Revista Română de Anatomie funcțională și clinică, macro- și microscopică și de AntropologieVol. XIII Nr. 2 2014 ORIGINAL PAPERS [601051]

148
Revista Română de Anatomie funcțională și clinică, macro- și microscopică și de AntropologieVol. XIII – Nr. 2 – 2014 ORIGINAL PAPERS
THE ROLE OF THE ENDOCARDIAL CUSHIONS
AND VESTIBULAR SPINE IN THE CARDIAC SEPTATION
C.M. Panțu1, R. Stănciulescu1, O. Enciu1, M. Enyedi1, C. Tătaru2, F .M. Filipoiu1
University of Medicine and Pharmacy “Carol Davila”, Bucharest
1. Anatomy Discipline
Department of Morphological Sciences
2. Emergency Clinical Hospital of Ophtalmology
Department of Ophtalmology
THE ROLE OF THE ENDOCARDIAL CUSHIONS AND VESTIBULAR SPINE IN THE CAR –
DIAC SEPTATION (Abstract): The cardiac septation involves the endocardial cushions of the
atrioventricular canal and mesenchymal cap of septum primum, classically called the vestibular
spine. The origin and the role of the endocardial cushions and of the vestibular spine represented
the aim of this paper. Dissections were performed on 4 embryonic hearts. The results were stained
using a Hematoxilin-Eozin method and after were photogarphied using a microscope imaging
software. The superior and inferior atrioventricular endocardial cushions develops from an epithe –
lial-to-mesenchymal transformation of the endocardial cells in the atrioventricular canal and unites
to form the septum intermedium. The formation of the atrioventricular septum and the closure of
the atrioventricular canal involves the mesenchymal cap covering the leading edge of the septum
primum. The cardiac jelly has an important role in the development of the endocardial cushions.
The migration of the endocardial cells in the cardiac jelly starts when an inductive stimulus is
produced by the myocardium. The closure of the atrioventricular canal results from the intracardi –
ally generated mesenchyme which starts to populate the free edge of septum primum, between the
two endocardial cushions. The vestibular spine is a band of mesenchyme which starts in the pul –
monary area and reaches the free edge of the septum primum. Its variabilty infuences the atrial
relationship to the middline extracardiac mesesenchyme. Spina vestibuli may also have a role in
functioning as an anchor in atrial septation process. Key words : ATRIOVENTRICULAR ENDO –
CARDIAL CUSHIONS, SPINA VESTIBULI, CARDIAC SEPTATION
INTRODUCTION
The cardiac septa develops between the 27
and the 31 days of embryonic life. The primary
atrial septum is a crescent-shaped muscular
septum and develops from the dorsal cranial
wall of the primitive atrium in the 6-th week.
Concomitant, the superior and the inferior en –
docardial cushions, formed at the atrioventric –
ular canal from mesenchymal cells, merge to
form the septum intermedium. Between the
septum primum and septum intermedium, is
the foramen primum, through which the atria
communicates. The vestibular spine (or spina
vestibuli) has been an arguable structure. It was
first described by His in 1880 as an intracar –
diac continuation of the extracardiac mesen -chyme which has the origin in the dorsal mes –
ocardium (1). Mesenchyme generated from
endocardium by epithelial-mesenchymal trans –
formation coats the leading edge of the septum
is called the vestibular spine (2, 3). The aim of
this paper is to highlight the importance of the
atrioventricular cushions and of the vestibular
spine, but also to bring together new informa –
tion, some contradictory, which puts in a whole
new light the complex mechanisms of the ca –
diac septation.
MATERIAL AND METHODS
The study involved the dissection on 4 hu –
man embryonic hearts, from 4 weeks to 10
weeks of de velopment. The specimens resulted

149
The Role of the Endocardial Cushions And V estibular Spine in the Cardiac Septationfrom the dissection were stained using a He –
matoxilin-Eozin technique and were photo –
graphed using a microscope imaging software.
The ages of the embryos were estimated accord –
ing to methods described by O’Rahilly and
Müller (4).
RESULTS
The endocardial cushions forms, starting
with the 4-th week in the human embryo, from
the epithelial-to-mesenchymal transformation
of the endocardial cells at the atrioventricular
canal. Finnaly, there will be a superior, an
inferior and two lateral endocardial cushions.
(fig. 1).
The superior and the inferior endocardial
cushions unites to form the septum intermedi –
um while the lateral cushions remains sepa –
rated and contributes in the development of the
tricuspid and mitral valve primordium. Septum
intermedium separates the atrioventricular ca –
nal in two oriffices, right and left (5). After the
septum intermedium is formed, the atrioven –
tricular canal expands to the right, resulting in
the alignment of the septum intermedium with
the septum primum and the interventricular
septum (6).
Between the endocardial cushions and the
dextrodorsal crest of the conus arteriosus mi –
grating cells of neuroectodermal origin are vis –
ible, which contributes to the regional differ –
entiation (fig. 2).
