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First published online 11 February 2009
doi: 10.1242/dev.034025
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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.
* Author for correspondence (e-mail: eric.olson{at}utsouthwestern.edu)
Accepted 13 January 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: bHLH, Hand2, Heart development, Limb development, Craniofacial development
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The HLH motif mediates the dimerization of tissue-specific
class B bHLH factors with ubiquitous class A bHLH proteins, called E-proteins,
juxtaposing their basic regions to form a bipartite DNA-binding domain that
recognizes the E box consensus sequence (CANNTG). Binding of bHLH dimers to
DNA results in either transcriptional activation or repression, depending on
the components of the dimer and the target gene. The basic regions of bHLH
proteins also participate in protein-protein interactions and, in the case of
the myogenic bHLH proteins of the MyoD family, have been proposed to
constitute a transcriptional code for muscle-specific gene activation
(Brennan et al., 1991;
Davis et al., 1990).
Hand1 (eHAND/Thing1/Hxt) and Hand2 (dHAND/Thing2/Hed) are bHLH
transcription factors expressed in a variety of embryonic tissues, including
the heart, branchial arches and limb buds
(Angelo et al., 2000;
Charite et al., 2000;
Cross et al., 1995;
Cserjesi et al., 1995;
Hollenberg et al., 1995;
Srivastava et al., 1995;
Yelon et al., 2000). During
mouse development, Hand1 and Hand2 are both expressed in the cardiac crescent.
As the heart tube forms, Hand1 expression becomes localized to the outer
curvature of the left ventricle (LV), derived from the primary heart field,
whereas Hand2 expression becomes restricted to the right ventricle (RV) and
derivatives of the secondary heart field. Mice lacking Hand1 die at
E8.5 of placental and extra-embryonic defects, precluding analysis of its
potential functions in the heart (Firulli
et al., 1998; Riley et al.,
1998). However, cardiac-specific deletion of the Hand1
gene in mice causes perinatal lethality with a spectrum of congenital heart
defects that reflect abnormalities in ventricular growth
(McFadden et al., 2005). Mice
homozygous for a Hand2 null mutation die between E9.5 and E10.5 from
right ventricular hypoplasia and defects in vascular development
(Srivastava et al., 1997;
Yamagishi et al., 2000). The
phenotypes of Hand1/Hand2 double mutant mice indicate dose-sensitive
functions of these transcription factors in the control of cardiac
morphogenesis (McFadden et al.,
2005). Further support for the essential role of Hand genes in
ventricular growth comes from the phenotype of the zebrafish mutant hands
off, which lacks the single Hand gene in that organism, and fails to form
a ventricular chamber (Yelon et al.,
2000).
Hand1 and Hand2 are expressed in the neural crest-derived mesenchyme of the
developing branchial arches, where their expression is regulated by endothelin
1 (ET-1; Edn1 – Mouse Genome Informatics) signaling
(Clouthier et al., 2000;
Cserjesi et al., 1995;
Thomas et al., 1998).
Hand2 null embryos display hypoplastic first and second branchial
arches, due to apoptosis, and fail to form the third and fourth arches before
lethality at E10.5 (Srivastava et al.,
1997; Thomas et al.,
1998). Hand2 is also expressed in the posterior region of the limb
mesenchyme that encompasses the zone of polarizing activity (ZPA)
(Charite et al., 2000), where
it is necessary to induce the expression of the morphogen sonic hedgehog (Shh)
(Charite et al., 2000).
Misexpression of Hand2 throughout the limb bud results in ectopic expression
of Shh and its target genes in the anterior compartment of the limb bud with
consequent preaxial polydactyly and mirror image duplications of posterior
digits (Charite et al., 2000;
Fernandez-Teran et al., 2000).
It was also shown that the transcriptional repressor Gli3 restricts Hand2
expression from the anterior mesenchyme, and Hand2 in turn excludes Gli3 and
Alx4 from posterior mesenchyme (te
Welscher et al., 2002). Hand2 also positively regulates posterior
expression of the BMP antagonist gremlin. These genetic interactions between
Gli3 and Hand2 polarize the nascent limb bud mesenchyme prior to Shh signaling
(te Welscher et al.,
2002).
