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
Author for correspondence (e-mail:
eric.olson{at}utsouthwestern.edu)
Accepted 1 November 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Mouse, Hand1, Hand2, Cardiac ventricles
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Heart development begins when mesodermal cells in a region of the embryo
known as the cardiac crescent become instructed to adopt a cardiac fate in
response to signals from adjacent tissues
(Marvin et al., 2001;
Schneider and Mercola, 2001
;
Schultheiss et al., 1997; Tzahor and
Lassar, 2001
) (reviewed by Olson, 2002). Cardiac precursors
proliferate and migrate to the embryonic midline to form a linear heart tube
that is segmentally patterned along its anteroposterior axis into regions
ultimately giving rise to the atrial and ventricular chambers. Rightward
looping of the linear heart tube followed by balloon-like growth of the outer
curvatures of the ventricular segments generates the right and left
ventricular chambers (Christoffels et al.,
2000
; Moorman et al.,
2000
). Notably, each cardiac chamber possesses distinct
physiological functions and patterns of gene expression.
Recent studies have revealed two populations of cardiac precursor cells
that contribute to different parts of the heart. The primary heart field is
thought to give rise to the atrial chambers and left ventricular region. A
second cardiogenic region, known as the anterior or secondary heart field,
lies anterior and dorsal to the linear heart tube. Cells from this region are
added to the developing heart tube and give rise to the outflow tract and
right ventricular region (Mjaatvedt et
al., 2001; Waldo et al.,
2001
) (reviewed by Kelly and
Buckingham, 2002
) (see also
Cai et al., 2003
). The
existence of these two distinct populations of cardiac progenitors provides a
potential explanation for many cardiac abnormalities in humans and model
organisms in which specific segments of the heart are underdeveloped or
deleted, leaving the remainder of the heart unaffected.
Several classes of transcription factors have been implicated in cardiac
morphogenesis and gene regulation (reviewed by
Bruneau, 2002;
Firulli and Thattaliyath,
2002
). Hand1 and Hand2 (also called eHAND/Thing-1/Hxt and
dHAND/Thing-2/Hed, respectively) are basic helix-loop-helix (bHLH)
transcription factors that display complimentary and overlapping expression
patterns in the developing heart (Cross et
al., 1995
; Hollenberg et al.,
1995
; Cserjesi et al.,
1995
; Srivastava et al.,
1995
). In mice, Hand2 is expressed throughout the linear
heart tube. Thereafter, its expression is highest in the developing right
ventricle (RV), with lower levels of expression in the atrial and left
ventricular chambers (Thomas et al.,
1998
). Targeted mutation of Hand2 in mice results in
lethality at embryonic day 10.5 (E10.5) from right ventricular hypoplasia and
vascular malformations (Srivastava et al.,
1997
; Yamagishi et al.,
2000
). By contrast, Hand1 is expressed in segments of the
linear heart tube destined to form the conotruncus and left ventricle (LV). At
the onset of cardiac looping, Hand1 expression becomes localized
primarily to the outer curvature of the LV and outflow tract, with lower
expression along the outer curvature of the developing RV
(Biben and Harvey, 1997
;
Thomas et al., 1998
). Mice
lacking Hand1 die at E8-8.5 from severe placental and extra-embryonic defects,
reflecting a role of Hand1 in trophoblast differentiation, and complicating
analysis of potential cardiac functions
(Firulli et al., 1998
;
Riley et al., 1998
).
Nevertheless, tetraploid aggregation experiments with wild-type and
Hand1 null embryonic stem (ES) cells have shown that mutant ES cells
fail to contribute to the LV of chimeric mouse embryos. Such embryos survive
until E10.5 when they exhibit abnormalities in cardiac looping
(Riley et al., 1998
;
Riley et al., 2000
).
Interpretation of the phenotype of such chimeric embryos is complicated by
possible extra-cardiac functions of Hand1 as well as the variable contribution
of Hand1 null cells to extra-embryonic tissues.
Studies in chick and zebrafish embryos have also revealed potential
functions of Hand genes in cardiac development. Exposure of chick embryos to
antisense oligonucleotides for Hand1 and Hand2 together, but not separately,
perturbs heart development at the looping stage, suggesting that these factors
act redundantly (Srivastava et al.,
1995). The zebrafish genome appears to encode only a single Hand
gene, most closely related to Hand2, and mutations in this gene in
hands off mutants, result in a dramatic reduction in ventricular
precursors (Yelon et al.,
2000
). This phenotype is more severe than that observed in
Hand2 knockout mice, possibly because of the lack of compensatory
activity of a second Hand gene in this organism.
To determine the function of Hand1 in mouse heart development
without complications from early lethality owing to extra-cardiac functions,
we generated mice harboring a conditional Hand1 allele flanked by Cre
recombinase loxP recognition sites, and deleted the gene specifically in the
developing heart using a cardiac-specific -myosin heavy chain
(
MHC) promoter, or the Nkx2.5 cardiac enhancer to express Cre
recombinase (Cre). Mice lacking myocardial expression of Hand1
survive until the perinatal period when they succumb to a spectrum of
congenital heart defects that reflect abnormalities in ventricular growth and
maturation. In addition, combination of the conditional Hand1
mutation with a Hand2 loss-of-function mutation revealed
dose-sensitive effects on heart development. These results identify novel
functions of Hand1 in heart development and demonstrate that Hand factors play
crucial and partially redundant roles in cardiac growth, morphogenesis and
gene expression.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The completed Hand1NEO-loxP targeting vector was linearized with NotI and electroporated into SM-1 ES cells. Following positive-negative selection with G418 and FIAU, resistant colonies were screened by Southern analysis of EcoRI digested genomic DNA using a probe (Fig. 1B) from the 3' flanking region. Recombination of the 5' arm was confirmed by EcoRI-KpnI double digestion of genomic DNA, and Southern blotting with the short arm of homology. Three correctly targeted clones (clones E12, C12 and C5) were expanded and injected into C57BL/6 blastocysts, and transferred into the uteri of pseudopregnant females. Chimeric males were bred onto a C57BL/6 or Black/Swiss background for germline transmission. Males from clone E12 transmitted the targeted allele through the germline, therefore mice derived from this line were used in all analyses.
|
Generation of Nkx2.5::Cre mice
A 2.5 kb fragment containing the Nkx2.5 basal promoter and cardiac
enhancer (Lien et al., 1999)
was cloned upstream of the NLS-Cre expression cassette (gift of J. Herz). This
vector was linearized using NotI and injected into fertilized oocytes
as previously described (McFadden et al.,
2000
). Founder transgenic mice were genotyped by hybridization of
EcoRV digested tail DNA to a Cre cDNA probe. Three
transgenic lines were obtained and intercrossed with ROSA26R
indicator mice (Soriano, 1999
)
in order to assess transgene expression and Cre-mediated recombination.
