© 1999 American Heart Association, Inc.
Original Contribution |
HIRA, a DiGeorge Syndrome Candidate Gene, Is Required for Cardiac Outflow Tract Septation
From the Developmental Biology Program, Institute of Molecular Medicine, Medical College of Georgia (M.J.F., H.S., K.T.W., R.L.H., R.W., L.L., M.L.K.), Augusta, Ga, and Institute of Child Health (P.S.), London, United Kingdom.
Correspondence to Margaret L. Kirby, PhD, at Institute of Molecular Medicine, Medical College of Georgia, Augusta, GA 30912-2640. E-mail mkirby{at}mail.mcg.edu
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Abstract—DiGeorge syndrome (DGS) is a congenital disease characterized by defects in organs and tissues that depend on contributions by cell populations derived from neural crest for proper development. A number of candidate genes that lie within the q11 region of chromosome 22 commonly deleted in DGS patients have been identified. Orthologues of the DGS candidate gene HIRA are expressed in the neural crest and in neural crest–derived tissues in both chick and mouse embryos. By exposing a portion of the premigratory chick neural crest to phosphorothioate end–protected antisense oligonucleotides, ex ovo, followed by orthotopic backtransplantation to the untreated embryos, we have shown that the functional attenuation of cHIRA in the chick cardiac neural crest results in a significantly increased incidence of persistent truncus arteriosus, a phenotypic change characteristic of DGS, but does not affect the repatterning aortic arch arteries, the ventricular function, or the alignment of the outflow tract.
Key Words: DiGeorge syndrome • HIRA • persistent truncus arteriosus
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On the basis of the organs and tissues affected and evidence from experiments on animal models, the congenital diseases DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) are believed to be caused by deficient contributions from the cranial neural crest.1 The most common distinct features of these syndromes include interrupted aortic arch, outflow tract malformation, hypoplastic thymus, and hypoparathyroidism.2 Significantly, the ablation of cardiac neural crest in chick embryos results in phenotypes that closely resemble those found in DGS patients.3 This implies that studies on chick cardiac neural crest may provide insight into the molecular mechanisms that underlie these diseases. Since >85% of DGS and VCFS patients carry microdeletions in the q11.2 region of chromosome 22, much effort has been devoted to identifying genes that lie in this region.4 5 6 7 8 9 10 11 12 A number of candidate genes, including HIRA, which encodes a putative chromosome remodeling protein, have been mapped to this region.3 13 14 15 It has been shown that HIRA orthologues are expressed in the neural crest and in neural crest–derived tissues in both chick and mouse embryos,6 15 making HIRA a particularly strong candidate gene. In the chick, cHIRA is expressed in the developing neural plate, the neural tube, the neural crest, and the mesenchyme of the head and pharyngeal arches.
Both DGS and VCFS are autosomal dominant genetic
diseases.16 Cases linked to the q11.2 region of chromosome
22 are apparently caused by a haploinsufficiency of 1 or more genes
that lie in this region. Although the deletions found in this region
are typically 
3 Mb, Gong et al17 have identified a
DiGeorge critical region that is 250 kb, on the basis of shortest
region of deletion overlap mapping. Complicating matters, a second
DiGeorge critical region, distinct from the first, has been identified
in a different patient population.18 Both critical regions
are at the proximal end of the typical 3-Mb deleted region. Perhaps
regulatory elements in 1 region affect gene expression in the other.
One patient with DGS in association with a balanced translocation
involving 22q11 sequences has been described, but this chromosome
rearrangement does not disrupt any protein-encoding gene,9
supporting the view that a long-range regulatory element lies in this
region of the genome.19 It seems likely, given the broad
scope of the phenotypes found for both DGS and VCFS, that the
hemizygosity of multiple genes in these regions is responsible for the
variations in phenotypic features observed in these syndromes. Several
genes mapped to chromosome 22q11.2 are now the subject of intense
investigation.
One way to determine whether a given candidate gene is actually involved in the etiology of a human genetic disease is to examine the phenotypic effect of functional disruption of the orthologous gene in an animal model. One popular model in which to study the function of a disrupted gene is the mouse, in which homologous recombination in embryonic stem cells allows any gene of interest to be targeted for disruption. Unfortunately, because of the pleiotropic effects of many genes and the subtle and complex manner in which pleiotropy affects development, the phenotypic effect in mice harboring this sort of gene disruption cannot always be related to lack of expression in a particular tissue.
