RXRalpha null mutant mice display ocular and cardiac malformations, liver developmental delay, and die from cardiac failure around embryonic day (E) 14.5 pc. To dissect the molecular basis of the RXRalpha-associated cardiomyopathy, we performed subtractive hybridization and systematically characterized putative downstream target genes that were selectively lacking in the mutant embryos, both at early (E10.5) and late (E13.5) stages of mouse embryonic development. Approximately 50% of the subtracted clones (61/115) encoded proteins involved in intermediary metabolism and electron transport, suggesting an energy deficiency in the RXRalpha−/− embryos. In particular, clone G1, which encodes subunit 14.5b of the NADH-ubiquinone dehydrogenase complex, displayed a dose-dependent expression in the wild-type, heterozygous and RXRalpha mutant mice. This gene was also downregulated in a retinoid-deficient rat embryo model. ATP content and medium Acyl-CoA dehydrogenase mRNA were lower in RXRalpha mutant hearts compared to wild-type mice. Ultrastructural studies showed that the density of mitochondria per myocyte was higher in the RXRalpha mutant compared to wild-type littermates. We propose a model whereby defects in intermediary metabolism may be a causative factor of the RXRalpha−/− phenotype and resembles an embryonic form of dilated cardiomyopathy.

REFERENCES

Arber
S.
,
Hunter
J. J.
,
Ross
J.
Jr.
,
Hongo
M.
,
Sansig
G.
,
Borg
J.
,
Perriard
J.-C.
,
Chien
K. R.
,
Caroni
P.
(
1997
)
MLP-deficient miceexhibit a disruption of cardiac myofibrillar organization, dilated cardiomyopathy, and heart failure.
Cell
88
,
393
403
Arizmendi
J. M.
,
Sketel
J. M.
,
Runswich
M. J.
,
Fearnley
I. M.
,
Walker
J. E.
(
1992
).
Complementary cDNA sequences of two 14.5 kDa subunits of NADH:ubiqunone oxidoredusctase from bovine heart mitochondria. Completion of the primary structure of the complex?.
FEBS Lett
313
,
80
84
Bouillet
P.
,
Oulad-Abdelghani
M.
,
Vacaire
S.
,
Garnier
J. M.
,
Schuhbaur
B.
,
Dolle
P.
,
Chambon
P.
(
1995
)
Efficient cloning of cDNAs of retinoic acid responsive genes in P19 embryonal carcinoma cell and characterization of a novel mouse gene, stra1 (mouse LERK-2/Eplg2).
Dev. Biol
170
,
420
433
Bradford
M.
(
1976
)
A rapid and sensitive method for the quantitaion of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem
72
,
248
254
Brouillet
E.
,
Hantraye
P.
,
Ferrante
R.J.
,
Dolan
R.
,
Leroy-Willig
A.
,
Kowall
N.W.
,
Flint-Beal
M.
(
1995
)
Chronic mitochondrial energy impairement produces selective striatal degeneration and abnormal choreiform movements in primates.
Proc. natl. Acad. Sci. USA
92
,
7105
7109
Buono
P.
,
Paolella
G.
,
Mancini
F. P.
,
Izzo
P.
,
Salvatore
F.
(
1988
)
The complete nucleotide sequence of the gene coding for the human aldolase C.
Nucleic Acids Res
16
,
4733
–.
Chen
Z.
,
Friedrich
G.A.
,
Soriano
P.
(
1994
)
Transcriptional enhancer factor 1 disruption by a retroviral gene trap leads to heart defects and embryonic lethality in mice.
Genes Dev
8
,
2293
2301
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.
,
Thaller
C.
,
Smith
S. M.
(
1997
)
Temporally-regulated retinoic acid depeletion produces specific neural crest, ocular and nervous system defects.
Development
124
,
3111
3121
Dyson
E.
,
Sucov
H. M.
,
Kubalak
S. W.
,
Schmid-Schönbein
G.
,
Delano
F. A.
,
Evans
R. M.
,
Ross
J.