Transgenic line Nk9 exhibited the earliest and most efficient recombination,
and was used in all subsequent experiments.
PCR genotyping
Tail and yolk sac DNA was isolated as previously described
(McFadden et al., 2000). PCR
reactions were used to detect Cre transgenes, and Hand1 and
Hand2 knockout loci (Firulli et
al., 1998
; Srivastava et al.,
1997
). Briefly, 1 µl of tail or yolk sac DNA was used a
template in 25 µl PCR reactions using Promega Taq polymerase and 4
mM MgCl2. Thermal cycle reactions were as follows: 2 minutes at
95°C, 30 cycles of 30 seconds at 95°C, 30 seconds at 55°C, 45
seconds at 72°C and a final 5 minute extension at 72°C. Reactions were
visualized on 1% agarose gels in TAE. Primer sequences are available upon
request.
RT-PCR
Left ventricles from E9.5 embryos were dissected and immediately frozen and
stored in liquid nitrogen until embryo and yolk sac DNA was isolated and
genotyped. Left ventricular tissue from eight mutant hearts was pooled and
total RNA was isolated using Trizol reagent and standard protocols. Total LV
RNA (150 ng) was used as a template for first strand cDNA synthesis using the
Superscript first strand synthesis kit from Invitrogen. Five percent of the
cDNA synthesis reaction was used as template for PCR reactions using Promega
Taq polymerase to detect Hand1 transcripts. Transcripts for
hypoxanthine phosphoribosyl transferase (HPRT) were detected as a control.
Thermal cycles were as follows: 94°C for 2 minutes, 28 cycles of 94°C
30 for seconds, 52°C for 30 seconds, 72°C for 30 seconds and a final 5
minute extension at 72°C. Reactions were visualized on 1% agarose gels in
TAE. Primer sequences are available upon request.
Histology
Embryos were harvested from timed matings and fixed overnight in 4%
paraformaldehyde in phosphate-buffered saline (PBS). Following fixation,
embryos were rinsed in PBS then dehydrated through graded ethanols and
embedded in paraffin wax as previously described
(Moller and Moller, 1994).
Histological sections were cut and stained with Hematoxylin and Eosin, or
nuclear Fast Red as previously described
(Moller and Moller, 1994
).
In situ hybridization
Section in situ hybridization was performed as described
(Shelton et al., 2000).
Whole-mount in situ hybridization was performed as previously described
(Riddle et al., 1993
).
Plasmids for in situ probes have been previously described and were linearized
and transcribed as follows: ANF, XhoI and T7
(Miller-Hance et al., 1993
);
connexin 40, Asp718 and T3
(Haefliger et al., 1992
);
Tbx5, SpeI and T7 (Bruneau et
al., 1999
); and mlc2V, BamHI and T7
(O'Brien et al., 1993
). The
coding regions of cited1 and cited2 were amplified as
ClaI-EcoRI fragments and subcloned into pBSK. Both plasmids
were linearized with XhoI and transcribed with T3 RNA polymerase.
Hand2- and Hand1-coding regions were amplified as
EcoRI-XbaI fragments and cloned into pBSK. Both plasmids
were linearized with EcoRI and transcribed with T7.
ß-Galactosidase staining
Embryos from timed matings were harvested and pre-fixed for 1-3 hours in 2%
paraformaldehyde, 0.25% glutaraldehyde in PBS. Staining for ß-gal
activity was performed as previously described
(McFadden et al., 2000).
TUNEL and immunohistochemistry
TUNEL staining was performed on paraffin wax embedded sections from E10.5
and E13.5 according to the Promega Fluorescein Apoptosis detection kit.
Embryos were harvested at E11.5 and fixed overnight in 4% paraformaldehyde
in PBS. Embryos were rinsed in PBS and equilibrated into 10% sucrose for 2
hours, followed by 30% sucrose overnight at 4°C. Embryos were transferred
into freezing medium and frozen in isopentane and liquid nitrogen. Blocks were
equilibrated to -20°C and serially sectioned. Sections were stored at
-80°C until antibody staining. Antibody staining was performed as
described (Frey et al., 2000).
Primary anti-phospho histone H3 antibody was diluted 1:200 in 1% BSA in
PBS.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In order to determine if the Hand1loxP allele might
function as a hypomorphic allele because of reduced expression, we bred mice
heterozygous for this allele with mice heterozygous for the
Hand1-null allele, referred to as Hand1lacZ,
described previously (Firulli et al.,
1998). Adult trans-heterozygous Hand1lacZ/loxP
mice bearing the two mutant alleles were overtly normal and fertile,
suggesting that expression from the Hand1loxP allele was
not significantly reduced. Homozygous Hand1loxP/loxP mice
were also phenotypically normal and fertile, and were used in subsequent
breedings.
Early embryonic recombination in MHC::Cre mice
In order to delete Hand1 specifically in the heart, we crossed
Hand1loxP/loxP females to Hand1lacZ/+
heterozygous male mice harboring a transgene that expresses Cre under
the control of the -myosin heavy chain (
MHC)
promoter. These mice have been reported to express Cre in the
embryonic and adult myocardium (Agah et
al., 1997
; Gaussin et al.,
2002
). In order to define precisely the onset of Cre-mediated
recombination during embryogenesis, we intercrossed
MHC::Cre
transgenic mice to ROSA26R indicator mice, which harbor a conditional
lacZ allele that requires Cre-mediated recombination for expression
(Soriano, 1999
).