Antisense oligonucleotides designed to hybridize to specific mRNA sequences have been used to attenuate the function of numerous genes both in vitro and in vivo.20 21 22 23 24 25 26 27 Recently, antisense oligonucleotides have been used to disrupt the function of developmentally regulated genes in specific tissues of chick embryos.28 29 This method can be particularly useful for studies on chick premigratory cardiac neural crest, in which techniques for explanting and backtransplanting tissue are well established.28 30 31 In the chick, gene function can be specifically attenuated in the cardiac neural crest by extirpation of the neural folds from the level of the otic placode to somite 3, placing the folds in a solution containing the antisense oligonucleotide for a short time and then backtransplanting them into the same embryo.28 Unlike typical mouse null mutants generated by homologous recombination, this technology allows gene function to be studied in a specific tissue, from a defined point in development, and may allow researchers to begin to tease out the genetic pathways involved in cardiac neural crest development.
By exposing the premigratory chick cardiac neural crest to antisense oligonucleotides against cHIRA, the chick HIRA orthologue, ex ovo, followed by orthotopic backtransplantation to untreated hosts, we have shown that the functional attenuation of cHIRA in the cardiac neural crest results in a phenotypic change characteristic of DGS. Specifically, treatment of chick cardiac neural crest with antisense oligonucleotides to cHIRA results in a significant portion of treated embryos developing persistent truncus arteriosus (PTA). This result generally supports the hypothesis that the cardiovascular features of DGS and VCFS are caused by deficient or defective contributions to this system by neural crest–derived cells.
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Materials and Methods |
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Operative Strategy and Explant Incubation
Fertilized Arbor Acre chicken eggs were incubated at 37°C and 70% humidity for about 30 hours or until they reached Hamburger-Hamilton stage 10. The eggs were windowed, and the embryos were stained with neutral red. The vitelline membrane was torn over the rhombencephalon, and the alar plate extending from the mid-otic placode to somite 4 was removed as a single piece. If the neural folds separated, the embryo was discarded. The alar plate was incubated for 1.5 hours at 37°C with 5% CO2 in 10 µL of
-MEM containing 2 nmol of oligonucleotide. After
this incubation, each alar plate was washed in saline containing
neutral red and replaced in situ, to the embryo from which it had been
explanted, with care to reestablish the correct craniocaudal and
dorsoventral orientation. The egg was returned to the incubator and
observed periodically until the graft was reincorporated, at which time
the window was sealed with cellophane tape and incubated until the
embryo was analyzed. For explant cultures, the alar plates were
removed and incubated in oligonucleotide. The tissue
was rinsed, plated in a 96-well plate with -MEM containing 5% horse
serum, and incubated at 37°C in a 5% CO2
incubator for 24 hours.
Oligonucleotides
Oligonucleotides were designed in accordance
with the principles set forth by Wagner.32 Four 20-mer
phosphorothioate end-protected oligonucleotides
(underlined) were used, as follows: as1,
5'-GCTAAGTGATTGTCCATCTG-3'; as2: 5',
ATCAGTTTGTCATCTCCCCC-3'; and mis2:
5'-CGATGAGTTAGTCTACCTGT-3'.
The oligonucleotides, obtained from the Midland
Certified Reagent Co, were suspended in distilled water at a
concentration of 1 nmol/µL. These stock solutions were frozen in
aliquots of 20 µL. To prepare working solutions, 2 µL of stock
solution was added to 8 µL of -MEM.
RNA Extraction and Reverse Transcriptase-Polymerase Chain
Reaction (RT-PCR)
For each oligonucleotide, 6 treated neural folds
were collected in a minimal volume of guanidinium isothiocyanate and
frozen in liquid nitrogen. After thawing, 0.5 mL RNAzol was added to
each set of folds and then incubated on ice for 1 hour. One-tenth
volume of chloroform was added, and the mix was incubated another 15
minutes on ice followed by centrifugation at
12 000g for 15 minutes. One-half volume of isopropanol and
10 µg of glycogen were added to the aqueous fraction, and the RNA was
precipitated. The RNA was washed twice with 1 mL of 85% alcohol and
then resuspended in 1 mL of 70% ethanol containing 120 mmol/L
sodium acetate. After overnight incubation at –70°C, the RNA was
precipitated and washed twice with 1 mL of 70% ethanol.