Despite detailed analysis of the consequences of loss-of-function Hand gene
mutations in mutant mice, relatively little is known of the mechanism of
action of Hand proteins during development. Like other bHLH proteins, Hand1
and Hand2 form heterodimers with E-proteins and can activate the transcription
of E-box-dependent reporter genes when overexpressed in vitro
(Firulli, 2003). However,
mutant forms of Hand proteins defective in DNA binding can also activate
transcription in overexpression assays (Xu
et al., 2003; Rychlik et al.,
2003). Similarly, a DNA binding-defective mutant of Hand2 was as
effective as the wild-type protein in inducing preaxial polydactyly and digit
duplications when misexpressed in the developing limb bud of transgenic mice
(McFadden et al., 2002). These
studies raise questions about the precise mechanism of action of Hand proteins
and whether they can function at physiological levels in vivo without binding
DNA.
In the present study, we used homologous recombination to introduce a mutation into the endogenous mouse Hand2 gene that abolished the DNA-binding activity of Hand2, but left the Hand2 protein otherwise intact. In contrast to a Hand2 null allele, which causes severe cardiac defects by E9.5 and lethality by 10.5, mice homozygous for the DNA-binding mutation, referred to as Hand2EDE, showed remarkably normal development of the heart and branchial arches until at least E11.5, when right ventricular growth became impaired and embryonic lethality ensued. However, similar to Hand2 null embryos, Hand2EDE/EDE embryos also showed underdeveloped limb buds and failed to express Shh and to restrict anterior gene expression, These findings suggest that Hand2 exerts at least a subset of its embryonic functions independently of DNA binding during the development of the heart and branchial arches, whereas during limb development the DNA-binding activity of Hand2 is indispensable for its function.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The completed targeting vector was linearized with AhdI and
electroporated into SM-1 ES cells. Following positive selection with G418 and
natural negative selection of the DTA cassette, resistant colonies were
screened by Southern blot analysis of NcoI-digested genomic DNA using
a probe from the 3' flanking region
(Fig. 1B). Homologous
recombination of the 5' arm was confirmed by Southern blotting of
NdeI-digested genomic DNA using a probe from the 5' flanking
region (Fig. 1B). Genomic DNA
from positive clones was also subjected to PCR and sequencing around the exon
1 region of the Hand2 gene to confirm the introduced mutations. Three
correctly targeted clones were expanded and injected into C57BL/6 blastocysts,
and transferred into the uteri of pseudopregnant female mice. Chimeric males
were bred to C57BL/6 mice to obtain germline transmission of the
Hand2EDE-neo allele. In order to remove the neomycin
resistance cassette in the germline, Hand2EDE-neo/+ mutant
mice were bred to hACTB::FLPe transgenic mice
(Rodriguez et al., 2000).
Removal of the neomycin resistance cassette was confirmed by PCR genotyping
(data not shown). The resulting Hand2EDE/+ heterozygous
mutants were bred to each other to obtain Hand2EDE/EDE
homozygous mutants.
PCR genotyping
Genotyping of Hand2EDE loci was performed by PCR with primers
flanking the inserted FRT sequences. Tail and yolk sac DNA was isolated as
previously described. One µl of tail or yolk sac DNA was used as a template
in 25 µl PCR reactions using CLP Taq polymerase and 2 mM MgCl2.
Thermal cycle reactions were as follows: 2 minutes at 95°C, 30 cycles of
30 seconds at 95°C, 30 seconds at 60°C, 20 seconds at 72°C and a
final 5 minutes extension at 72°C. Reactions were visualized on 2%
agarose/TAE gels. The wild-type allele gave rise to a 244-bp fragment, whereas
the Hand2EDE allele gave rise to a 390-bp fragment. Primer
sequences are available upon request.
RNA isolation, RT-PCR and real-time RT-PCR
Whole embryos or embryonic hearts at E9.5 were dissected and immediately
frozen and stored in liquid nitrogen until yolk sac DNA was isolated and
genotyped. Hearts of the same genotypes were pooled as one sample before RNA
isolation. Total RNA was isolated using Trizol reagent and standard protocols.
Total RNA (10 µg) was used as a template for reverse transcription with
random hexamer primers. cDNA (25 ng) was used as template for PCR reactions
using Promega Taq polymerase to detect Hand2 transcripts and
transcripts for hypoxanthine phosphoribosyl transferase (HPRT) as a control.