ß-Galactosidase (ß-gal) activity from the ROSA26 locus was
detected as early as E8.5 and by E9.0 expression was detected throughout the
embryonic myocardium as well as in the outflow tract
(Fig. 1C). At E11.5, both
atrial and ventricular myocardium expressed high levels of lacZ. We
did not detect lacZ expression elsewhere in these embryos.
To confirm that the Hand1loxP allele was efficiently
recombined in vivo, we analyzed Hand1 expression in
MHC::Cre; Hand1loxP/lacZ embryos at E10.5 by
whole-mount in situ hybridization. As shown in
Fig. 1D, Hand1
transcripts were specifically absent from the embryonic heart of these
embryos, whereas expression of Hand1 was not affected in the
branchial arches and lateral mesoderm. The absence of Hand1
transcripts in the LV by E9.5 was also confirmed by RT-PCR of cardiac RNA from
MHC::Cre; Hand1loxP/lacZ embryos
(Fig. 1E). These results
demonstrated that efficient cardiac-specific removal of Hand1
transcripts occurred before E9.5.
Congenital heart defects resulting from cardiac deletion of Hand1
Genotyping of litters from intercrosses of
Hand1loxP/loxP to MHC::Cre;
Hand1lacZ/+ mice revealed that offspring with the
MHC::Cre; Hand1loxP/lacZ genotype were born at
Mendelian ratios (data not shown), but the majority became cyanotic and died
within 3 days of birth. This suggested that loss of Hand1 in the embryonic
myocardium resulted in perinatal lethality. Newborn mice of the
MHC::Cre; Hand1loxP/lacZ genotype nursed
normally. At postnatal day 10 (P10), only 4% of offspring had the
MHC::Cre; Hand1loxP/lacZ genotype
(Table 1). Only three mice of
the
MHC::Cre; Hand1loxP/lacZ genotype survived to
adulthood (representing less than 2% of offspring from the above intercross,
or less than 1/10 of the predicted number of such mice).
|
|
Cardiac defects generated with an Nkx2.5::Cre transgene
Expression of Hand1 is initially detected in the cardiac crescent
at E7.75 (Cserjesi et al.,
1995). Because the
MHC::Cre transgene does not
direct high levels of Cre expression until after E8.5, it is possible
that Hand1 has an important role earlier in cardiac development, in
which case the cardiac phenotype we observed in
MHC::Cre;
Hand1loxP/lacZ mice could reflect the incomplete deletion of
Hand1 during early cardiogenesis. In an effort to eliminate cardiac
Hand1 expression at an earlier stage, we expressed Cre recombinase
fused to a nuclear localization signal (NLS) under control of Nkx2.5
regulatory sequences, which direct expression throughout the heart tube from
the onset of cardiac commitment (Lien et
al., 1999
; Reecy et al.,
1999
). By E8.5, activity of these Nkx2.5 regulatory sequences is
restricted to the developing ventricles and outflow tract. These sequences
also direct expression in the thyroid primordium, and regions of the pharynx
where Hand1 is not expressed.
We generated six Nkx2.5::Cre transgenic lines, and crossed three
lines into the ROSA26R heterozygous background to examine the
efficiency and tissue-specificity of Cre-mediated recombination. All three
lines exhibited a similar pattern of ß-gal expression, which included
heart, pharynx and a subset of cells within the liver (data not shown). We
used a transgenic line (line 9) that directed the highest levels of
recombination in the heart for all subsequent experiments. Efficient
recombination was detected within the myocardium of Nkx2.5::Cre
transgenic mice at the linear heart tube stage, and recombination occurred
throughout the heart tube by E8.5 (Fig.
3A,B). Serial sections through stained E8.5 embryos revealed that
the majority of cells in the right and left ventricular myocardium underwent
recombination. At E10.5, the majority of cells within the LV were ß-gal
positive; however, some cells failed to express lacZ, presumably
owing to a lack of Cre expression (data not shown). This may reflect
downregulation of the Nkx2.5 cardiac enhancer in the LV at later
stages of cardiac development (Lien et
al., 1999) or mosaicism of transgene expression. At E12.5, high
efficiency of Cre-mediated recombination was observed in the RV and LV.
Interestingly, the outflow tract failed to undergo recombination
(Fig. 3E), which may reflect
contribution of a secondary heart field not derived from the cardiac crescent
to the outflow tract myocardium (Kelly et
al., 2001
; Waldo et al.,
2001
).
|
Dose-sensitive requirements of Hand1 and Hand2 for left ventricular growth
We next addressed the possibility that Hand1 and Hand2
act in a functionally redundant fashion during cardiac development. In
contrast to Hand1, which is expressed specifically in the developing
LV and conotruncus, Hand2 is expressed throughout the atrial and
ventricular myocardium with highest levels of expression in the RV
(Thomas et al., 1998). In
Hand2 mutant embryos, the RV is hypoplastic, but the LV forms, albeit
with fewer trabeculations (Srivastava et
al., 1995
). Because of their overlapping expression in the LV, it
is possible that Hand2 may compensate for loss of Hand1 in
this region of the developing heart. To address this possibility, we reduced
the level of Hand2 expression by generating Hand1
cardiac-KO/KO; Hand2 KO/+ embryos from timed matings. Whereas
Hand1 cardiac-KO/KO mice survived until birth and Hand2 KO/+
mice are normal, no embryos of the combined genotype were observed in litters
harvested after E10.5. At E10.5, Hand1 cardiac-KO/KO; Hand2
KO/+ embryos were observed at Mendelian ratios, but appeared slightly delayed
relative to Hand1 cardiac-KO/KO littermates. Expression of
lacZ from the Hand1lacZ allele was decreased in
the LVs of Hand1 cardiac-KO/KO embryos, and was further decreased in
Hand1 cardiac-KO/KO; Hand2 KO/+ embryos, suggesting that
expansion of the left ventricular chamber was perturbed by the loss of Hand
genes in a dose-sensitive manner (Fig.