Approximately 1 µg of total RNA isolated from either antisense or control oligonucleotide–treated explants, as spectrometrically determined, was reverse transcribed using random hexamer primers and a Gibco-BRL SuperScript RNase H Reverse Transcriptase kit (catalogue No. 18053-017), according to the manufacturer's instructions. One µL of the first-strand cDNA produced was used as a template for the PCR reactions. PCRs were performed in 30-µL reactions containing both Hoxb4 and Hira primers, shown below, at a final concentration of 1 µmol/L. A touchdown protocol was followed, initially annealing at 61°C and proceeding in 1°C steps to 56°C followed by 22 cycles at this annealing temperature. This number of cycles produced product in the linear range. Reactions were run on 2.5% NuSieve agarose gels, blotted, and hybridized with radiolabeled Hoxb4 and Hira probes. Filters were exposed to phosphor imaging screens, and signals were quantified using ImageQuant software (Molecular Dynamics). Primers on which PCR was performed were the following: Hira (forward), 5'-TGGAATATGGCTCCTGTCCTG-3'; Hira (reverse), 5'-CACTACAGGATGCCAGCCAAG-3'(taken from chick Hira sequence, GenBank accession No. X99375); Hoxb4 (forward), 5'-CCAAGCAGCGGTGTTATCCC-3'; and Hoxb4 (reverse), 5'-CTTTCTTCCATTTCATCCTCCTG-3'.
Immunocytochemistry
Neural folds that had been explanted, treated with control or
antisense oligonucleotides, and cultured for 24 hours
at 37°C in -MEM containing 5% horse serum were placed in methanol
at –20°C overnight. After washing twice in PBS, cells were incubated
in 5% goat serum/PBS for 30 minutes followed by a 1-hour incubation in
the HIRA antibody (a gift from J.H.C. Meijers, University Hospital,
Utrecht, The Netherlands) diluted in PBS at 1:200 or in
-smooth muscle actin antibody (Sigma) diluted at 1:1000. The fixed
neural crest cells were washed in PBS before a 30-minute incubation in
FITC-labeled goat anti-mouse antibody (Southern Biotechniques) diluted
at 1:50 in PBS. Finally, the neural crest cells were washed once in PBS
before Gel Mount (Fisher) was applied to preserve the
fluorescence.
Ink Injections
Pelikan India ink was injected into a vitelline vein using a
capillary pipette pulled to a fine diameter. The ink was ejected using
gentle, positive pressure until it filled the intravascular
compartment. The embryos were dissected from surrounding membranes and
immersion fixed in neutral buffered formalin overnight, dehydrated, and
cleared in benzyl benzoate:methylsalicylate (1:1).
Microcinephotography
Real-time video analysis was used to determine the
shortening fractions, ejection fractions, ventricular
diastolic and systolic areas, and cardiac outputs
as described by Leatherbury et al33 for neural
crest–ablated chick embryos. The chick embryos were selected for
videomicroscopy at stage 18, when the heart is a looped tube with a
well-formed outflow tract and bilaterally symmetrical aortic arch
arteries 2, 3, and 4. Videophotography was performed using a
high-speed, digital video camera (Kodak 100 HRC digital imager) mounted
on a stereoscopic microscope (Olympus Corporation of America). The
heart cycle was recorded at a frame rate of 100 frames per second,
for 10 seconds, resulting in 40 frames per cardiac cycle for a typical
embryo. Cardiac measurements were made according to the methods
previously described by Leatherbury et al.33 Cardiac
indices were then calculated. These included the shortening fraction,
ejection fraction, stroke volume, heart rate, and cardiac output of the
cardiac outflow segment. All image analysis was performed
independently by 2 investigators, who were blinded to the experimental
status of the embryos. Parameters that were significantly
different by ANOVA were further tested by Tukey's honest significant
difference procedure. Significant interactions were considered to
be P<0.05.
Western Blot Hybridizations
Embryos (one 10.5-day-old mouse embryo, 22 stage 14 chick embryo
heads, and 10 stage 22 whole embryos) were collected in PBS and then
homogenized in an equal volume of lysis buffer (50
mmol/L Tris, pH 7.4, containing 0.25% deoxycholate, 125 mmol/L
NaCl, 1 mmol/L EGTA, 1 mmol/L PMSF, and 1 µg/mL aprotinin).