Reactions were visualized on 1% agarose/TAE gels and Hand2 RT-PCR products
were gel-isolated and subjected to sequencing. Primer sequences are available
upon request.
For quantitative real-time PCR, cDNA (25 ng) was amplified in each reaction by using the TaqMan Universal PCR Master Mix Kit (Applied Biosystems, Foster City, CA). Mean relative gene expression was calculated by using standard curves from serial dilutions of cDNA from wild-type embryos and normalized to GAPDH (n=3 per group).
Western blotting analysis
Hearts from E9.5 embryos were dissected and immediately frozen and stored
in liquid nitrogen until yolk sac DNA was isolated and genotyped. Hearts from
the same genotypes were pooled as one sample before protein isolation. Equal
amounts of protein were loaded onto 4-20% SDS-PAGE gels and western blotting
was performed according to standard protocols. The antibody against Hand2
(Santa Cruz, 1:100) was described previously
(Zhao et al., 2005).
Whole-mount in situ hybridization
Embryos were harvested at the indicated age and fixed in 4%
paraformaldehyde in PBS overnight at 4°C. Whole-mount in situ
hybridizations were performed as previously described
(Clouthier et al., 2000) using
digoxigenin-labeled riboprobes.
Histology and skeletal analysis
Embryos were harvested from timed matings and fixed overnight in 4%
paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4°C.
Following fixation, embryos were rinsed in PBS and then embedded in paraffin
as previously described. Sections were cut and stained with Hematoxylin and
Eosin. For skeletal analysis, postnatal day 1 (P1) mice were collected,
prepared and stained with Alizarin Red and Alcian Blue for visualization of
bone and cartilage formation, respectively
(Yanagisawa et al., 1998).
TUNEL and immunohistochemistry
TUNEL staining was performed on paraffin-embedded sections according to the
Roche in situ cell death detection kit. Phospho-histone H3 antibody staining
was performed as described previously (Xin
et al., 2006). Briefly, sections were deparaffinized in xylene,
rehydrated through graded ethanol to PBS, and permeabilized in 0.3% Triton
X-100 in PBS. Sections were then blocked by 1.5% normal horse serum in PBS
followed by incubation with rabbit anti-phosphohistone H3 (Upstate Cell
Signaling Solutions, Charlottesville, VA) at a 1:200 dilution in 0.1% BSA in
PBS overnight at 4°C. Sections were washed in PBS, and
fluorescein-conjugated secondary antibodies (Vector Laboratories, Burlingame,
CA) were applied at a 1:200 dilution in 1% normal horse serum for 1 hour.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The mutant Hand2 protein was also expressed at the same
level as the wild-type protein in transfected cells, dimerized with E12, and
was localized to the nucleus (McFadden et
al., 2002) (data not shown). The mouse Hand2 gene contains two exons, and the basic domain is encoded by exon 1 (Fig. 1A). Our targeting strategy involved insertion of a neomycin-resistance cassette flanked by sites for the FLP recombinase (Frt) into the intron of the Hand2 gene (Fig. 1B). The targeting vector was electroporated into ES cells and positive clones containing the targeted allele were identified by Southern blot analysis (Fig. 1C). Injection of heterozygous ES cells into mouse blastocysts yielded chimeric male mice, which transmitted the mutant allele through the germline. The heterozygous mice were bred to mice expressing the FLPe recombinase to remove the neomycin-resistance cassette, leaving only a single Frt site in the intron (Fig. 1B). Successful removal of the neomycin-resistance cassette was detected by PCR analysis of tail DNA (data not shown).
To confirm that the Frt site in the intron did not alter Hand2 expression, we performed RT-PCR on RNA isolated from E9.5 embryos using primers that flank the junctions of exons 1 and 2 of the Hand2 gene. As shown in Fig. 1D, RNA from wild-type and Hand2EDE/EDE embryos yielded the expected PCR fragments, and DNA sequencing confirmed the presence of the EDE mutation in homozygous mutant embryos (Fig. 1E). The junction sequences between exons 1 and 2 in the homozygous embryos were also correct in the mutant embryos, indicating that the mutant allele was correctly spliced. Quantitative real-time PCR confirmed that wild-type and mutant Hand2 transcripts were expressed at comparable levels (Fig. 1F). Western blotting on E9.5 embryonic hearts also showed that the mutant Hand2 protein is expressed at a comparable level to the wild-type Hand2 protein (Fig. 1G). These results indicate that the mutations in the basic region did not alter protein stability or subcellular localization.