4). Histological sectioning of Hand1 cardiac-KO/KO;
Hand2 KO/+ embryos revealed a thin and poorly trabeculated myocardium
(Fig. 4I). Thus, removal of one
Hand2 allele in the absence of Hand1 generates an embryonic
lethal defect in cardiac growth.
|
|
Dysregulation of ventricular gene expression in the absence of Hand genes
Several genes are expressed specifically along the outer curvature of the
embryonic ventricles in patterns partially overlapping that of Hand1.
Upregulation of these genes is thought to reflect the expansion of the chamber
myocardium from the ventral surface of the more primitive linear heart tube
myocardium (Christoffels et al.,
2000). Nkx2.5-null embryos die at E10.5 from LV defects
and fail to express Hand1 in the heart, which has led to the
suggestion that Hand1 might act as an important downstream mediator of Nkx2.5
function during cardiac morphogenesis
(Biben and Harvey, 1997
;
Lyons et al., 1995
;
Tanaka et al., 1999
). Thus, we
examined expression of potential Nkx2.5 target genes and other markers of
chamber myocardium in Hand mutant embryos to determine whether they were
sensitive to the level of Hand gene expression.
Atrial natriuretic factor (Anf; Nppa - Mouse Genome
Informatics) is expressed in the embryonic LV in a pattern similar to that of
Hand1 and is downregulated in the hearts of Nkx2.5-null
embryos (Lyons et al., 1995).
Expression of ANF in the LV was slightly reduced in hearts from
Hand1 cardiac-KO/KO embryos, but was dramatically upregulated in the
RV of these mutant embryos (Fig.
6A). As Hand1 is not expressed in the RV of wild-type
embryos, we believe the upregulation of Anf expression in the RV of
the mutant may reflect a secondary response, perhaps to cardiac stress.
Expression of Anf was completely absent in the ventricles of both
Hand1 cardiac-KO/KO; Hand2 KO/+
(Fig. 6B) and Hand1
cardiac-KO/KO; Hand2 KO/KO (data not shown) embryos. Thus, left
ventricular expression of Anf displayed a dose-dependency on the
level of Hand1/2 expression, but was not absolutely dependent on
Hand1.
|
|
Connexin 40 (Cx40; Gja5 - Mouse Genome Informatics),
connexin 43 (Cx43; Gja1 - Mouse Genome Informatics),
sarcoplasmic reticulum Ca2+ ATPase (Serca2a) and
Tbx5 also show LV-restricted expression patterns. The expression of
Serca2a was unaffected in Hand1 cardiac-KO/KO;
Hand2 KO/+ embryos (data not shown). By contrast, expression of
Cx40 was slightly decreased in Hand1 cardiac KO/KO embryos
and was completely absent from the ventricular myocardium in Hand1
cardiac-KO/KO; Hand2 KO/+ embryos
(Fig. 6A,B). It is notable in
this regard that mice lacking Tbx5 also fail to express Anf
and Cx40 (Bruneau et al.,
2001). Therefore, to determine if Hand1 and
Hand2 might act genetically upstream of Tbx5, we examined
expression of Tbx5 in embryos lacking combinations of the Hand genes
and found it to be unaffected (Fig.
6, Fig. 7B). We
conclude that Hand1 and Hand2 regulate Cx40 and Anf
expression through mechanisms independent of Tbx5 transcription.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Hand1 expression is excluded from all but a small subpopulation of
septal myocytes, suggesting that it plays a non-cell autonomous role in
definition of the septal boundary, or that septal defects are secondary to
abnormal growth and morphogenesis of the LV. A recent study provides evidence
that Hand1 is indeed an important regulator of the interventricular boundary
(Togi et al., 2004). Using
homologous recombination, Hand1 was targeted to the Mlc2v
locus, which is expressed throughout the ventricular and septal myocardium.
Hand1 knock-in mice died at midgestation and completely lacked an
interventricular septum. Thus, overexpression of Hand1 eliminates the
interventricular septum, while cardiac deletion of Hand1 causes expansion of
this region of the heart.
Valve defects resulting from cardiac Hand1 deletion
Embryos that lack cardiac expression of Hand1 also displayed
abnormally thickened AV valves and hyperplastic endocardial cushions. The
endocardial cushion malformations in these mutant mice are interesting because
the MHC::Cre transgene does not direct Cre expression
in the endocardial cushions or cardiac valves
(Agah et al., 1997
;
Gaussin et al., 2002
). This
suggests that Hand1 regulates a myocardium-derived signal that controls
endocardial cushion morphogenesis. Crosstalk between the myocardium and
endocardium is well documented and has been shown to involve BMP- and
TGFß-mediated signals (Brown et al.,
1999
; Eisenberg and Markwald,
1995
; Gaussin et al.,
2002
; Kim et al.,
2001
). Mice that lack TGFß2 or Smad6, a BMP effector, also
exhibit hyperplastic cardiac valves
(Galvin et al., 2000
;
Bartram et al., 2001
). However,
Bmp2, Bmp4, Smad6, Smad7 and Tgfb expression is unaltered in
Hand1 mutants (data not shown), suggesting that Hand1 regulates other
signaling pathways. It should also be noted that early myocardial function
profoundly influences endocardial cushion development
(Bartman et al., 2004
). Thus,
abnormalities in cardiac contractility or morphogenesis in embryos lacking
cardiac Hand1 expression could indirectly influence the formation of
cardiac cushions.
Redundancy of mammalian Hand genes
The cardiac phenotype resulting from cardiac deletion of Hand1 was
much less severe than that of Hand2 mutant embryos in which the
entire right ventricular region of the heart is absent
(Srivastava et al., 1997).
These differences in ventricular phenotypes are likely to reflect important
distinctions in the expression patterns of Hand1 and Hand2.
Hand1 is expressed specifically in the outer curvatures of the embryonic
LV, RV and outflow tract. By contrast, Hand2 is expressed throughout
the left and right ventricular chambers, although its expression is highest in
the RV (Srivastava et al.,
1997
; Biben and Harvey,
1997
). Therefore, in the absence of Hand1, residual
Hand2 expression in the LV and outflow tract may partially compensate
for the loss of Hand1. By contrast, in the absence of Hand2
there is a more complete lack of Hand factors in the presumptive RV
(Srivastava et al., 1997
).
