the CAM
acting as an extraembryonic membrane
mediating gas and nutrient exchanges
until hatching (Romanoff, 1960), may be
considered an outer organ and not an
intrinsic organ of the developing embryo
in which the mechanisms regulating the
development of the vascular tree are
different.

18
ED

Fig. 8. Percentage change of expression of VEGF-A, FGF-2, ANG-1, HGF, HIF-1a, HIF-2a, and endostatin between CAM and optic tectum at ED7, 12, 14 and 18. A progressive positive percentage
change is displayed by VEGF-A, FGF-2 and HGF with a reduction at ED18. HIF-1a and HIF-2a showed
different percentage change trend with a positive and a negative variation respectively in the embryonic development. ANG-1 reached a positive percentage peak at the end of embryonic development
while endostatin showed a decrease from positive at ED7 to negative at ED12, ED14 and ED18. List
of abbreviations: CAM, chorioallantoic membrane; VEGF-A, vascular endothelial growth factor-A; FGF-2,
fibroblast growth factor-2; ANG-1, angiopoietin-1; HGF, hepatocyte growth factor; HIF-1a and HIF-2a,
hypoxia inducible factor 1 alpha and 2 alpha; ED, embryonic day.

Materials and Methods

Chorioallantoic membrane, heart, liver and
optic tectum tissue sampling
Two hundred fertilized White Leghorn
chicken eggs were incubated at 37C at constant humidity (60%). On day 3 of incubation
a square window was opened in the egg shell
after removal of 2-3 ml of albumen to detach
the developing CAM from the shell. The window was sealed with glass and the eggs were
returned to the incubator. Embryo manipulation
did not alter its development. The possibility
to look inside the embryo through the window
allows to verify the vitality of the embryo. Ten
chick at embryonic day (ED) 7, 12, 14 and 18
of development were sacrificed. Ten hearts,
10 livers, 10 optic tecta and 10 CAM specimens from each chick embryo at each above
mentioned stages were isolated and immersed
in RNA Later Solution (Ambion, Austin, TX,

Angiogenic and anti-angiogenic molecules in the chick embryo 915

TABLE 1
SPECIFIC PRIMERS FOR CHICKEN TRANSCRIPTS
FOR REAL-TIME PCR
Factor

Primer sequence (5- 3)