The homogenate was centrifuged at 5°C at
15 800g for 5 minutes. Protein concentrations were
determined on the supernatant using the Bradford protein assay with BSA
as a standard. Twenty micrograms of each extract were fractionated on a
12% SDS-polyacrylamide gel and transferred electrophoretically
to nitrocellulose. The membrane was blocked overnight at 5°C in PBS
containing 0.1% Tween 20, 5% BSA, and 5% nonfat milk and then
incubated for 3 hours at room temperature in a 1:1000 dilution of
primary antibody in PBS containing 0.1% Tween-20 and 0.5% BSA. After
washing with 0.1 mol/L Tris, pH 7.4, containing 150 mmol/L NaCl
and 0.05% Tween-20, the membrane was incubated in a goat anti-rabbit
secondary antibody with an alkaline phosphate tag for 30 minutes at
room temperature. After washing, color was developed using NBT and
BCIP.
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Phenotypic Change Caused by cHIRA Antisense Treatment of Cardiac Neural Crest
To determine the functional role played by cHIRA in the cardiac neural crest, neural folds at the level of midotic placode to somite 3 were explanted into 10 µL of
-MEM containing 2 nmol of 1 of 2
phosphorothioate-protected antisense oligonucleotides
(as1 and as2) designed to hybridize to the portion of the mRNA that
spans the ATG encoding the first amino acid in the cHIRA protein. A
phosphorothioate-protected missense oligonucleotide of
the same nucleotide composition as as1, but with nearest
neighboring nucleotides swapped, (mis2) was used in control
experiments.
Examination of day 8 embryos, 7 days after backtransplantation of
neural folds treated with the cHIRA antisense
oligonucleotides, revealed a significant increase in
PTA as compared with embryos in which the neural folds had been treated
with a missense oligonucleotide (Figure 1
). Eight of 12 embryos in which the
neural folds had been treated with as2 and 11 of 17 embryos treated
with as1 developed PTA (Figure 1). Only 1 of 8 embryos treated
with mis2 developed PTA (Figure 1). In total, 66% of the
embryos in which folds had been treated with an antisense
oligonucleotide developed PTAs, whereas 12.5% of the
embryos in which folds had been treated with a control
oligonucleotide and 25% of the embryos treated with
medium alone developed PTAs (Figure 1). These results should be
considered in light of the fact that only 90% of chicks with complete
cardiac neural crest ablation develop PTAs.
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While a high incidence of PTAs was found in embryos in which the chick
neural crest had been treated with the cHIRA antisense
oligonucleotide, the percentage of those embryos in
which the common outflow vessel arose completely from the right
ventricle was much lower than is typically found after cardiac neural
crest ablation. Seven hearts from the antisense group that had
developed PTA were sectioned and examined microscopically to determine
outflow tract alignment. Of these hearts, 5 (71%) had normally aligned
outflow tracts (Figure 2). The outflow
tracts of the other 2 hearts were found to arise entirely from the
right ventricle. This result contrasts with that found in a study in
chick neural crest–ablated embryos in which >80% of outflow
tracts originated from the right ventricle.34 In our
study, it appeared that smaller ventricular septal
defects (VSDs) located immediately beneath the semilunar valves
correlated with normal alignment, while large VSDs were found in hearts
in which the outflow vessel arose completely from the right ventricle.
The PTAs examined by histology were all type 1, in which a short main
pulmonary trunk branched from the truncus and gave rise to 2
pulmonary arteries.35 The single
arterial valve had 5 cusps, which is expected in avian
PTA.36
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) remains open and is known to be populated
by cardiac neural crest cells normally.
To determine whether aortic arch artery formation was affected by the
antisense treatments, ink injections were performed on stage 22 embryos
(Figure 3). Although some minor
variations were observed, no significant differences were observed
between embryos in the antisense and missense groups, suggesting that
the anomalies were induced by technical procedures that are common to
both groups, rather than attributable to the antisense treatment. In
addition, aortic arch artery anomalies that were observed at early
stages appeared to resolve by day 8, since patterning of the great
arteries was normal.