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). By
contrast, homozygous Hand2EDE/EDE embryos were
morphologically indistinguishable from wild-type embryos at E9.5, except for a
slightly smaller right ventricle and outflow tract
(Fig. 2A). At E10.5, we
obtained 57 viable Hand2EDE/EDE embryos out of 244 embryos
examined from Hand2EDE/+ heterozygous crosses,
representing Mendelian ratios (Fig.
1H). At this stage, these mutant embryos appeared grossly normal
compared with wild-type embryos (Fig.
2B, parts a-d). We have never observed viable
Hand2KO/KO embryos at E10.5 from several hundred embryos
analyzed from heterozygous Hand2KO/+ intercrosses. At
E11.5, 16 Hand2EDE/EDE embryos were obtained out of 117
embryos collected from heterozygous crosses and many showed multifocal
hemorrhages, indicative of cardiovascular defects
(Fig. 2B). The forelimb and
hindlimb buds of Hand2EDE/EDE embryos at this stage were
hypoplastic, and the face and jaws were also obviously abnormal
(Fig. 2B, parts e,f). At E12.5, we obtained one viable Hand2EDE/EDE embryo out of 22 embryos collected from heterozygous crosses. This mutant embryo showed no signs of hemorrhage, but its limb buds were underdeveloped relative to those of wild-type embryos (Fig. 2B). No viable Hand2EDE/EDE embryos were obtained after E12.5. A summary of homozygous Hand2EDE/EDE embryos obtained from timed matings is shown in Fig. 1H. We conclude that the homozygous Hand2EDE/EDE mutation results in lethality between E10.5 and E12.5 due to cardiovascular defects, one to three days later than does the knockout of the Hand2 gene in Hand2KO/KO embryos.
Cardiac abnormalities of Hand2EDE/EDE embryos
At E9.5, Hand2KO/KO embryos have an extremely
hypoplastic right ventricle and outflow tract
(Fig. 2A,
Fig. 3A)
(Srivastava et al., 1997). By
contrast, the RV and outflow tract appeared to develop nearly normally in the
Hand2EDE/EDE embryos at this stage, although they were
slightly smaller relative to those of wild-type littermates
(Fig. 3A). At E10.5, the RV of
Hand2EDE/EDE embryos continued to develop and contained
trabeculations (Fig. 3A). The
endocardial cushions appeared slightly disorganized in the
Hand2EDE/EDE mutant embryos at this stage, indicative of a
potential delay in their formation. At E11.5, the
Hand2EDE/EDE mutant embryos showed enlarged and
disorganized endocardial cushions, and the RV showed reduced trabeculation
compared with wild type (Fig.
3A). The LV of the Hand2EDE/EDE embryos was
also abnormally thin with few trabeculations. The interventricular septum
(IVS) in wild-type embryos was apparent, as evidenced by the thickening and
alignment of cardiomyocytes in this region. In
Hand2EDE/EDE mutants, the interventricular groove began to
form but its development was delayed and myocytes failed to thicken
(Fig. 3A). The cushion defect
together with the underdeveloped RV and dilated LV probably cause embryonic
lethality in Hand2EDE/EDE mutants. At E12.5, the heart of
the surviving Hand2EDE/EDE embryo appeared normal, except
for a slightly hypoplastic RV and IVS, and less trabeculation in the RV
(Fig. 3A).
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;
Srivastava et al., 1997;
Thattaliyath et al., 2002;
Yamagishi et al., 2001). To
further define the functions of the Hand2EDE protein, we analyzed
the expression of these genes by quantitative real-time PCR in E9.5 hearts. As
shown in Fig. 3B, expression of
Gata4, ANP and Hand1 was unchanged in the hearts of mutant embryos compared
with wild type, but expression of Irx4 was slightly decreased. These results
suggest that Hand2 regulates expression of Gata4, ANP and Hand1 in a
DNA-binding-independent manner, whereas regulation of Irx4 expression by Hand2
requires DNA binding.