Jr.
,
Chien
K. R.
(
1995
)
Atrial-phenotype is associated with embryonic ventricular failure in retinoid X receptor/ mice.
Proc. Natl. Acad. Sci. USA
92
,
7386
7390
Evans
R. M.
(
1988
)
The steroid and thyroid hormone receptor superfamily.
Science
240
,
889
895
Fishman
M.C.
,
Chien
K. R.
(
1997
)
Fashioning the vertebrate heart: the earliest embryonic decisions.
Development
124
,
2099
2117
Gobel
H.H.
,
Heipertz
R.
,
Scholz
W.
,
Iqbal
K.
,
Tellez-Nagel
I.
(
1978
)
Juvenile Huntington chorea: clinical, ultrastructural and biochemical studies.
Neurology
28
,
23
31
Green
S.
,
Chambon
P.
(
1988
)
Nuclear receptors enhance our understanding of transcription regulation.
Trends Genet
4
,
309
314
Gruber
P. J.
,
Kubalak
S. W.
,
Pexieder
T.
,
Sucov
H. M.
,
Evans
R. M.
,
Chien
K. R.
(
1996
)
RXRdeficiency confers susceptibility for aortic sac, conotruncal, atrioventricularcushion, and ventricular muscle defects in mice.
J. Clin. Invest
98
,
1332
1343
Gulick
T.
,
Cresci
S.
,
Caira
T.
,
Moore
D. D.
,
Kelly
D. P.
(
1991
)
The peroxisome proliferator-activated receptor regulates mitochondrial fatty acid oxidative enzyme gene expression.
Proc. Natl. Acad. Sci. USA
91
,
11012
11016
Jaber
M.
,
Koch
W.J.
,
Rockman
H.A.
,
Smith
B.
,
Bood
R.A.
,
Sulik
K.
,
Ross
J.
Jr
,
Lefkowitz
R.J.
,
Caron
M.C.
,
Giros
B.
(
1996
)
Essential role of beta adrenergic receptor kinase in cardiac development and function.
Proc. Natl. Acad. Sci. USA
93
,
12974
12979
Jiang
Y.
,
Evans
T.
(
1996
)
The Xenopus GATA-4,5,6 genes are associated with cardiac specification and can regulate cardiac-specific transcription during embryogenesis.
Dev. Biol
174
,
258
270
Jonk
L. J.
,
Jonge
M. E.
,
Vervaat
J. M.
,
Wissink
S.
,
Kruijer
W.
(
1994
)
Isolation and developmental expression of retinoic-acid induced genes.
Dev. Biol
161
,
604
614
Kastner
P.
,
Grondona
J.
,
Mark
M.
,
Gansmuller
A.
,
LeMeur
M.
,
Decimo
D.
,
Vonesch
J. L.
,
Dolle
P.
,
Chambon
P.
(
1994
)
Genetic analysis ofRXRdevelopmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis.
Cell
78
,
987
1003
Kastner
P.
,
Mark
M.
,
Chambon
P.
(
1995
)
Nonsteroid nuclear receptors: What are the genetic studies telling us about their role in real life?.
Cell
83
,
859
869
Kastner
P.
,
Mark
M.
,
Ghysenlicck
N.
,
Krezel
W.
,
Dupe
V.
,
Grondona
J. M.
,
Chambon
P.
(
1997
)
Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development.
Development
124
,
313
326
Kelly
D.P.
,
Gordon
J.I.
,
Alperts
R.
,
Strauss
A.W.
(
1989
)
The tissue-specific expression and developmental regulation of two nuclear genes encoding rat mitochondrial proteins.
J. Biol. Chem
264
,
18921
18925
Kelly
D. P.
,
Strauss
A. W.
(
1994
)
Inherited cardiomyopathies.
New Engl. J. Med
330
,
913
919
Kreldberg
J. A.
,
Sartola
H.
,
Loring
J. M.
,
Maeda
M.
,
Pelleder
J.
,
Housman
D.
