Retinoic acid (RA), the active derivative of vitamin A, has been implicated in various steps of cardiovascular development, but its contribution to early heart morphogenesis has not been clearly established in a mammalian system. To block endogenous RA synthesis, we have disrupted the gene encoding RALDH2, the first retinaldehyde dehydrogenase whose expression has been detected during early mouse post-implantation development. We describe here the heart abnormalities of the RA-deficient Raldh2 mutants that die in utero at gestational day 10.5. The embryonic heart tube forms properly, but fails to undergo rightward looping and, instead, forms a medial distended cavity. Expression of early heart determination factors is not altered in mutants, and the defect in heart looping does not appear to involve the Nodal/Lefty/Pitx2 pathway. Histological and molecular analysis reveal distinct anteroposterior components in the mutant heart tube, although posterior chamber (atria and sinus venosus) development is severely impaired. Instead of forming trabeculae, the developing ventricular myocardium consists of a thick layer of loosely attached cells. Ultrastructural analysis shows that most of the ventricular wall consists of prematurely differentiated cardiomyocytes, whereas undifferentiated cells remain clustered rostrally. We conclude that embryonic RA synthesis is required for realization of heart looping, development of posterior chambers and proper differentiation of ventricular cardiomyocytes. Nevertheless, the precise location of this synthesis may not be crucial, as these defects can mostly be rescued by systemic (maternal) RA administration. However, cardiac neural crest cells cannot be properly rescued in Raldh2(−/−)embryos, leading to outflow tract septation defects.

Reference

Barak
Y.
,
Nelson
M. C.
,
Ong
E. S.
,
Jones
Y. Z.
,
Ruiz-Lozano
P.
,
Chien
K. R.
,
Koder
A.
,
Evans
R. M.
(
1999
)
PPARis required for placental, cardiac, and adipose tissue development.
Mol. Cell
4
,
585
595
Berggren
K.
,
McCaffery
P.
,
Dräger
U.
,
Forehand
C. J.
(
1999
)
Differential distribution of retinoic acid synthesis in the chicken embryo as determined by immunolocalization of the retinoic acid synthetic enzyme, RALDH-2.
Dev. Biol
210
,
288
304
Biben
C.
,
Harvey
R.
(
1997
)
Homeodomain factor NKx2-5 controls left/right asymmetric expression of the bHLH gene eHand during murine heart development.
Genes Dev
11
,
1357
1369
Bruneau
B. G.
,
Logan
M.
,
Davis
N.
,
Levi
T.
,
Tabin
C. J.
,
Seidman
J. G.
,
Seidman
C. E.
(
1999
)
Chamber-specific cardiac expression of Tbx5 and heart defects in Holt-Oram syndrome.
Dev. Biol
211
,
100
108
Campione
M.
,
Steinbeisser
H.
,
Schweickert
A.
,
Deissler
K.
,
van Bebber
F.
,
Lowe
L. A.
,
Nowotschin
S.
,
Viebahn
C.
,
Haffter
P.
,
Kuehn
M. R.
,
Blum
M.
(
1999
)
The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping.
Development
126
,
1225
1234
Capdevila
J.
,
Vogan
K. J.
,
Tabin
C. J.
,
Izpisua-Belmonte
J. C.
(
2000
)
Mechanisms of left-right determination in vertebrates.
Cell
101
,
9
21
Chazaud
C.
,
Chambon
P.
,
Dolle
P.
(
1999
)
Retinoic acid is required in the mouse embryo for left-right asymmetry determination and heart morphogenesis.
Development
126
,
2589
2596
Chen
J.
,
Kubalak
S. W.
,
Chien
K. R.
(
1998
)
Ventricular muscle-restricted targeting of the RXRalpha gene reveals a non-cell-autonomous requireent in cardiac chamber morphogenesis.
Development
125
,
1943
1949
Christoffels
V. M.
,
Habets
P. E. M. H.
,
Franco
D.
,
Campione
M.
,
de Jong
F.
,
Lamers
W. H.
,
Bao
Z. Z.
,
Palmer
S.
,
Biben
C.
,
Harvey
R. P.
,
Moorman
A. F. M.
(
2000
)
Chamber formation and morphogenesis in the developing mammalian heart.
Dev. Biol
223
,
266
278
Christoffels
V. M.
,
Keijser
A. G. M.
,
Houweling
A. C.
,
Clout
D. E. W.
,
Moorman
A. F. M.
(
2000
)
Patterning the embryonic heart: identification of five mouse Iroquois homeobox genes in the developing heart.
Dev. Biol
224
,
263
274
Collignon
J.
,
Varlet
I.
,
Robertson
E. J.
(
1996
)
Relationship between asymmetric nodal expression and the direction of embryonic turning.
Nature
381
,
155
158
Dersch
H.