Limb development in Hand2EDE/EDE embryos
During limb outgrowth and patterning, Hand2 is expressed in the posterior
region of the limb bud, and it is necessary and sufficient to induce the
expression of Shh (Charite et al.,
2000). Hand2 was shown to be repressed by the transcriptional
repressor Gli3 in the anterior mesenchyme of the limb buds, and Hand2 in turn
represses the anterior genes Gli3 and Alx4 from being expressed in the
posterior region (te Welscher et al.,
2002). Hand2 is necessary for expression of the posterior gene
gremlin (te Welscher et al.,
2002). The repressive interactions between Gli3 and Hand2
prepattern the limb bud mesenchyme prior to Shh signaling
(te Welscher et al., 2002). In
Hand2 null mice, the forelimbs are hypoplastic, variably malformed
and fail to upregulate Shh (Charite et al.,
2000). Expression of Gli3 and Alx4 is no longer restricted to the
anterior-most mesenchyme, but is expanded posteriorly in Hand2 null
limb buds (te Welscher et al.,
2002).
A developmental delay in the growth of the limb buds was apparent in the
majority of Hand2EDE/EDE mutant embryos by E10.5 and
became more extreme by E11.5 (Fig.
2B; Fig. 4A,B). The
growth defects were apparent in both the anteroposterior (AP) and
proximodistal dimensions of the fore- and hind-limbs at E11.5
(Fig. 4A,B). Additionally, the
anterior margin of the forelimb buds appeared to be shifted posteriorly
compared with that of wild-type embryos
(Fig. 2B;
Fig. 4A,C), a phenotype also
seen in Hand2 null limb buds
(Charite et al., 2000).
Using Fgf8 expression as a marker for the apical ectodermal ridge (AER)
(Lewandoski et al., 2000;
Moon and Capecchi, 2000), we
examined the length of the AER in Hand2EDE/EDE mutant limb
buds. We found that, at E10.5, the AER of Hand2EDE/EDE
forelimbs was shorter along the AP axis, but the AER of the hindlimbs was
comparable to that of the wild-type hindlimbs
(Fig. 4C). At E11.5, the AER
was significantly shorter in both forelimbs and hindlimbs of
Hand2EDE/EDE mutant embryos
(Fig. 4C). The shortened AERs
in Hand2EDE/EDE mutant limb buds are likely to be due to
disruption of the Shh/gremlin/Fgf loop that is important for maintenance of
the AER (Panman et al., 2006;
Sun et al., 2000;
Zuniga et al., 1999). We also
analyzed early chondrocyte condensation by Sox9 expression and observed no
difference between wild-type and Hand2EDE/EDE mutant
embryos at E10.5 (Fig. 4D).
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Taken together, the result that Hand2EDE/EDE mutant embryos resemble Hand2 null embryos in terms of limb defects suggests that the DNA-binding activity of Hand2 is necessary for the regulation of Hand2 target genes during limb outgrowth and patterning.
Branchial arch development in Hand2EDE/EDE mutant embryos
In Hand2 null embryos, the first and second branchial arches are
severely hypoplastic at E9.5 due to extensive apoptosis, and the third and
fourth arches fail to form (Srivastava et
al., 1997; Thomas et al.,
1998). At E9.5 and E10.5, the majority of
Hand2EDE/EDE embryos had relatively normal branchial
arches, with all four (first to fourth) being formed
(Fig. 5A). However, at E11.5 in
some Hand2EDE/EDE embryos, the left and right mandibular
components of the first branchial arch of the Hand2EDE/EDE
embryos failed to fuse, leaving a cavity in the center of the mandible
(Fig. 5A). Hand2 was shown to
regulate Msx1 expression during branchial arch growth, and, in
Hand2-null embryos, no expression of Msx1 is detectable in the
branchial arches (Thomas et al.,
1998). Interestingly, in Hand2EDE/EDE embryos,
Msx1 expression was not affected in the branchial arches at E10.5, as detected
by whole-mount in situ hybridization (Fig.
5B). Other markers of the neural-crest-derived ectomesenchyme were
expressed normally in branchial arches of Hand2EDE/EDE
embryos at E10.5 (data not shown).