VEGF-A

5-TCAGAGTCAGCACATAGC-3
5-CCTTTCCTCGCTTTGATTT-3

FGF-2

5-GGCTATGAAGGAGGATGG-3
5-TGCCACATACCAATCAGA-3

ANG-1

5-CAGTGGGAGAAGATTTAACC-3
5-CTGAAGAGCGTTAGTGTTG-3

HIF-1

5-CTTCAGCAGACTCAGACA-3
5-ACAGGAGATGATGGAACAA-3

HIF-2

5-CAGCAGAGTCTAAGTAGCA-3
5-GTTGAAGAACACCGATGATAG-3

HGF

5-TTCAGAGAACCAGATGTTAGTA-3
5-GGAGTTGTGTCACCAGTA-3

Endostatin

5-ATGAAGTCATCCTCTACCTG-3
5-GAGCCTTCTTCTAGTTCCA-3

-Actin

5-CTTCCAGCCATCTTTCTT-3
5-ATATCCACATCACACTTCAT-3

USA) for subsequent RNA extraction. The experiments were performed in

accordance with the international directions for the protection of animals
used for scientific purposes and approved by the local ethical committee.
RNA extraction and Real-time RT-PCR
Total RNA was extracted from heart, liver and CAM samples using
RNeasy mini kit (Quiagen, Hilden, Germany), while the RNeasy Lipid mini
kit (Quiagen, Hilden, Germany) was used to extract total RNA from optic
tectum samples. Total RNA was then reverse-transcribed into cDNAs using
iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manifacturers instructions. The cDNAs were used to amplify
a panel of six pro-angiogenic and one anti-angiogenic factors with the iQ
SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). PCR
amplification and Real-Time detection were performed using the Chromo4
Real-Time PCR Detection System (Bio-Rad Laboratories). Samples were
normalized to chick beta-actin. Table 1 shows the sequences of chicken
primers (Invitrogen) for each factor analyzed. Primers were designed
using the software Beacon Designer 8 (PREMIER Biosoft International,
Palo Alto, CA, USA).
All the experiments were performed with 60C as the annealing temperature. The amplification process included 35 cycles, each consisting
of three steps: incubation at 95C for 3 minutes; incubation at 95C for
15s; annealing and extension at 60C for 30 s. The melting curve analysis
was performed at 55-95C interval with 0.5C temperature increases per
reading step.
Real time RT-PCR relative expression analysis
The relative expression (fold change) of the pro- and anti-angiogenic
factors in each stage of the chick embryo development was determined
with the comparative Cycle threshold method (2-Ct). Ct values from
RT-PCR were normalized with the housekeeper gene b-actin and then
compared with the latest stage considered of the CAM development at
ED18. CAM of chick embryo at ED18 was chosen as the reference stage
for expression analysis to establish a comparison between its angiogenic
gene expression pattern and the one of other organs examined. Resulting
Fold Change was plotted using GraphPad Prism 5 (GraphPad Software,
Inc, San Diego, CA, USA).
Percentage change data analysis
Relative expression of heart, liver and optic tectum from each stage
of development were compared with relative expression of CAM at the
same stage of development of the above mentioned organs as percentage increase or decrease from CAM expression. Resulting percentage
changes were plotted using GraphPad Prism 5 (GraphPad Software, Inc,
San Diego, CA, USA).

Statistical analysis
Fold change data are reported as means SD. Newman-Keuls multiple comparison post-test was used to compare all developmental stages
following one-way ANOVA. The Graph Pad Prim 5.0 statistical package
(GraphPad Software, San Diego, CA, USA) was used for the analysis and
P<0.05 was considered as the limit for statistical significance.
Acknowledgements
The research leading to these results received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant
agreement n278570 to DR.

References
AUSPRUNK, D.H., KNIGHTON, D.R. and FOLKMAN, J. (1974). Differentiation of
vascular endothelium in the chick chorioallantois: a structural and autoradiographic
study. Dev Biol 38: 237-248.
BAR, T. (1980). The vascular system of the cerebral cortex. Adv Anat Embryol Cell
Biol 59: I-VI,1-62.
BAUTCH, V.L. and JAMES, J.M. (2009). Neurovascular development: The beginning
of a beautiful friendship. Cell Adh Migr 3: 199-204.
BREIER, G., ALBRECHT, U., STERRER, S. and RISAU, W. (1992). Expression of
vascular endothelial growth factor during embryonic angiogenesis and endothelial
cell differentiation. Development 114: 521-532.
BUSSOLINO, F., DI RENZO, M.F., ZICHE, M., BOCCHIETTO, E., OLIVERO, M.,
NALDINI, L., GAUDINO, G., TAMAGNONE, L., COFFER, A. and COMOGLIO, P.M.
(1992). Hepatocyte growth factor is a potent angiogenic factor which stimulates
endothelial cell motility and growth. J Cell Biol 119: 629-641.
CLEAVER, O. and MELTON, D.A. (2003). Endothelial signaling during development.
Nat Med 9: 661-668.
CONSIGLI, S.A. and JOSEPH-SILVERSTEIN, J. (1991). Immunolocalization of
basic fibroblast growth factor during chicken cardiac development. J Cell Physiol
146: 379-385.
CRIVELLATO, E., NICO, B. and RIBATTI, D. (2007). Contribution of endothelial
cells to organogenesis: a modern reappraisal of an old Aristotelian concept. J
Anat 211: 415-427.
DEFOUW, D.O., RIZZO, V.J., STEINFELD, R. and FEINBERG, R.N. (1989). Mapping of the microcirculation in the chick chorioallantoic membrane during normal
angiogenesis. Microvasc Res 38: 136-147.
DUSSEAU, J.W. and HUTCHINS, P.M. (1989). Microvascular responses to chronic
hypoxia by the chick chorioallantoic membrane: a morphometric analysis. Microvasc Res 37: 138-147.
GUADIX, J.A., CARMONA, R., MUNOZ-CHAPULI, R. and PEREZ-POMARES,
J.M. (2006). In vivo and in vitro analysis of the vasculogenic potential of avian
proepicardial and epicardial cells. Dev Dyn 235: 1014-1026.
JUNG, J., ZHENG, M., GOLDFARB, M. and ZARET, K.S. (1999). Initiation of mammalian liver development from endoderm by fibroblast growth factors. Science
284: 1998-2003.
LECOUTER, J., MORITZ, D.R., LI, B., PHILLIPS, G.L., LIANG, X.H., GERBER, H.P.,
HILLAN, K.J. and FERRARA, N. (2003). Angiogenesis-independent endothelial
protection of liver: role of VEGFR-1. Science 299: 890-893.
ATSUMOTO, K., YOSHITOMI, H., ROSSANT, J. and ZARET, K.S. (2001). Liver
organogenesis promoted by endothelial cells prior to vascular function. Science
294: 559-563.
NAKAMURA, T., NISHIZAWA, T., HAGIYA, M., SEKI, T., SHIMONISHI, M., SUGIMURA,
A., TASHIRO, K. and SHIMIZU, S. (1989). Molecular cloning and expression of
human hepatocyte growth factor. Nature 342: 440-443.
PARDANAUD, L. and DIETERLEN-LIEVRE, F. (1999). Manipulation of the angiopoietic/
hemangiopoietic commitment in the avian embryo. Development 126: 617-627.
PARSONS-WINGERTER, P., LWAI, B., YANG, M.C., ELLIOTT, K.E., MILANINIA, A.,
REDLITZ, A., CLARK, J.I. and SAGE, E.H. (1998). A novel assay of angiogenesis in the quail chorioallantoic membrane: stimulation by bFGF and inhibition
by angiostatin according to fractal dimension and grid intersection. Microvasc