The genome of the zebrafish encodes a single Hand gene, and
loss-of-function mutations (hands off) result in a near complete
absence of ventricular precursors. This has led to the speculation that mouse
Hand1 and Hand2 act redundantly during ventricular
differentiation (Yelon et al.,
2000). Our results are consistent with this notion. Removal of one
copy of the Hand2 gene in the setting of cardiac-specific deletion of
Hand1 exacerbated the Hand1 cardiac phenotype and resulted
in embryonic lethality at midgestation; removal of both Hand2 genes
caused severe ventricular hypoplasia and lethality at yet an earlier
developmental stage. However, the cardiac phenotype of our Hand1
cardiac KO/KO; Hand2 KO/KO is less severe than that of zebrafish
hands off mutants. Although these results must be interpreted in
light of the technical limitations of Cre-mediated gene deletion, this may
also suggest that mammalian Hand genes have acquired unique functions during
the evolution of the four-chambered heart.
Hand genes regulate ventricular expansion
The morphogenesis of the single-chamber embryonic heart into the adult
four-chamber heart is crucially dependent on the expansion, or `ballooning' of
the outer curvature of the right and left ventricular chambers
(Christoffels et al., 2000).
Expression of Hand1 along the outer curvature of the LV and outflow
tract is consistent with a role in the expansion of chamber myocardium. The
expression patterns of several genes mark the expansion of this chamber
myocardium from the outer curvatures of the ventricles
(Christoffels et al., 2000
).
We unexpectedly found that ventricular expansion and gene expression was
strictly dependent on the gene dose of Hand1 and Hand2. In
Hand1 cardiac KO/KO; Hand2 KO/+ embryos, expression of
connexin 40 and Anf, both of which mark the outer curvature of the
LV, was abolished. In addition, the ventricles failed to expand
morphologically, and the embryos died at midgestation. Ventricular fate is
specified in these embryos as evidenced by robust Mlc2v expression;
in addition, the expression of Tbx5 indicates that cardiac chamber
specification occurred. These findings suggest that the ventricular expansion
program is abnormal in these embryos. In addition, Hand1 cardiac
KO/KO; Hand2 KO/KO embryos display an even more severe ventricular
phenotype, forming only a single immature ventricle presenting abnormal
cellular morphology in some segments of the ventricular myocardium. The
exquisite sensitivity of Hand1 mutant hearts to Hand2 gene
dose underscores the crucial role of these genes during ventricular
expansion.
What mechanism(s) might account for the severe ventricular hypoplasia seen
in mice lacking cardiac expression of Hand1 and Hand2?
Because we did not detect significant differences in apoptosis or
proliferation of ventricular myocytes in wild-type and double Hand
mutant embryos, it is unlikely that abnormalities in these events account for
the severe deficiency of ventricular myocytes in the mutant. Therefore, we
speculate that the absence of both Hand genes results in a deficiency in
specification of cardiac myocytes at an early stage of cardiogenesis. A
similar mechanism has been proposed to account for the lack of cardiomyocytes
in the hands off mutant (Yelon et
al., 2000).
The expression pattern of Hand1 in the developing heart is nearly
identical to that of Cited1, which encodes a transcriptional
co-activator (Biben and Harvey,
1997; Dunwoodie et al.,
1998
). The downregulation of Cited1 in Hand1 mutant
embryos suggests that Hand1 acts upstream of Cited1 during
cardiac development. It is notable, in this regard, that cited1 and Hand1 are
also co-expressed in trophoblastic tissues of the placenta, and mice lacking
either gene display lethal defects in placental development
(Rodriguez et al., 2004
;
Riley et al., 1998
;
Firulli et al., 1998
).
Analysis of heart morphology has not been described in Cited1 mutants
(Rodriguez et al., 2004
), but
mice lacking Cited2 display numerous congenital heart defects that
overlap with those observed in Hand1 cardiac KO animals
(Bamforth et al., 2001
).
However, we detected no difference in Cited2 expression between
wild-type and Hand1 mutant mice (data not shown). It remains possible
that subtle cardiac defects are indeed present in Cited1-null mice,
or differences in genetic background between Hand1 and
Cited1 mutant mouse lines account for the lack of obvious cardiac
defects in Cited1-null mice. Regardless, it is likely that downstream
targets in addition to Cited1 contribute to the heart defects
observed in Hand1 cardiac KO animals.
The relationship between Hand1 and Nkx2.5
In mice lacking Nkx2.5, the left ventricular chamber fails to
expand following cardiac looping, and expression of several markers of cardiac
differentiation is reduced throughout the remaining myocardium
(Lyons et al., 1995;
Tanaka et al., 1999
;
Yamagishi et al., 2001
).
Interestingly, Hand1 expression is abolished in the hearts of
Nkx2.5 mutant embryos (Biben and
Harvey, 1997
). Therefore, it has been proposed that loss of Hand1
contributes to abnormal cardiac morphogenesis of Nkx2.5 mutant hearts
(see Fig. 8). The data
presented here suggest that lack of Hand1 is not solely responsible for left
ventricular hypoplasia in Nkx2.5-null mice. First, loss of
Hand1 expression in the early heart tube results in only a modest and
transient decrease in size of the left ventricular chamber in the embryo, much
less severe than in Nkx2.5 mutants. Second, in contrast to
Nkx2.5 null mice, Anf and Mlc2v are expressed at
high levels in the LV of Hand1 mutants. Both Anf and
Mlc2v are also normally expressed in
Hand1lacZ/lacZ null mice
(Firulli et al., 1998
),
demonstrating that expression of these markers in Hand1 conditional
knockout mice is not due to inefficient or delayed excision of Hand1.
In addition, markers of the left ventricular chamber, including Tbx5
and Cx40, are expressed normally in the absence of Hand1.