,
Zile
M. H.
(
1993
)
Induction of normal cardiovascular development in the vitamin A-deprived quail embryo by natural retinoids.
Dev. Biol
160
,
424
433
Dickman
E. D.
,
Smith
S. M.
(
1996
)
Selective regulation of cardiomyocyte gene expression and cardiac morphogenesis by retinoic acid.
Dev. Dyn
206
,
39
48
Drysdale
T. A.
,
Patterson
K. D.
,
Saha
M.
,
Krieg
P. A.
(
1997
)
Retinoic acid can block differentiation of the myocardium after heart specification.
Dev. Biol
188
,
205
215
Duester
G.
(
2000
)
Families of retinoid dehydrogenases regulating vitamin A function: production of visual pigment and retinoic acid.
Eur. J. Biochem
267
,
4315
4324
Echelard
Y.
,
Vassileva
G.
,
McMahon
A. P.
(
1994
)
Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS.
Development
120
,
2213
2224
Epstein
J. A.
,
Li
J.
,
Lang
D.
,
Chen
F.
,
Brown
C. B.
,
Jin
F.
,
Lu
M. M.
,
Thomas
M.
,
Liu
E. C. J.
,
Wessels
A.
,
Lo
C. W.
(
2000
)
Migration of cardiac neural crest cells in Splotch embryo.
Development
127
,
1869
1878
Ghyselinck
N. B.
,
Wendling
O.
,
Wessaddeq
N.
,
Dierich
A.
,
Lampron
C.
,
Decimo
D.
,
Viville
S.
,
Chambon
P.
,
Mark
M.
(
1998
)
Contribution of retinoic acid receptorisoforms to the formation of the conotruncal septum of the embryonic heart.
Dev. Biol
198
,
303
318
Gruber
P. J.
,
Kubalak
S.
,
Pexieder
T.
,
Sucov
H.
,
Evans
R.
,
Chien
K.
(
1996
)
RXRdeficiency confers genetic susceptibility for aortic sac, atrioventricular cushion and ventricular defects in mice.
J. Clin. Invest
98
,
1332
1343
Heine
U. I.
,
Roberts
A. B.
,
Munoz
E. F.
,
Roche
N. S.
,
Sporn
M. B.
(
1985
)
Effects of retinoid deficiency on the development of the heart and vascular system of the quail embryo.
Virchows Arch. Cell Pathol
50
,
135
152
Jiang
X.
,
Rowitch
D. H.
,
Soriano
P.
,
McMahon
A. P.
,
Sucov
H. M.
(
2000
)
Fate of the mammalian cardiac neural crest.
Development
127
,
1607
1616
Kastner
P.
,
Grondona
J.
,
Mark
M.
,
Gansmuller
A.
,
LeMeur
M.
,
Decimo
D.
,
Vonesch
J. L.
,
Dolle
P.
,
Chambon
P.
(
1994
)
Genetic analysis of RXRdevelopmental function: convergence of RXR and RAR signalling pathways in heart and eye morphogenesis.
Cell
78
,
987
1003
Kastner
P.
,
Messaddeq
N.
,
Mark
M.
,
Wendling
O.
,
Grondona
J.M.
,
Ward
S.
,
Ghyselinck
N.
,
Chambon
P.
(
1997
)
Vitamin A deficiency and mutations of RXR, RXR and RARlead to early differentiation of embryonic ventricular cardiomyocytes.
Development
124
,
4749
4758
Klein
E. S.
,
Wang
J. W.
,
Khalifa
B.
,
Gavignan
S. A.
,
Chandraratna
R. A. S.
(
2000
)
Recruitement of nuclear receptor corepressor and coactivator to the retinoic acid receptor by retinoid ligands.
J. Biol. Chem
275
,
19401
19408
Kostetskii
I.
,
Jiang
Y.
,
Kostetskaia
E.
,
Yuan
S.
,
Evans
T.
,
Zile
M.
(
1999
)
Retinoid signaling required for normal heart development regulates GATA-4 in a pathway distinct from cardiomyocyte differentiation.
Dev. Biol
206
,
206
218
Kuo
C. T.
,
Morrisey
E. E.
,
Anandappa
R.
,
Sigrist
K.
,
Lu
M. M.
,
Parmacek
M. S.
,
Soudais
C.
,
Leiden
J. M.
(
1997
)
GATA4 transcription factor is required for ventral morphogenesis and heart tube formation.
Genes Dev
11
,
1048
1060
Li
H.
,
Wagner
E.
,
McCaffery
P.
,
Smith
D.
,
Andreadis
A.
,
Dräger
U.
(
2000
)
A retinoic acid synthesizing enzyme in vental retina and telencephalon of the embryonic mouse.