916

C. Marinaccio et al.

Res 55: 201-214.

PUGH, C.W. and RATCLIFFE, P.J. (2003). Regulation of angiogenesis by hypoxia:
role of the HIF system. Nat Med 9: 677-684.
RIBATTI, D. (2006). Genetic and epigenetic mechanisms in the early development
of the vascular system. J Anat 208: 139-152.
RIBATTI, D. (2009). Endogenous inhibitors of angiogenesis: a historical review. Leuk
Res 33: 638-644.
RIBATTI, D., MANCINI, L., RONCALI, L., NICO, B. and BERTOSSI, M. (1989). Morphometric analysis on the effects of hypoxia during the central nervous system
vasculogenesis. Int J Microcirc Clin Exp 8: 135-142.
RIBATTI, D., NICO, B., CRIVELLATO, E., ROCCARO, A.M. and VACCA, A. (2007).
The history of the angiogenic switch concept. Leukemia 21: 44-52.
RIBATTI, D., URBINATI, C., NICO, B., RUSNATI, M., RONCALI, L. and PRESTA,
M. (1995). Endogenous basic fibroblast growth factor is implicated in the vascularization of the chick embryo chorioallantoic membrane. Dev Biol 170: 39-49.
RISAU, W. (1986). Developing brain produces an angiogenesis factor. Proc Natl
Acad Sci USA 83: 3855-3859.
RISAU, W. (1995). Differentiation of endothelium. FASEB J 9: 926-933.
RISAU, W. and FLAMME, I. (1995). Vasculogenesis. Annu Rev Cell Dev Biol 11: 73-91.
RISAU, W., GAUTSCHI-SOVA, P. and BOHLEN, P. (1988). Endothelial cell growth
factors in embryonic and adult chick brain are related to human acidic fibroblast
growth factor. EMBO J 7: 959-962.
ROMANOFF, A.L. (1960). The extraembryonic membranes. In The Avian Embryo
Structural and Functional Development. Mac Milian, New York, pp.1039-1141.
RONCALI, L., RIBATTI, D. and AMBROSI, G. (1985). Vasculogenesis in the chick

embryo optic tectum. Acta Anat (Basel) 122: 229-234.