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Attenuated Expression of cHIRA in Cardiac Neural Crest
To show that the increased occurrence of PTA correlated to a
decrease in cHIRA expression, experiments were carried out to determine
the effect of antisense treatment on both cHIRA mRNA and protein levels
in the cardiac neural crest. Six hours after backtransplantation, when
the treated neural crest had reattached to the embryo, the level of
cHIRA mRNA expression in the cardiac neural crest was
examined. At this stage, cHIRA expression in the cardiac
neural crest, as determined by in situ hybridization, was low, making
comparison of cHIRA expression in antisense- and
missense-treated cardiac neural crest by this method
problematic (data not shown). However, quantitative RT-PCR
performed on mRNA isolated from neural folds immediately following
oligonucleotide treatment showed a 26% decrease in
cHIRA mRNA levels in antisense-treated folds (Table 1) as determined by
autoradiodensitometry (Figure 4). These
folds were treated exactly as those that were used in
backtransplantations. RNA was isolated from the folds immediately
following the 1.5-hour oligonucleotide treatment in
-MEM. The 26% decrease in cHIRA mRNA in neural folds
treated with antisense oligonucleotide, while
significant, may not completely reveal the extent to which the
antisense oligonucleotide attenuates functional
cHIRA in the cardiac neural crest. In addition to effects on
mRNA levels, the cHIRA antisense oligonucleotides may
significantly reduce protein levels by inhibiting translation of the
cHIRA mRNA. To determine the effect of antisense treatment on cHIRA
protein expression, neural folds treated with antisense or missense
oligonucleotides were maintained in culture medium for
24 hours. At 24 hours, the neural folds were fixed, and the level of
cHIRA expression in the neural folds was assayed by
immunocytochemistry (Figure 5). The
antibody used in these assays was made against the mouse HIRA protein,
but Western blot analysis showed that this antibody also
recognized a chick protein of the correct size (Figure 5a). A
second smaller chick protein recognized by the antibody is likely to be
a degradation product of cHIRA. Compared with neural folds treated
with the missense oligonucleotide, those neural folds
treated with the antisense oligonucleotide showed a
marked decrease in the level of cHIRA protein as detected by
immunocytochemistry in migrating neural crest cells (Figure 5b
through 5e). To ensure that the effect of antisense
oligonucleotides on cHIRA protein expression was not
nonspecific, –smooth muscle actin expression was analyzed
similarly in cultured neural crest cells after
oligonucleotide treatment. In culture, –smooth
muscle actin is generally expressed in both smooth muscle cells and
neural crest cells.37 No differences in expression of
–smooth muscle actin were observed between the antisense– and
missense–treated cells (data not shown).
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Ventricular Function
No significant differences in the hemodynamic
variables were observed in embryos with attenuated cHIRA
in the cardiac neural crest (Table 2).
Although there was a significant difference in the heart rates observed
in the 3 groups that underwent backtransplantations of the premigratory
neural crest versus the sham-operated control group, this does not
support the view that attenuation of cHIRA gene expression
affects embryonic hemodynamics, since there was no
evidence of decreased contractility caused by
attenuation of cHIRA expression, as is seen following neural
crest ablation in the chick.
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Discussion |
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HIRA, initially called TUPLE1, was isolated through positional cloning and has been mapped to the DiGeorge critical region.5 A number of attributes make HIRA a particularly strong DGS candidate gene. HIRA is hemizygous in all but 1 DGS patient with a known 22q11 deletion38 and maps within the more distal of the 2 published critical regions. The chick and mouse orthologues of HIRA are expressed at relatively high levels in embryonic regions that contain migrating neural crest cells, particularly those emanating from rhombomeres 4 and 6.6 15 cHIRA is expressed throughout the rostral mesenchyme, but with particularly high levels of expression in regions that contain neural crest cells.15 At later stages, expression is strong in neural crest–derived regions of the head and in the pharyngeal arches and pouches.15 This expression pattern is intriguing, given the fact that the cardiovascular and glandular structures affected in DGS depend on contributions from pharyngeal arches 3 and 4.