Mech. Dev
95
,
283
289
Liberatore
C. M.
,
Searcy-Schrick
R. D.
,
Yutzey
K. E.
(
2000
)
Ventricular expression of tbx5 inhibits normal heart chamber development.
Dev. Biol
223
,
169
180
Lin
Q.
,
Schwarz
J.
,
Bucana
C.
,
Olson
E. N.
(
1997
)
Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C.
Science
276
,
1404
1407
Lin
C. R.
,
Kioussi
C.
,
O'Connell
S.
,
Briata
P.
,
Szeto
D.
,
Liu
F.
,
Izpisua-Belmonte
J. C.
,
Rosenfeld
M. G.
(
1999
)
Pitx2 regulates lung asymmetry, cardiac positioning and pituitary and tooth morphogenesis.
Nature
401
,
279
282
Linask
K. K.
,
Lask
J. W.
(
1988
)
A role for fibronectin in the migration of avian precardiac cells. I. Dose-dependent effects of fibronectin antibody.
Dev. Biol
129
,
315
323
Lyons
I.
,
Parson
L. M.
,
Hartley
L.
,
Li
R.
,
Andrews
J. E.
,
Robb
L.
,
Harvey
R. P.
(
1995
)
Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5.
Genes Dev
9
,
1654
1666
Maconochie
M.
,
Krishnamurthy
R.
,
Nonchev
S.
,
Meier
P.
,
Manzanares
M.
,
Mitchell
P. J.
,
Krumlauf
R.
(
1999
)
Regulation of Hoxa2 in cranial neural crest cells involves members of the AP-2 family.
Development
126
,
1483
1494
Mark
M.
,
Lufkin
T.
,
Vonesch
J. L.
,
Ruberte
E.
,
Olivo
J. C.
,
Dolle
P.
,
Gorry
P.
,
Lumsden
A.
,
Chambon
P.
(
1993
)
Two rhombomeres are altered in Hoxa-1 mutant mice.
Development
119
,
319
338
Mascrez
B.
,
Mark
M.
,
Dierich
A.
,
Ghyselinck
N. B.
,
Kastner
P.
,
Chambon
P.
(
1998
)
The RXRoigand-dependent activation function (AF-2) is important for mouse development.
Development
125
,
4691
4707
McCaffery
P.
,
Dräger
U.
(
2000
)
Regulation of retinoic acid signaling in the embryonic nervous system: a master differentiation factor.
Cytokine Growth Factor Rev
11
,
233
249
Means
A. L.
,
Guda
L. J.
(
1995
)
The roles of retinoids in vertebrate development.
Annu. Rev. Biochem
64
,
201
233
Mendelsohn
C.
,
Lohnes
D.
,
Decimo
D.
,
Lufkin
T.
,
LeMeur
M.
,
Chambon
P.
,
Mark
M.
(
1994
)
Function of the retinoic acid receptors (RARs) during development. II. Multiple abnormalities at various stages of organogenesis in RAR double mutants.
Development
120
,
2749
2771
Molkentin
J. D.
,
Lin
Q.
,
Duncan
S. A.
,
Olson
E. N.
(
1997
)
Requirement of the transcription factor GATA4 for heat tube formation and ventral morphogenesis.
Genes Dev
11
,
1061
1072
Moss
J. B.
,
Xavier-Neto
J.
,
Shapiro
M. D.
,
Nayeem
S. M.
,
McCaffery
P.
,
Dräger
U. C.
,
Rosenthal
N.
(
1998
)
Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart.
Dev. Biol
199
,
55
71
Niederreither
K.
,
McCaffery
P.
,
Dräger
U. C.
,
Chambon
P.
,
Dolle
P.
(
1997
)
Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development.
Mech. Dev
62
,
67
78
Niederreither
K.
,
Subbarayan
V.
,
Dolle
P.
,
Chambon
P.
(
1999
)
Embryonic retinoic acid synthesis is essential for early mouse post-implantation development.
Nat. Genet
21
,
444
448
Niederreither
K.
,
Vermot
J.
,
Schuhbaur
B.
,
Chambon
P.
,
Dolle
P.
(
2000
)
Retinoic acid synthesis and hindbrain patterning in the mouse embryo.
Development
127
,
75
85
Osmond
M. K.
,
Butler
A. J.
,
Voon
F. C. T.
,
Bellairs
R.
(
1991
)
The effects of retinoic acid on heart formation in the early chick embryo.
Development
113
,
1405
1417
Oulad-Abdelghani
M.
,
Chazaud
C.
,
Bouillet
P.
,
Mattei
M. G.
,
Dolle
P.
,
Chambon
P.
(
1998
)
Stra3/lefty, a retinoic acid-inducible novel member of the transforming growth factor-beta superfamily.
Int. J. Dev. Biol
42
,
23
32
Rossant
J.
,
Zirnbigl
R.
,
Cado
D.