We previously showed that mice deficient in Nkx2.5 and Hand2 formed only a
single cardiac chamber, molecularly defined as the atrium
(Yamagishi et al., 2001). By
contrast, in the absence of cardiac Hand expression, ventricular
cardiomyocytes are evident as shown by expression of Mlc2v, however
ballooning of the ventricular chamber is abrogated. The less severe phenotype
of the Hand1/Hand2 mutant again suggests that Nkx2.5
regulates genes in addition to Hand1 and that the lack of
Hand1 expression is only partially responsible for cardiac defects
and embryonic lethality observed in Nkx2.5-null mice.
Contributions of primary and secondary heart fields to the developing heart
It is interesting to consider the roles of Hand1 and Hand2 in the context
of the contributions of the primary and secondary heart fields to the
developing heart. The primary heart field is believed to give rise to the
atria and left ventricular chambers, while the secondary (or anterior) heart
field contributes primarily to the outflow tract and right ventricular region
of the heart (reviewed by Kelly et al.,
2001; Kelly and Buckingham,
2002
; Abu-Issa et al.,
2004
) (see also Cai et al.,
2003
). The absence of Hand2 results in the deletion of the right
ventricular regions of the heart
(Srivastava et al., 1997
),
suggesting that it is an essential component of the pathway for development of
the secondary heart field. The cardiac phenotype of Nkx2.5 mutant
embryos is complementary to that of Hand2 mutants, i.e. a lack of the
left ventricular region (Lyons et al.,
1995
; Tanaka et al.,
1999
). Consistent with the idea that Hand2 and Nkx2.5 regulate
growth of complementary regions of the heart, mice lacking both genes form
only a primitive cardiac rudiment
(Yamagishi et al., 2001
). It
is notable that the zebrafish heart has only a single ventricle and no
evidence of a secondary heart field. The presence of only a single Hand gene
in that organism raises the possibility that a primordial Hand gene may have
been duplicated during evolution such that each Hand gene fell under separate
temporal and spatial control as a means of generating two heart fields that
contribute to the different ventricular chambers.
Implications
The types of cardiac abnormalities observed in mice lacking either Hand1 or
Hand2 are reminiscent of congenital heart defects in humans. Most congenital
heart defects in humans that have been linked to mutations in specific genes
represent haploinsufficiency often influenced by genetic or environmental
modifiers. By contrast, most cardiac defects studied in the mouse arise owing
to homozygous gene deletion. Given the diversity of cardiac abnormalities that
can result from Hand gene mutations (ventricular hypoplasia, VSDs,
valve defects, outflow tract abnormalities), we anticipate that the Hand genes
will prove to be crucial for heart development and congenital heart disease in
humans.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abu-Issa, R., Waldo, K. and Kirby, M. L. (2004). Heart fields: one, two or more? Dev. Biol. 272,281 -285.[CrossRef][Medline]
Agah, R., Frenkel, P. A., French, B. A., Michael, L. H.,
Overbeek, P. A. and Schneider, M. D. (1997). Gene
recombination in postmitotic cells. Targeted expression of Cre recombinase
provokes cardiac-restricted, site-specific rearrangement in adult ventricular
muscle in vivo. J. Clin. Invest.
100,169
-179.
Angelo, S., Lohr, J., Lee, K. H., Ticho, B. S., Breitbart, R. E., Hill, S., Yost, H. J. and Srivastava, D. (2000). Conservation of sequence and expression of Xenopus and zebrafish dHAND during cardiac, branchial arch and lateral mesoderm development. Mech. Dev. 95,231 -237.[CrossRef][Medline]
Bamforth, S. D., Braganca, J., Eloranta, J. J., Murdoch, J. N., Marques, F. I. R., Kranc, K. R., Farza, H., Henderson, D. J., Hurst, H. C. and Bhattacharya, S. (2001). Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator. Nat. Genet. 29,469 -474.[CrossRef][Medline]
Bartman, T., Walsh, E. C., Wen, K.-K., McKane, M., Ren, J., Alexander, J., Rubenstein, P. A. and Stainier, D. Y. R. (2004). Early myocardial function affects endocardial cushion development in zebrafish. PloS Biol. 2, 673-681.[CrossRef]
Bartram, U., Molin, D. G. M., Wisse, L. J., Mohamad, A.,
Sanford, L. P., Doetschman, T., Speer, C. P., Poelmann, R. E. and
Gittenberger-de Groot, A. C. (2001). Double-outlet right
ventricle and overriding tricuspid valve reflect disturbances of looping,
myocardialization, endocardial cushion differentiation, and apoptosis in
TGF-beta(2)-knockout mice. Circulation
103,2745
-2752.
Biben, C. and Harvey, R. P. (1997). Homeodomain factor Nkx2-5 controls left/right asymmetric expression of bHLH gene eHand during murine heart development. Genes Dev. 11,1357 -1369.[Abstract]
Brown, C. B., Boyer, A. S., Runyan, R. B. and Barnett, J. V.
(1999). Requirement of type III TGF-beta receptor for endocardial
cell transformation in the heart. Science
283,2080
-2082.
Bruneau, B. G. (2002). Transcriptional
regulation of vertebrate cardiac morphogenesis. Circ.
Res. 90,509
-519.
Bruneau, B. G., Logan, M., Davis, N., Levi, T., Tabin, C. J., Seidman, J. G. and Seidman, C. E. (1999). Chamber-specific cardiac expression of Tbx5 and heart defects in Holt-Oram syndrome. Dev. Biol. 211,100 -108.[CrossRef][Medline]
Bruneau, B. G., Nemer, G., Schmitt, J. P., Charron, F., Robitaille, L., Caron, S., Conner, D. A., Gessler, M., Nemer, M., Seidman, C. E. et al. (2001). A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106,709 -721.[CrossRef][Medline]
Cai, C. L., Liang, X., Shi, Y., Chu, P. H., Pfaff, S. L., Chen, J. and Evans, S. (2003). Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev. Cell 5, 877-889.[CrossRef][Medline]
Christoffels, V. M., Habets, P., Franco, D., Campione, M., de Jong, F., Lamers, W. H., Bao, Z. Z., Palmer, S., Biben, C., Harvey, R. P. et al. (2000). Chamber formation and morphogenesis in the developing mammalian heart. Dev. Biol. 223,266 -278.[CrossRef][Medline]
Cross, J. C., Flannery, M. L., Blanar, M. A., Steingrimsson, E.,
Jenkins, N. A., Copeland, N. G., Rutter, W. J. and Werb, Z.