The hypothesis that DGS is due to a defective contribution to the affected structures by neural crest–derived cells is supported by several lines of evidence. The neural crest, which originates in the neural folds at the dorsalmost aspect of the neural tube, contributes to the development of numerous neural and nonneural structures affected in DGS patients. Studies on neural crest migration have shown that the rostral neural crest makes significant contributions to the formation of the developing head and pharyngeal arches, in which the neural crest–derived cells differentiate into mesenchymal cells.39 40 41 42 43 Studies on chick-quail chimeras have shown that neural crest cells that originate from the level of the otic placode to somite 3 can also migrate into the cardiac outflow tract.44 45 Migration of the cardiac neural crest into the pharyngeal arches, around the arch arteries, and finally into the outflow tract of the heart is required for normal development of the heart and great arteries.3 Removal of the neural crest at this level before migration leads to defects in the embryonic cardiovascular system that include PTA.3 Incomplete removal of neural crest at this level leads to less severe defects such as VSDs, double outlet right ventricle, overriding aorta and mispatterning of the great arteries.46 Of particular interest is the fact that all of the heart defects seen after neural crest ablation are also part of the DGS phenotype. PTA, which is the hallmark of cardiac neural crest ablations, is rare in humans with nonsyndromic congenital heart disease.
When the cHIRA gene expression is attenuated in the premigratory chick cardiac neural crest, PTAs are produced in a significant portion of chick embryos. Therefore, HIRA remains a viable candidate gene to underlie the neural crest–related conotruncal defects found in DGS patients. Cardiac neural crest absence during early cardiovascular development results in a wide spectrum of cardiovascular abnormalities. Previous in vivo microcinephotography studies have shown that neural crest–ablated embryos have morphological and functional changes long before structural congenital heart defects, such as truncus arteriosus, are apparent.33 Morphologically, these embryos often exhibit incomplete looping of the cardiac tube, altered conotruncal shape, and dilated ventricles at stage 18.33 The most striking functional abnormality observed in neural crest–ablated chick embryos is decreased ventricular contractility.33 Recently, 2 aspects of early myocardial development, excitation–contraction coupling and the assembly of the contractile apparatus, have been shown to be defective in cardiac neural crest–ablated embryos, indicating that early myocardial development is influenced by the cardiac neural crest. This influence of the neural crest on myocardial development is both temporally and spatially isolated from the role of the neural crest in outflow tract septation.
cHIRA gene attenuation in the cardiac neural crest causes an increased incidence of PTA, a structural defect associated with cardiac neural crest ablation, but it does not affect ventricular function, arch-artery development, or outflow tract alignment. Our results indicate that outflow tract alignment does not depend on septation and is more likely due to defective myocardial development. The absence of ventricular functional abnormalities or outflow misalignment in these hearts suggests that attenuation of cHIRA expression in the cardiac neural crest does not affect the role played by neural crest in early myocardial development. Furthermore, the fact that outflow tract alignment tends to be normal, in this case, in the absence of outflow tract septation, supports the hypothesis that early myocardial development plays a major role in outflow tract alignment.
In previous experiments, antisense oligonucleotides to a paraloguous Hox gene family were shown to affect development of the aortic arch arteries significantly but did not cause a high incidence of PTA,28 showing that arch-artery development and conotruncal septation are not dependent on one another. The current experiments showing that defective outflow septation can occur in the absence of arch-artery anomalies supports this finding. It would be interesting to determine whether outflow tract alignment or ventricular function is affected in embryos with isolated arch-artery anomalies.
The homology of HIRA to proteins known to play roles in regulating
chromatin assembly suggests a possible role for HIRA in the regulation
of transcription through alterations in chromatin structure. HIRA
encodes a protein with no apparent DNA-binding domain, but with a
number of WD (Trp-Asp) repeats, helical protein structures
forming propeller-like structures and thought to be involved in
protein-protein interactions.13 47 HIRA is most closely
related to the yeast transcriptional corepressors HIR1 and HIR2, which
appear to interact with chromatin components to regulate histone gene
synthesis in phase with the cell cycle.13 The strong
homology between HIRA and HIR1 and HIR2 suggests that HIRA may also
function in the regulation of histone synthesis. However, the dynamic
HIRA expression pattern and its homology to other genes,
such as TUP-1, suggest a possible role in the global
regulation of gene transcription via transcription factor/histone
interactions.47 48 49 TUP-1 is known to
interact with histones and with the homeodomain protein 2. Recently,
HIRA has been shown to interact directly with the histones H3 and
H2B,50 supporting the view that HIRA may act as a
regulator of chromatin structure.