SONG, W., MAJKA, S.M. and MCGUIRE, P.G. (1999). Hepatocyte growth factor
expression in the developing myocardium: evidence for a role in the regulation of
the mesenchymal cell phenotype and urokinase expression. Dev Dyn 214: 92-100.
SUGI, Y. and MARKWALD, R.R. (1996). Formation and early morphogenesis of endocardial endothelial precursor cells and the role of endoderm. Dev Biol 175: 66-83.
SUGISHITA, Y., TAKAHASHI, T., SHIMIZU, T., YAO, A., KINUGAWA, K., SUGISHITA,
K., HARADA, K., MATSUI, H. and NAGAI, R. (2000). Expression of genes encoding
vascular endothelial growth factor and its Flk-1 receptor in the chick embryonic
heart. J Mol Cell Cardiol 32: 1039-1051.
SURI, C., JONES, P.F., PATAN, S., BARTUNKOVA, S., MAISONPIERRE, P.C., DAVIS,
S., SATO, T.N. and YANCOPOULOS, G.D. (1996). Requisite role of angiopoietin-1,
a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87: 1171-1180.
TOMANEK, R.J., HOLIFIELD, J.S., REITER, R.S., SANDRA, A. and LIN, J.J. (2002).
Role of VEGF family members and receptors in coronary vessel formation. Dev
Dyn 225: 233-240.
TOMANEK, R.J., LOTUN, K., CLARK, E.B., SUVARNA, P.R. and HU, N. (1998).
VEGF and bFGF stimulate myocardial vascularization in embryonic chick. Am J
Physiol 274: H1620-H1626.
TOMANEK, R.J., ZHENG, W., PETERS, K.G., LIN, P., HOLIFIELD, J.S. and SUVARNA,
P.R. (2001). Multiple growth factors regulate coronary embryonic vasculogenesis.
Dev Dyn 221: 265-273.
WELLS, J.M. and MELTON, D.A. (1999). Vertebrate endoderm development. Annu
Rev Cell Dev Biol 15: 393-410.
WIKENHEISER, J., DOUGHMAN, Y.Q., FISHER, S.A. and WATANABE, M. (2006).
Differential levels of tissue hypoxia in the developing chicken heart. Dev Dyn
235: 115-123.

Further Related Reading, published previously in the Int. J. Dev. Biol.

Zebrafish enhancer trap line recapitulates embryonic aquaporin 1a expression pattern in vascular endothelial cells
Kira Rehn, Kuan Shen Wong, Darius Balciunas and Saulius Sumanas
Int. J. Dev. Biol. (2011) 55: 613-618
http://dx.doi.org/10.1387/ijdb.103249kp
Zebrafish embryo, a tool to study tumor angiogenesis
Chiara Tobia, Giulia De Sena and Marco Presta
Int. J. Dev. Biol. (2011) 55: 505-509
http://dx.doi.org/10.1387/ijdb.103238ct
Anti-angiogenesis in cancer; met and unmet goals - an interview with Robert Kerbel
Francesco Bertolini
Int. J. Dev. Biol. (2011) 55: 395-398
http://dx.doi.org/10.1387/ijdb.103217fb
A brief history of angiogenesis assays
Anca-Maria Cimpean, Domenico Ribatti and Marius Raica
Int. J. Dev. Biol. (2011) 55: 377-382
http://dx.doi.org/10.1387/ijdb.103215ac
ADAM17 overexpression promotes angiogenesis by increasing blood vessel sprouting
and pericyte number during brain microvessel development
Juntang Lin, Cornelius Lemke, Christoph Redies, Xin Yan, Eilhard Mix, Arndt Rolfs and
Jiankai Luo
Int. J. Dev. Biol. (2011) 55: 961-968
http://dx.doi.org/10.1387/ijdb.103210jl
Tryptase and chymase are angiogenic in vivo in the chorioallantoic membrane assay
Domenico Ribatti, Girolamo Ranieri, Beatrice Nico, Vincenzo Benagiano and Enrico Crivellato
Int. J. Dev. Biol. (2011) 55: 99-102
http://dx.doi.org/10.1387/ijdb.103138dr
Development of a fast, objective, quantitative methodology to monitor angiogenesis
in the chicken chorioallantoic membrane during development
Eva Verhoelst, Bart De Ketelaere, Veerle Bruggeman, Eduardo Villamor, Eddy Decuypere
and Josse De Baerdemaeker
Int. J. Dev. Biol. (2011) 55: 85-92
http://dx.doi.org/10.1387/ijdb.103119ev
5 yr ISI Impact Factor (2011) = 2.959