The caracteristics of the cellular organites
changes during the merging process of the cush -ions. The cells starts to accumulate Golgi com –
plexes and granular endoplasmic reticuli while
the nucleus becomes irregular in shape. These
changes first occurs in the cardiac jelly cells (7).
The right side of the superior endocardial
cushion unites with the aortic vestibule and
with the wall of the presumtive right atrium.
The fusion area will form the membranous
atrioventricular septum. The development of
the atrioventricular septum (fig.3 ) is a decisive
factor in the formation of the septal and pos –
teroinferior cusps of the tricuspid valve and of
the the anterior cusp of the mitral valve (8).
DISCUSSION
The cardiac jelly has an important role in
the development of the endocardial cushions
due to its rich content in highly charged hydro –
philic glycosaminoglycans which aids the en –
docardial cells to invade the endocardial cush –
ions (9). The cardiac jelly is secreted by the
myocardium and expands at the junction of the
atrium and primitive left ventricle before cush –
ions are formed (10). Studies on the Tie2-Cre/
lacZ double transgenic mouse with permanent –
ly marked endothelial cells demonstrated that
the atrioventricular cushion mesenchyme was
derived from endocardium (1 1).
The migration of the endocardial cells in the
cardiac jelly starts when an inductive stimulus
produced by the myocardium is received and
Nfatc1 nuclear factor is translocated from the
cytoplasm to the nucleus of the endothelial
cells. Aft er this the cells grows in size, and due
to a decrease in different adhesion molecules
Fig. 1 . Superior (SEC) and inferior (IEC)
atrioventricular endocardial cushions. Conus
arteriosus (CA) and its dextrodorsal and sintroven –
tral endocardial crests (DD-EC; SV-EC) are
visible. The crests will form the spiral septum.
The primitive ventricle (PV) presents mesenchy –
mal trabecullae.
Fig. 2 . Atrioventricular endocardial cushions
(SEC; IEC) in the process of unification. The
migrating cells are visible between the two
cushions. This population of cells was considered
to be spina vestibuli. Recent studies demonstrated
that these are modified endocardial cells.

150
C.M. Panțu et al.like N-Cam, VE-cadherin, and Pecam1, detach
themselves from the endocardial epithelium
(12, 13). Finally, the free endocardial cells ex –
tend filopodia and enters the cardiac jelly (14).
In the atrioventricular canal of the mouse the
formation occurs between 21 and 28 somites,
and at stages 25–28 somites in the chick (15).
The mesenchymal cap covering the leading edge
of the septum primum, which was thought to
be the spina vestibuli but now it was demon –
strated to be intracardially generated mesen –
chyme (16), merges with the superior endocar –
dial cushion. Following this event, the fusion
area undergos a myocardial transformation (2).
Using lineage tracing it was discovered that
the mesenchymal cap is formed by epithelial-
to-mesenchymal transformation from the endo –
cardium covering the septum. This newelly
formed mesenchyme is continuous with the
mesenchyme of the vestibular spine situated in
the right pulmonary ridge (16).
The mesenchym of the spine and the mes –
enchymal cap of septum primum have common
structural and biochemical properties with the
endocardial cushions (17). The difference be –
tween the endocardial cells and the spina ves –
tibuli cells is that spinal cells does not express
the 9G9 epitope (18).
The location of the spine is also considered
to be important for the development of the septum secundum. Its ventro-caudal margin
develops from the vestibular spine (2). The
vestibular spine has role in the septation of the
venous pole of the heart which is similar to that
of the spiral septum, derived from the neural
crest, in the arterial pole of the heart. There –
fore, the spine may have a role in acting as
conduit for migrating cells with neural crests
origin (19).
CONCLUSIONS
The atrial septation apears to due the septum
primum development. This unites with the
atrioventricular endocardial cushions. The clo –
sure of the atrioventricular canal results from
the mesenchyme which starts to populate the
free edge of septum primum, between the two
endocardial cushions (20). The septal atrial
defects, like those seen in the Holt-Oram syn –
dome, have the origin in the failure of the
mesenchymal cap to fuse the endocardial cush –
ions (21).
Classic theories cosidered that the mesen –
chymal cap of septum primum is an intracardiac
continuation of the extracardiac mesenchyme
which has the origin in the dorsal mesocardium
and named this mesencyme the vestibular spine.
It was demonstrated that this is an intracardi –
ally generated mesenchyme (22).
The dorsal mesenchyme contains a band of
mesenchyme which starts in the pulmonary area
and reaches the free edge of the septum primum.
This band is called the vestibular spine (23).
The vestibular spine infuences the atrial re –
lationship to the extracardiac meseenchyme
from the middline (fig. 4).
The area where the primitive atrium comes
Fig. 3 . Formation of the atrioventricular septum.
(marked with star). SEC-superior endocardial
cushion; AO-aortic vestibule; P A- primitive
ventricle.
Fig. 4 . Spina vestibuli-schematic representation (23)

151
The Role of the Endocardial Cushions And V estibular Spine in the Cardiac Septationin contact with the vestibular spine forms the
pulmonary pit. This is the point where the prim –
itive pulmonary vein enters the left atrium. Any
abnormality in this connection leads to positional
abnormalities of the pulmonary veins (24).Spina vestibuli may also have a role in function –
ing as an anchor in atrial septation process (2).