,
Shago
M.
,
Giguere
V.
(
1991
)
Expression of a retinoic acid response element- hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis.
Genes Dev
5
,
1333
1344
Sapin
V.
,
Dolle
P.
,
Hindelang
C.
,
Kastner
P.
,
Chambon
P.
(
1997
)
Defects of the choriallantoic placenta in mouse RXRnull fetuses.
Dev. Biol
191
,
29
41
Sapin
V.
,
Ward
S. J.
,
Bronner
S.
,
Chambon
P.
,
Dolle
P.
(
1997
)
Differential expression of transcripts encoding retinoid binding proteins and retinoic acid receptors during placentation of the mouse.
Dev. Dyn
208
,
199
210
Sucov
H. M.
,
Dyson
E.
,
Gumeringer
C. L.
,
Price
J.
,
Chien
K. R.
,
Evans
R. M.
(
1994
)
RXRmutant mice establish a genetic basisforvitamin A signaling in heart morphogenesis.
Genes Dev
8
,
1007
1018
Tsukui
T.
,
Capdevila
J.
,
Tamura
K.
,
Ruiz-Lozano
P.
,
Rodriguez-Esteban
C.
,
Yonei-Tamura
S.
,
Magallon
J.
,
Chandraratna
R. A.
,
Chien
K.
,
Blumberg
B.
,
Evans
R. M.
,
Izpisua-Belmonte
J. C.
(
1999
)
Multiple left-right asymmetry defects in the Shh/ mutant mice unveil a convergence of the shh and retinoic acid pathways in the control of Lefty-1.
Proc. Natl. Acad. Sci. USA
96
,
11376
11381
Ulven
S. M.
,
Gundersen
T. E.
,
Weedon
M. S.
,
Landaas
V. O.
,
Sakhi
A. K.
,
Fromm
S. H.
,
Geronimo
B. A.
,
Moskaug
J. O.
,
Blomhoff
R.
(
2000
)
Identification of endogenous retinoids, enzymes, binding proteins, and receptors during early postimplantation development in mouse: important role of retinal dehydrogenase type 2 in synthesis of all-trans-retinoic acid.
Dev. Biol
220
,
379
391
Vermot
J.
,
Fraulob
V.
,
Dolle
P.
,
Niederreither
K.
(
2000
)
Expression of enzymes synthetizing (Aldh1 and Raldh2) and metabolizing (CYP26)retinoic acid in the mouse female reproductive system.
Endocrinology
141
,
3638
3645
Wasiak
S.
,
Lohnes
D.
(
1999
)
Retinoic acid affects left-right patterning.
Dev. Biol
215
,
332
342
Wendling
O.
,
Chambon
P.
,
Mark
M.
(
1999
)
Retinoid X receptors are essential for early mouse development and placentogenesis.
Proc. Natl. Acad. Sci. USA
96
,
547
551
Wilson
J. G.
,
Warkany
J.
(
1949
)
Aortic arch and cardiac anomalies in the offspring of vitamin A deficient rats.
Am. J. Anat
85
,
113
155
Xavier-Neto
J.
,
Neville
C. M.
,
Shapiro
M. D.
,
Houghton
L.
,
Wang
G. F.
,
Nikovits
W.
Jr
,
Stockdale
F. E.
,
Rosenthal
N.
(
1999
)
A retinoic acid-inducible transgenic marker of sino-atrial development in the mouse heart.
Development
126
,
2677
2687
Yutzey
K. E.
,
Rhee
J. T.
,
Bader
D.
(
1994
)
Expression of the atrial-specific myosin heavy chain AMHC1 and the establishment ofanteroposterior polarity in the developing chicken heart.
Development
120
,
871
883
Zhao
D.
,
McCaffery
P.
,
Ivins
K. J.
,
Neve
R. L.
,
Hogan
P.
,
Chin
W. W.
,
Dräger
U. C.
(
1996
)
Molecular identification of a major retinoic acid-synthesizing enzyme, a retinaldehyde dehydrogenase.
Eur. J. Biochem
15
,
15
22
Zheng
W. L.
,
Sierra-Rivera
E.
,
Luan
J.
,
Osteen
K. G.
,
Ong
D. E.
(
1999
)
Retinoic acid synthesis and expression of cellular retinol-binding protein and cellular retinoic acid-binding protein type II are concurrent with decidualization of rat uterine stromal cells.
Endocrinology
141
,
802
808
Zile
M. H.
,
Kostetskii
I.
,
Yuan
S.
,
Kostetskaia
E.
,
St. Amand
T. R.
,
Chen
Y. P.
,
Jiang
W.
(
2000
)
Retinoid signaling is required to complete the vertebrate cardiac left/right aymmetry pathway.
Dev. Biol
223
,
323
338
This content is only available via PDF.