,
Jaenkseh
R.
(
1993
)
WT-1 is required for early kidney development.
Cell
74
,
679
691
Kuo
C. T.
,
Morrisey
E. E.
,
Anandappa
R.
,
Sigrist
K.
,
Lu
M. M.
,
Parmacek
M. S.
,
Soudais
C.
,
Leiden
J. M.
(
1997
)
GATA-4 transcription factor is required for ventral morphogenesis and heart tube formation.
Genes Dev
11
,
1048
1060
Lee
R.Y.
,
Luo
J.
,
Evans
R.M.
,
Giguere
V.
,
Sucov
H.M.
(
1997
)
Compartment-selective sensitivity of cardiovascular morphogenesis to combinations of retinoic acid receptor gene mutations.
Circulation Res
80
,
757
764
Leone
T. C.
,
Cresci
S.
,
Carter
M. E.
,
Zhang
Z.
,
Lala
D. S.
,
Strauss
A. W.
,
Kelly
D. P.
(
1995
)
The human medium chain Acyl-CoA dehydrogenase gene promoter consists of a complex arrangement of nuclear receptor response elements and Sp1 binding sites.
J. Biol. Chem
270
,
16308
16314
Lints
T. J.
,
Parsons
L. M.
,
Hartley
L.
,
Lyons
I.
,
Harvey
R. P.
(
1993
).
Nkx2.5, a novel murine homeobox expressed in early heart progenitor cells and their myogenic descendants.
Development
119
,
419
431
Lok
S.
,
Kuijper
J. L.
,
Jelinek
L. J.
,
Kramer
J. M.
,
Whitmore
T. E.
,
Sprecher
C. A.
,
Mathewes
S.
,
Grant
F. J.
,
Biggs
S. H.
,
Rosenberg
G. B.
(
1994
)
The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization.
Gene
140
,
203
209
Lyons
L.
,
Parsons
L. M.
,
Hartley
L.
,
Andrews
J. E.
,
Robb
L.
,
Harvey
R. P.
(
1995
).
Myogenic and morphogenic defects in the heart tubes of murine embryos lacking the homeobox gene Nkx2.5.
Genes Dev
9
,
1654
1656
Lyons
G. E.
,
Micales
B. K.
,
Schwarz
J.
,
Martin
J. F.
,
Olson
E. N.
(
1995
)
Expression of Mef2 genes in the mouse central nervous system suggests a role in neuronal maturation.
J. Neurosci
15
,
5727
5738
MacNeil
D. J.
,
Occi
J. L.
,
Hey
P. J.
,
Strader
C. D.
,
Graziano
M. P.
(
1994
)
Cloning and expression of a human glucagon receptor.
Biochem. Biophys. Res. Commun
198
,
328
334
Mangelsdorf
D. J.
,
Thummel
C.
,
Beato
M.
,
Herrlich
P.
,
Shutz
G.
,
Umesono
K.
,
Blumberg
B.
,
Kastner
P.
,
Mark
M.
,
Chambon
P.
,
Evans
R. M.
(
1996
)
The nuclear receptor superfamily: The second decade.
Cell
83
,
835
839
Moens
C.B.
,
Stanton
B.R.
,
Parada
L.F.
,
Rossant
J.
(
1993
)
Defects in heart and lung development in compound heterozygotes for two different targeted mutations at the N-myc locus.
Development
119
,
485
499
Molkentin
J. D.
,
Lin
Q.
,
Duncan
S. A.
,
Olson
E. N.
(
1997
)
Requirement of the trancription factor GATA-4 for heart tube formation and ventral morphogenesis.
Genes Dev
11
,
1061
1072
Murphy
A. M.
,
Thompson
W. R.
,
Peng
L. F.
,
Jones
L.
(
1997
)
Regulation of the rat cardiac troponin I gene by the transcription factor GATA-4.
Biochem. J
322
,
393
401
Neely
J. R.
,
Rovetto
M. J.
,
Oram
F. J.
(
1972
)
Myocardial utilization of carbohydrate and lipids.