(1995). Hxt encodes a basic helix-loop-helix transcription factor
that regulates trophoblast cell development.
Development 121,2513
-2523.
Cserjesi, P., Brown, D., Lyons, G. E. and Olson, E. N. (1995). Expression of the novel basic helix-loop-helix gene eHAND in neural crest derivatives and extraembryonic membranes during mouse development. Dev. Biol. 170,664 -678.[CrossRef][Medline]
Dunwoodie, S. L., Rodriguez, T. A. and Beddington, R. S. (1998). Msg1 and Mrg1, founding members of a gene family, show distinct patterns of gene expression during mouse embryogenesis. Mech. Dev. 72,27 -40.[CrossRef][Medline]
Eisenberg, L. M. and Markwald, R. R. (1995).
Molecular regulation of atrioventricular valvuloseptal morphogenesis.
Circ. Res. 77,1
-6.
Firulli, A. B. and Thattaliyath, B. D. (2002). Transcription factors in cardiogenesis: the combinations that unlock the mysteries of the heart. Int. Rev. Cytol. 214, 1-62.[Medline]
Firulli, A. B., McFadden, D. G., Lin, Q., Srivastava, D. and Olson, E. N. (1998). Heart and extra-embryonic mesodermal defects in mouse embryos lacking the bHLH Transcription factor Hand1. Nat. Genet. 18,266 -270.[CrossRef][Medline]
Fishman, M. C. and Chien, K. R. (1997).
Fashioning the vertebrate heart: earliest embryonic decisions.
Development 124,2099
-2117.
Frey, N., Richardson, J. A. and Olson, E. N.
(2000). Calsarcins, a novel family of sarcomeric
calcineurin-binding proteins. Proc. Natl. Acad. Sci.
USA 97,14632
-14637.
Galvin, K. M., Donovan, M. J., Lynch, C. A., Meyer, R. I., Paul, R. J.,Lorenz, J. N., Fairchild-Huntress, V., Dixon, K. L., Dunmore, J. H., Gimbrone, M. A. et al. (2000). A role for Smad6 in development and homeostasis of the cardiovascular system. Nat. Genet. 24,171 -174.[CrossRef][Medline]
Gaussin, V., van de Putte, T., Mishina, Y., Hanks, M. C.,
Zwijsen, A., Huylebroeck, D., Behringer, R. R. and Schneider, M. D.
(2002). Endocardial cushion and myocardial defects after cardiac
myocyte-specific conditional deletion of the bone morphogenetic protein
receptor ALK3. Proc. Natl. Acad. Sci. USA
99,2878
-2883.
Haefliger, J. A., Bruzzone, R., Jenkins, N. A., Gilbert, D. J.,
Copeland, N. G. and Paul, D. L. (1992). Four novel members of
the connexin family of gap junction proteins. Molecular cloning, expression,
and chromosome mapping. J. Biol. Chem.
267,2057
-2064.
Harvey, R. P. (2002). Patterning the vertebrate heart. Nat. Rev. Genet. 3, 544-556.[CrossRef][Medline]
Hoffman, J. I. (1995). Incidence of congenital heart disease: II. Prenatal incidence. Pediatr. Cardiol. 16,155 -165.[Medline]
Hoffman, J. I. and Kaplan, S. (2002). The incidence of congenital heart disease. J. Am. Coll. Cardiol. 39,1890 -1900.[CrossRef][Medline]
Hollenberg, S. M., Sternglanz, R., Cheng, P. F. and Weintraub, H. (1995). Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system. Mol. Cell. Biol. 15,3813 -3822.[Abstract]
Kelly, R. G. and Buckingham, M. E. (2002). The anterior heart-forming field: voyage to the arterial pole of the heart. Trends Genet. 18,210 -216.[CrossRef][Medline]
Kelly, R. G., Brown, N. A. and Buckingham, M. E. (2001). The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm. Dev. Cell 1,435 -440.[Medline]
Kim, R. Y., Robertson, E. J. and Solloway, M. J. (2001). Bmp6 and Bmp7 are required for cushion formation and septation in the developing mouse heart. Dev. Biol. 235,449 -466.[CrossRef][Medline]
Lien, C. L., Wu, C., Mercer, B., Webb, R., Richardson, J. A. and
Olson, E. N. (1999). Control of early cardiac-specific
transcription of Nkx2-5 by a GATA-dependent enhancer.
Development 126,75
-84.
Lyons, I., Parsons, L. M., Hartley, L., Li, R., Andrews, J. E., Robb, L. and 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.[Abstract]
Marvin, M. J., di Rocco, G., Gardiner, A., Bush, S. M. and
Lassar, A. B. (2001). Inhibition of Wnt activity induces
heart formation from posterior mesoderm. Genes Dev.
15,316
-327.
McFadden, D. G. and Olson, E. N. (2002). Heart development: learning from mistakes. Curr. Opin. Genet. Dev. 12,328 -335.[CrossRef][Medline]
McFadden, D. G., Charite, J., Richardson, J. A., Srivastava, D.,
Firulli, A. B. and Olson, E. N. (2000). A GATA-dependent
right ventricular enhancer controls dHAND transcription in the developing
heart. Development 127,5331
-5341.
Miller-Hance, W. C., LaCorbiere, M., Fuller, S. J., Evans, S.
M., Lyons, G., Schmidt, C., Robbins, J. and Chien, K. R.
(1993). In vitro chamber specification during embryonic stem cell
cardiogenesis. Expression of the ventricular myosin light chain-2 gene is
independent of heart tube formation. J. Biol. Chem.
268,25244
-25252.