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), there was no difference in the number of apoptotic
cells observed by TUNEL assay in the branchial arches of the wild-type and
Hand2EDE/EDE embryos at E10.5 (data not shown). Analysis
of cell proliferation at E10.5 by staining with anti-phosphohistone H3
antibody also failed to reveal differences in the number of proliferating
cells (data not shown). These results, together with results from whole-mount
in situ hybridization, confirmed that, at least until E10.5, development of
branchial arches was normal in the Hand2EDE/EDE embryos,
indicating that regulation of the initial events of branchial arch development
by Hand2 does not require its DNA-binding activity.
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).
Homozygous Hand2BA/BA mice die at postnatal day 1 from
failure to suckle and exhibit craniofacial abnormalities, including cleft
palate and malformations of the mandible and Meckel's cartilage
(Yanagisawa et al., 2003) (see
also Fig. 6B,E,H,K).
Hand2EDE/BA mice also died at P1 from failure to suckle
and exhibited cleft palate. The mandibles of Hand2BA/BA
mice (Fig. 6B, arrow), like
those of Hand2EDE/BA mice
(Fig. 6C, arrow), were
hypoplastic and foreshortened compared with wild-type mice
(Fig. 6A, arrow). Close
examination showed that the angle between the right and left mandibular bones
was also wider in both Hand2BA/BA and
Hand2EDE/BA mutants than in wild-type mice
(Fig. 6D-F). In wild-type mice,
bilateral palatine processes extend horizontally and fuse to form the
secondary palate (Fig. 6G,
dotted vertical line). By contrast, the palatine processes of the
Hand2EDE/BA and Hand2BA/BA mice did
not appear to be elevated and thus the secondary palate was not formed
(Fig. 6H,I, dotted vertical
line), causing the underlying presphenoid bone to be visible in ventral view
(ps in Fig. 6H,I). In addition,
the tympanic rings of the Hand2EDE/BA mouse
(Fig. 6L) were shortened and
deformed compared with those of the wild-type mouse
(Fig. 6J), a phenotype also
observed in the Hand2EDE/BA mouse
(Fig. 6K). In summary, the
Hand2EDE/BA mouse exhibited similar craniofacial defects
to the Hand2BA/BA mouse at P1, suggesting that
Hand2EDE protein cannot support late craniofacial development.
Thus, the DNA-binding activity of Hand2 is essential for its role in
patterning and development of the cranial neural crest. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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DNA-binding-dependent and -independent functions of Hand2
Tissue-specific (class B) bHLH proteins dimerize with ubiquitous bHLH
proteins (E proteins) to form a bipartite DNA-binding domain that recognizes
the E box consensus sequence (Massari and
Murre, 2000). The basic regions of bHLH proteins are required not
only for binding to target DNA, but also for protein-protein interactions and
tissue-specific gene activation (Brennan et
al., 1991; Davis et al.,
1990).
It is striking that Hand2 is able to partially function in vivo in the
absence of DNA binding. Although we cannot formally rule out the possibility
that the Hand2EDE mutant protein retains residual DNA binding
activity in vivo, we feel this is unlikely because we have detected no
DNA-binding activity of this protein with a high-affinity binding site in
vitro and because the replacement of basic residues with acidic residues in
the basic regions of other bHLH proteins completely abolishes DNA-binding
activity (McFadden et al.,
2002).
How might Hand2 function in the absence of DNA binding? We suggest two
possibilities. (1) Hand2 might interact with other transcriptional activators
that are bound to DNA, thereby establishing a multi-protein transcriptional
complex. There is evidence for such a mechanism from transfection assays
(Rychlik et al., 2003;
Xu et al., 2003). (2) Hand2
might act by titrating out repressor proteins, possibly through the HLH
region, that negatively regulate specific gene programs.
SCL (also known as Tal1), a bHLH protein required for hematopoiesis and
vascular development, has also been shown to possess DNA-binding-independent
functions such that a DNA-binding-defective mutant can rescue hematopoiesis in
SCL–/– ES cells, and can restore hematopoiesis and
vasculogenesis when expressed in zebrafish
(O'Neil et al., 2001;
Porcher et al., 1999).
Inhibition of the pro-osteogenic activity of the Runx2 transcription factor by
the Twist family of bHLH proteins has also been shown to occur independently
of DNA binding, at least in vitro (Bialek
et al., 2004). Together, these studies suggest that bHLH proteins
may operate through DNA-binding-dependent and -independent mechanisms. The
present study is the first to analyze the requirement of bHLH protein DNA
binding in vivo through the expression of a DNA binding-defective mutant from
the endogenous gene locus at physiological levels.