Prog. Cardiovasc. Dis
15
,
289
329
Neubauer
S.
,
Horn
M.
,
Cramer
M.
,
Harre
K.
,
Newell
J.B.
,
Peters
W.
,
Pabst
T.
,
Ertl
G.
,
Hahn
D.
,
Ingwall
J.S.
,
Kochsiek
K.
(
1997
)
Myocardial phosphocreatine-to-ATP ratio is a predictor of mortality in patients with dilated cardiomyopathy.
Circulation
96
,
2190
2196
Noack
K.
,
Zardoya
R.
,
Meyer
A.
(
1996
)
The complete mitochondrial DNA sequence of the bichir (Polypterus ornatipinnis), a basal ray-finned fish: ancient establishment of the consensus vertebrate gene order.
Genetics
144
,
1165
1180
Pilkington
S. J.
,
Arizmendi
J. M.
,
Fearnley
I. M.
,
Runswich
M. J.
,
Skehel
J. M.
,
Walker
J. E.
(
1993
)
Structural organization of complex I from bovine mitochondria.
Biochem. Society Transaction
21
,
26
31
Poole
D. C.
,
Mathieu-Costello
O.
(
1996
)
Relationship between fiber capillarization and mitochondrial volume density in control and trained rat soleus and plantaris muscles.
Microcirculation
3
,
175
186
Rossant
J.
(
1996
)
Mouse mutants and cardiac development: new molecular insights into cardiogenesis.
Circ. Res
78
,
349
353
Srivastava
D.
,
Cerjesi
P.
,
Olson
E.N.
(
1995
)
New subclass of bHLH proteins required for cardiac morphogenesis.
Science
270
,
1995
1999
Srivastava
D.
,
Thomas
T.
,
Lin
Q.
,
Kirby
M.L.
,
Brown
D.
,
Olson
E.N.
(
1997
)
Regulation of cardiac mesodermal and neural crest development by the HLH transcription factor dHAND.
Nat. Genetics
16
,
154
160
Steeghs
K.
,
Benders
A.
,
Oelermans
F.
,
de Haan
A.
,
Heerschap
A.
,
Ruitenbeck
W.
,
Jost
C.
,
vanDeursen
J.
,
Peryman
B.
,
Pette
D.
, et al. 
(
1997
)
Altered Ca2+responses in muscles with combined mitochondrial and cytosolic creatine kinase deficiencies.
Cell
89
,
93
103
Sucov
H. M.
,
Dyson
E.
,
Gumeringer
C. L.
,
Price
J.
,
Chien
K. R.
,
Evans
R. M.
(
1994
)
RXRmutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis.
Genes Dev
8
,
1007
1018
Usui
H.
,
Falk
J. D.
,
Dopazo
A.
,
deLecea
L.
,
Erlander
M. G.
,
Sutcliffe
J. G.
(
1994
)
Isolation of clones of rat striatum-specific mRNAs by directional tag PCR subtraction.
J. Neurosci
14
,
4915
4926
Walker
J.E.
,
Skenel
J.M.
,
Buchanan
S.K.
(
1995
)
Structural analysis of NADH;ubiquinone oxidoreductase from bovine heart mitochondria.
Methods Enzymol
260
,
14
34
Wilson
J. G.
,
Warkany
J.
(
1949
)
Aortic-arch and cardiac abnormalities in the offspring of vitamin A deficient rats.
Am. J. Anat
85
,
113
155
Zhou
Q. Y.
,
Quaife
C. J.
,
Palmiter
R. D.
(
1995
)
Targeted disruption of the tyrosine hydroxylase gene reveals that catecholamines are required for mouse fetal development.
Nature
374
,
640
643
Zou
Y.
,
Evans
S.
,
Chen
J.
,
Kuo
H.-C.
,
Harvey
R. P.
,
Chien
K. R.
(
1997
)
CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2-5 homeobox gene pathway.
Development
124
,
793
804
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