Mjaatvedt, C. H., Nakaoka, T., Moreno-Rodriguez, R., Norris, R. A., Kern, M. J., Eisenberg, C. A., Turner, D. and Markwald, R. R. (2001). The outflow tract of the heart is recruited from a novel heart-forming field. Dev. Biol. 238,97 -109.[CrossRef][Medline]
Moller, W. and Moller, G. (1994). Chemical dehydration for rapid paraffin embedding. Biotech. Histochem. 69,289 -290.[Medline]
Moorman, A. F. M., Schumacher, C. A., de Boer, P. A. J., Hagoort, J., Bezstarosti, K., van den Hoff, M. J. B., Wagenaar, G. T. M., Lamers, J. M. J., Wuytack, F., Christoffels, V. M. et al. (2000). Presence of functional sarcoplasmic reticulum in the developing heart and its confinement to chamber myocardium. Dev. Biol. 223,279 -290.[CrossRef][Medline]
O'Brien, T. X., Lee, K. J. and Chien, K. R. (1993). Positional specification of ventricular myosin light chain 2 expression in the primitive murine heart tube. Proc. Natl. Acad. Sci. USA 90,5157 -5161.[Abstract]
Olson, E. N. and Schneider, M. D. (2003).
Sizing up the heart: development redux in disease. Genes
Dev. 17,1937
-1956.
Olsen, E. N. (2001). Development: the path to
the heart and the road not taken. Science
291,2327
-2328.
Reecy, J. M., Li, X., Yamada, M., DeMayo, F. J., Newman, C. S.,
Harvey, R. P. and Schwartz, R. J. (1999). Identification of
upstream regulatory regions in the heart-expressed homeobox gene Nkx2-5.
Development 126,839
-849.
Riddle, R. D., Johnson, R. L., Laufer, E. and Tabin, C. (1993). Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75,1401 -1416.[Medline]
Riley, P. R., Anson-Cartwright, L. and Cross, J. C. (1998). The Hand1 bHLH transcription factor is essential for placentation and cardiac morphogenesis. Nat. Genet. 18,271 -275.[CrossRef][Medline]
Riley, P. R., Gertsenstein, M., Dawson, K. and Cross, J. C. (2000). Early exclusion of hand1-deficient cells from distinct regions of the left ventricular myocardium in chimeric mouse embryos. Dev. Biol. 227,156 -168.[CrossRef][Medline]
Rodriguez, C. I., Buchholz, F., Galloway, J., Sequerra, R., Kasper, J., Ayala, R., Stewart, A. F. and Dymecki, S. M. (2000). High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat. Genet. 25,139 -140.[CrossRef][Medline]
Rodriguez, T. A., Sparrow, D. B., Scott, A. N., Withington, S.
L., Preis, J. I., Michalicek, J., Clements, M., Tsang, T. E., Shioda, T.,
Beddington, R. S. and Dunwoodie, S. L. (2004). Cited1 is
required in trophoblasts for placental development and for embryo growth and
survival. Mol. Cell. Biol.
24,228
-244.
Schneider, V. A. and Mercola, M. (2001). Wnt
antagonism initiates cardiogenesis in Xenopus laevis. Genes
Dev. 15,304
-315.
Shultheiss, T. M., Burch, J. B. and Lassar, A. B. (1997). A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes Dev. 11,451 -462.[Abstract]
Shelton, J. M., Lee, M. H., Richardson, J. A. and Patel, S.
B. (2000). Microsomal triglyceride transfer protein
expression during mouse development. J. Lipid Res.
41,532
-537.
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Srivastava, D. (2001). Genetic assembly of the heart: implications for congenital heart disease. Annu. Rev. Physiol. 63,451 -469.[CrossRef][Medline]
Srivastava, D., Cserjesi, P. and Olson, E. N. (1995). A subclass of bHLH proteins required for cardiac morphogenesis. Science 270,1995 -1999.[Abstract]
Srivastava, D., Thomas, T., Lin, Q., Kirby, M. L., Brown, D. and Olson, E. N. (1997). Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND. Nat. Genet. 16,154 -160.[Medline]
Tanaka, M., Chen, Z., Bartunkova, S., Yamasaki, N. and Izumo,
S. (1999). The cardiac homeobox gene Csx/Nkx2.5 lies
genetically upstream of multiple genes essential for heart development.
Development 126,1269
-1280.
Thomas, T., Yamagishi, H., Overbeek, P. A., Olson, E. N. and Srivastava, D. (1998). The bHLH factors, dHAND and eHAND, specify pulmonary and systemic cardiac ventricles independent of left-right sidedness. Dev. Biol. 196,228 -236.[CrossRef][Medline]
Togi, K., Kawamoto, T., Yamauchi, R., Yoshida, Y., Kita, T. and
Tanaka, M. (2004). Role of Hand1/eHAND in the dorso-ventral
patterning and interventricular septum formation in the embryonic heart.
Mol. Cell. Biol. 24,4627
-4635.
Tzahor, E. and Lassar, A. B. (2001). Wnt
signals from the neural tube block ectopic cardiogenesis. Genes
Dev. 15,255
-260.
Waldo, K. L., Kumiski, D. H., Wallis, K. T., Stadt, H. A., Hutson, M. R., Platt, D. H. and Kirby, M. L. (2001). Conotruncal myocardium arises from a secondary heart field. Development 128,3179 -3188.[Medline]
Yamagishi, H., Olson, E. N. and Srivastava, D.
(2000). The basic helix-loop-helix transcription factor, dHAND,
is required for vascular development. J. Clin. Invest.
105,261
-270.
Yamagishi, H., Yamagishi, C., Nakagawa, O., Harvey, R. P., Olson, E. N. and Srivastava, D. (2001). The combinatorial activities of Nkx2.5 and dHAND are essential for cardiac ventricle formation. Dev. Biol. 239,190 -203.[CrossRef][Medline]
Yelon, D., Ticho, B., Halpern, M. E., Ruvinsky, I., Ho, R. K.,
Silver, L. M. and Stainier, D. Y. (2000). The bHLH
transcription factor hand2 plays parallel roles in zebrafish heart and
pectoral fin development. Development
127,2573
-2582