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Hand2EDE/EDE mutant embryos display disorganized and
enlarged endocardial cushions and a delay in formation of the IVS. We cannot
rule out the possibility that these defects are secondary to delayed RV
development, but we favor the interpretation that Hand2 is important in the
formation of the endocardial cushions and the IVS, because embryos that lack
cardiac expression of Hand1, which shares partially redundant functions with
Hand2, also displayed a thickened and disorganized ventricular septum and
hyperplastic endocardial cushions
(McFadden et al., 2005). It
has also been reported that overexpression of Hand2 in the ventricles results
in a complete absence of the IVS, indicative of a negative role of Hand2 in
the formation of this cardiac structure
(Togi et al., 2006).
Functions of Hand2 in the limb buds
Limb bud patterning and outgrowth along the three axes are controlled by
distinct but interdependent signaling pathways from both ectoderm and
mesodermal mesenchyme (Mariani and Martin,
2003; Niswander,
2002). For example, limb outgrowth along the proximodistal axis is
regulated by signals from the Fgf family in the AER (AER-Fgfs)
(Boulet et al., 2004;
Sun et al., 2002), and AP
patterning is controlled by Shh secreted from the ZPA
(Riddle et al., 1993). Precise
interactions between the AER and the ZPA via the Fgf/gremlin/Shh positive loop
and the Fgf/gremlin negative loop determine limb bud size and shape during
limb outgrowth and termination (Panman et
al., 2006; Sun et al.,
2000; Verheyden and Sun,
2008; Zuniga et al.,
1999). During initiation of limb bud outgrowth, prepatterning
between anterior and posterior mesenchyme through genetic repression between
Gli3/Alx4 and Hand2 determines posterior identity, which is essential for
differential mesenchymal responsiveness to future Shh signaling
(te Welscher et al.,
2002).
Although elegant genetic studies have been performed, it remains unclear at
the molecular level how Hand2 activates Shh and gremlin and represses Gli3 and
Alx4 expression. The finding that Hand2EDE/EDE embryos
display growth defects in limb buds suggests that Hand2 regulates limb growth
through a DNA-binding-dependent mechanism. Our results also show that the
regulation of Shh, Gli3, Alx4 and gremlin by Hand2 requires a functional
DNA-binding domain. We showed previously that overexpression of the
Hand2EDE mutant in the limb buds of transgenic mice can
induce ectopic digit formation as effectively as the wild-type Hand2 protein
(McFadden et al., 2002). The
disparity between these phenotypes suggests either that regulation of digit
patterning and growth by Hand2 are indeed independent of DNA binding, whereas
earlier functions in limb bud outgrowth require DNA binding, or that the
overexpressed Hand2EDE protein acts through a non-physiological
mechanism to induce ectopic digits.
Functions of Hand2 in the branchial arches
Hand2 controls development of the branchial arches via a signaling pathway
involving ET-1, Hand2 and Msx1 (Charite et
al., 2001; Thomas et al.,
1998). Our results suggest that the DNA-binding activity of Hand2
is dispensable during early branchial arch development, but is required for
the development of mandibular components of the first branchial arch, as
evidenced by failure in fusion of the left and right mandibular components in
mutant embryos at E11.5. Interestingly, Hand2EDE/BA mutant
mice display craniofacial defects at P1 that include shortened jaw (derived
from mandibular components of the first branchial arch) and cleft palate, a
phenotype similar to that of the Hand2BA/BA mice
(Yanagisawa et al., 2003).
These findings indicate that the DNA-binding activity of Hand2 is required for
late craniofacial development, especially jaw formation.
Implications
In conclusion, the results of this study demonstrate both
DNA-binding-dependent and -independent functions of the Hand2 protein during
mouse embryonic development, underscoring the complexity of mechanisms by
which this bHLH protein regulates such a diverse spectrum of developmental
processes. Thus, the complete set of regulatory influences of Hand2 is likely
to reflect not only direct protein-DNA interactions, but also protein-protein
interactions, both positive and negative, in different cell types. We
anticipate that this level of complexity is shared by other bHLH proteins that
regulate the cell growth, specification and differentiation of other cell
types.
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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