Most intriguing is the report of an interaction between HIRA and the transcription factor Pax3.51 A Pax3 mutation in the Splotch mouse results in a cardiovascular phenotype that closely resembles DGS. Mice that are homozygous for this Pax3 mutation die in utero or neonatally with cardiovascular and neural tube defects. Haploinsufficiency of HIRA in DGS patients, by causing altered stoichiometries in transcription factor complexes, may be crucial to the etiology of the disease.
The cardiovascular phenotype seen in the chicks
in which cHIRA had been functionally attenuated in the cardiac neural
crest is similar to that found in the Sox-4–null mutant
mouse.52 Sox-4 is a member of the sex-determining region
Y–related high-mobility group transcription factors, which
regulate transcription by altering chromatin structure. Since Sox-4 is
expressed in the endocardial ridges rather than neural crest, in this
instance, it has been suggested that the defective development of the
outflow tract septum is due to a disturbance of inductive
processes between the ingrowing neural crest–derived cells and the
endocardial ridges. Although Hira and Sox-4 expression do not coincide,
each may play a role in any inductive process between the endocardial
ridges and the cardiac neural crest. Other structures that depend on
contributions from the cardiac neural crest for proper development are
unaffected in these mice. Null mutations in 2 other gene families show
a wider spectrum of cardiovascular defects, which are
probably caused by deficiencies in the cardiac neural crest.
Transforming growth factor (TGF)-ß2–null mice have a similar
although milder spectrum of defects, including a large percentage of
mutant mice that harbor VSDs.53 Other aspects of the
phenotype, such as craniofacial defects, including cleft
palate, support the view that a primary cause of the heart defects
found in these mice is deficient or defective contributions by cranial
neural crest–derived cell populations to
cardiovascular development. TGF-ßs are known to
affect the migration, differentiation, and cell adhesion of cranial
neural crest cells and also to modulate production of certain
components of the extracellular matrix as well as to specifically
enhance proliferation of neural crest–derived smooth muscle cells.
Abnormal development of the aortic arch arteries is not found in either
the Sox-4–null mouse or the TGF-ß2–null mouse.52 53
Retinoic acid receptor (RAR) /RAR(ß or 
) and retinoid X
receptor-/RAR(, ß, or ) compound mutant mice have a spectrum
of phenotypes affecting a variety of structures that depend on
neural crest–derived cells for proper development, including
conotruncal defects and defective patterning of the great
arteries.54 The conotruncal defects in these mice include
PTAs. It will be interesting to determine whether HIRA expression is
affected in any of these mutants or, alternatively, whether attenuation
of HIRA expression affects expression of any of these genes.
Abnormal remodeling of the aortic arch arteries and right ventricular origin of the common arterial trunk are hallmarks of >90% of embryos with cardiac neural crest ablation in our experience.34 Männer et al55 performed the neural crest extirpation in unstained embryos after dorsal closure of the neural folds and found somewhat different results. The embryos had a high incidence of abnormal remodeling of the aortic arch arteries. Since the hearts were analyzed by scanning electron microscopy, comparisons with the present results are difficult. They typically showed high subpulmonary VSDs, with only 30% of the embryos having PTA. In some of the embryos with high VSDs, a fissure was seen between 2 well-formed semilunar valves, each having 3 leaflets. This same type of fissure was seen in high VSDs produced by unilateral vitelline vein ligation.56 This fissure, which does not represent PTA because of the presence of 2 well-formed semilunar valves, is not comparable with the PTAs produced after cHIRA treatment of the cardiac neural crest because of the presence of a single valve with 5 leaflets.
Using antisense oligonucleotides, we have shown that attenuation of cHIRA expression in chick cardiac neural crest results in a high incidence of PTA, a phenotypic defect characteristic of DGS. These results support the view that attenuated expression of HIRA is involved in the etiology of DGS and that the heart defects associated with DGS can be attributed to contributions from the cardiac neural crest. Our results, in conjunction with the correlation of microdeletions with the broad spectrum of phenotypes seen in these diseases, suggests that more than 1 gene may play a role in the etiologies of DGS and VCFS. Alternatively, deficient HIRA expression in other tissues may be required to generate the other cardiovascular defects observed in DGS patients.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL36059, HL51533, and HD17063 (to M.L.K.) and by the British Heart Foundation and Birth Defects Foundation (to P.S.). M.J.F. was supported by National Research Service Award 1 F32 HG00163-01. We thank Donna Kumiski for help with immunocytochemistry.
Received February 10, 1998; accepted October 23, 1998.
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