First published online 2 December 2004
doi: 10.1242/dev.01562
Development 132, 189-201 (2005)
Published by The Company of Biologists 2005
The Hand1 and Hand2 transcription factors regulate expansion of the embryonic cardiac ventricles in a gene dosage-dependent manner
David G. McFadden1,*,
Ana C. Barbosa1,*,
James A. Richardson2,
Michael D. Schneider3,
Deepak Srivastava1,4 and
Eric N. Olson1,
1 Department of Molecular Biology, University of Texas Southwestern Medical
Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA
2 Department of Pathology, University of Texas Southwestern Medical Center, 6000
Harry Hines Boulevard, Dallas, TX 75390-9148, USA
3 Center for Cardiovascular Development, Department of Medicine, Molecular and
Cellular Biology, and Molecular Physiology and Biophysics, Baylor College of
Medicine, Houston, TX 77030-3498, USA
4 Department of Pediatrics, University of Texas Southwestern Medical Center,
6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA
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Fig. 3. Generation of Nkx2.5::Cre mice. (A,B) X-gal stained embryos
showing staining in the linear heart tube (arrowhead) at E8.0 (A) and
throughout the ventricular myocardium at E8.5 (B). (C,D) Nuclear Fast Red
counterstained serial sections through the embryo in B. There are high levels
of ß-gal staining throughout the ventricular myocardium and in a subset
of endocardial cells. (E) X-gal stained E12.5 embryo showing efficient
ventricular recombination and minimal recombination in the outflow tract (oft)
and atria (a). lv, left ventricle; rv, right ventricle; la, left atria; ra,
right atria.
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Fig. 4. Abnormal cardiac morphogenesis in E10.5 embryos with compound mutations in
Hand1 and Hand2. (A-F) E10.5 embryos stained for
lacZ activity expressed from the Hand1lacZ
allele. Left lateral view (A-C); frontal view (D-F). (G-I) Nuclear Fast Red
counterstained transverse sections of the hearts of embryos in A-F. Genotypes
of embryos are shown above each set of panels. There is severe reduction in
Hand1-expressing cells in the LV and the hypoplastic ventricular
chambers in H and I, and an absence of the interventricular groove and
ventricular septation in I. lv, left ventricle; rv, right ventricle.
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Fig. 5. Abnormal cardiac morphogenesis in E9.0 embryos with compound mutations in
Hand1 and Hand2. (A-L) E9.0 embryos stained for
lacZ activity expressed from the Hand1lacZ
allele. Left lateral view (A-F); frontal view of the heart (G-L). a, atrium;
lv, left ventricle; rv, right ventricle; v, ventricular chamber. The asterisk
in F shows the thin outflow tract present in the Hand1 cardiac-KO/KO;
Hand2 KO/KO embryos. (M-X) Nuclear Fast Red counterstained transverse
sections of embryos in A-L at anterior (M-R) and middle (S-X) levels of the
heart. Genotypes of embryos are shown above each set of panels. Cardiac
abnormalities increase in severity from left to right panels.
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Fig. 6. Dysregulation of cardiac genes in Hand1 and Hand2 mutant
embryos. (A) Transcripts for Anf, Mlc2v, Cited1, connexin 40 (Cx40)
andTbx5 were detected by whole-mount in situ hybridization on
wild-type and Hand1 cardiac-KO/KO embryos at E10.5. The reduction in
Cited1 expression in the LV and the upregulation of Anf in
the RV of mutant embryos is shown. lb, limb bud; lv, left ventricle; rv, right
ventricle. (B) Transcripts for Anf, connexin 40 and Tbx5
were detected by whole-mount in situ hybridization on wild-type and
Hand1 cardiac-KO/KO; Hand2 KO/+ embryos at E9.5.
Anf expression is absent in the LV of the mutant embryo (arrows).
Expression of Cx40 in the LV of the mutant is also lost (arrows), but
normal levels of expression are maintained in the dorsal aorta and
vasculature. Expression of Tbx5 is unaffected in the heart of the
mutant (arrows).
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Fig. 7. Expression of Mlc2v and Tbx5 in embryos with compound
mutations in Hand1 and Hand2. Expression of (A)
Mlc2v and (B) Tbx5 was examined by in situ hybridization to
transverse or sagittal sections of E9.0 embryos of the indicated genotypes.
Silver grains are pseudocolored in red. a, atrium; lv, left ventricle; v,
ventricular chamber.
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Fig. 8. The roles of Hand genes in ventricular development. Knockout mice lacking
Hand2 have demonstrated the essential role of this gene in formation
of the RV. Knockout mice lacking Nkx2.5 show a loss of the LV and
downregulation of Hand1. Knockout mice lacking Hand1 show
relatively minor abnormalities in the LV, suggesting that the loss of
Hand1 alone is insufficient to account for the more severe LV defects
in Nkx2.5 mutant embryos. Knockout mice lacking both Hand1
and Hand2 show severe ventricular hypoplasia, suggesting redundant
functions of the Hand genes in the developing LV.
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© The Company of Biologists Ltd 2005
MHC promoter or
Nkx2.5 cardiac enhancer. Positions of the probe used for Southern
analysis and the primers (a and b) used for RT-PCR are shown. Coding regions
are shown in white and non-coding regions are shown in gray. (B) Detection of
all four Hand1 alleles by Southern blot analysis of
EcoRI-digested genomic DNA, using the 3' probe shown in A. (C)