From the center of the spine the tendon of
Todaro forms, as the only part of the spine
which does’t myocardialize (25).
REFERENCES
1. His W . Die area interposita, die Eustachi’sche klappe und die spina vestibuli. In: Anatomie Menschli –
cher Embryonen . Leipzig, Germany: V ogel, 149–152, 1880
2. Kim JS, Viragh S, Moorman AF , Anderson RH, Lamers WH. Development of the myocardium of the
atrioventricular canal and the vestibular spine in the human heart. Circ.Res . 88:295–402, 2001.
3. Tasaka H, Krug EL, Markwald RR.. Origin of the pulmonary venous orifice in the mouse and its rela –
tion to the morphogenesis of the sinus venosus, extracardiac mesenchyme (spina vestibuli), and atrium.
Anat. Rec . 246:107–1 13, 1996
4. O’Rahilly R, Müller F . Developmental stages in human embryos . Washington, DC: Carnegie Institu –
tion of Washington, publication No. 637, 1987.
5. Person AD, Klewer SE, Runyan RB.. Cell biology of cardiac cushion development. Int. Rev. Cytol .
243:287–335, 2005
6. Wessels A, Sedmera D. Developmental anatomy of the heart: a tale of mice and man. Physiol. Genom .
15:165–176, 2003.
7. Hay DA, Low FN.. The fusion of dorsal and ventral endocardial cushions in the embryonic chick heart:
A study in fine structure. Am. J. Anat ., 133: 1–23. doi: 10.1002/aja.1001330102, 1972
8. Markwald RR, Fitzharris TP , Manasek FJ.. Structural development of endocardial cushions. Am. J.
Anat. 148:85–1 19, 1977
9. Manasek FJ. Sulfated extracellular matrix production in the embryonic heart and adjacent tissues. J.
Exp. Zool . 174:415–439, 1970.
10. Krug EL, Runyan RB, Markwald MR. Protein extracts from early embryonic hearts initiate cardiac
endothelial cytodifferentiation. Dev. Biol . 1 12:414–426, 1985.
1 1. Kisanuki YY , Hammer RE, Miyazaki JI, Williams SC, Richardson JA, Y anagisawa M. Tie2-cre trans –
genic mice: a new model for endothelial cell-lineage analysis in vivo. Dev. Biol . 230:230–242, 2001.
12. Baldwin HS, Shen HM, Y an HC, et al. Platelet endothelial cell adhesion molecule-1 (PECAM-1/
CD31): alternatively spliced, functionally distinct isoforms expressed during mammalian cardiovascu –
lar development. Development 120:2539–2553, 1994.
13. Timmerman, L. A., J. Grego-Bessa, A. Raya, et al. Notch promotes epithelial-mesenchymal transition
during cardiac development and oncogenic transformation. Genes Dev . 18:99–1 15, 2004.
14. Person AD, Klewer SE, Runyan RB. Cell biology of cardiac cushion development. Int. Rev. Cytol .
243:287–335, 2005.
15. Delot EC, Bahamonde ME, Zhao MX, Lyons KM. BMP signaling is required for septation of the
outflow tract of the mammalian heart. Development 130:209–220, 2003.
16. Mommersteeg, MT, Soufan AT, de Lange FJ, et al. Two distinct pools of mesenchyme contribute to
the development of the atrial septum. Circ. Res . 99:351–353, 2006.
17. Gerety M, Watanabe M. Polysialyted NCAM expression on endocardial cells of the chick primary
atrial septum. Anat Rec . 247:71–84, 1997
18. Wessels A, Anderson RH, Markwald RR, et al. Atrial development in the human heart: an immuno –
histochemical study with emphasis on the role of mesenchymal tissues. Anat Rec . 259:288–300, 2000
19. Poelmann RE, Gittenberger-de Groot AC. A subpopulation of apoptosis-prone cardiac neural crest cells
targets to the venous pole: multiple functions in heart development. Dev Biol . 207:271–286, 1999
20. Puerta Fonolla AJ, Orts Llorca F .. Origin and development of the septum primum. Acta Anat . (Basel).
100:250–257, 1978
21. Li QY , Newbury-Ecob RA, Terrett JA, et al. Holt-Oram syndrome is caused by mutations in TBX5, a
member of the Brachyury (T) gene family. Nat. Genet . 15:21–29, 1997.
22. Filipoiu FM. Cordul – Anatomie, repere embriologice si notiuni de infrastructura a miocardului. Atlas
explicitat si comentat , Editura Prior & Books Bucuresti, 2012;
23. Kirby ML, Cardiac Development , Oxford University Press, 2007
24. Webb S, Anderson RH, Lamers WH, Brown NA. Mechanisms of deficient cardiac septation in the
mouse with trisomy 16. Circ. Res . 84:897–905, 1999.
25. Webb S, Brown NA, Anderson RH. Formation of the atrioventricular septal structures in the normal
mouse. Circ Res . 82:645–656, 1998