Cardiovascular Research

Cardiovascular Research 1997 36(2):163-173; doi:10.1016/S0008-6363(97)00172-7
© 1997 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Conway, S. J Articles by Copp, A. J Search for Related Content PubMed PubMed Citation Articles by Conway, S. J Articles by Copp, A. J Social Bookmarking          What's this?

Copyright © 1997, European Society of Cardiology

Development of a lethal congenital heart defect in the splotch (Pax3) mutant mouse

Simon J Conway^a,b,*, Deborah J Henderson^b, Margaret L Kirby^a, Robert H Anderson^c and Andrew J Copp^b

^aDevelopmental Biology Program, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-2640, USA
^bNeural Development Unit, Institute of Child Health, University of London, 30 Guilford Street, London WC1N 1EH, UK
^cNational Heart and Lung Institute, Imperial College School of Medicine, University of London, London, UK

^* Corresponding author. Tel.: +1 706 7218775; Fax: +1 706 7218685; E-mail: sconway@mail.mcg.edu

Received 4 April 1997; accepted 2 May 1997

	Abstract

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Objective: The splotch (Sp^2H) mutation disrupts the Pax3 gene and is lethal in homozygotes. The aim of the present study was to investigate the cause of lethality. Methods and results: Using the splotch (Sp^2H) mouse mutant, we demonstrated that approximately 60% of Sp^2H homozygotes die in utero at 13.5–14.5 days of gestation. All these embryos have cardiac malformations involving partial or complete failure of septation of the outflow tract. Although the cause of death in utero is unknown, the dying embryos are edematous, their superior caval veins are over-expanded, and the fetal liver is enlarged and engorged with blood, all signs of cardiac failure. The remaining Sp^2H homozygotes die around the time of birth, and these embryos have grossly normal hearts. All Sp^2H homozygotes have neural tube defects, either spina bifida, exencephaly, or both. Although these defects clearly do not cause death in utero, they are very likely responsible for the perinatal death of homozygotes that survive to late gestation. There is no correlation between the presence or absence of a cardiac defect and the type of neural tube defect. On the other hand, there is a striking correlation between presence of a cardiac defect and reduction or absence of dorsal root ganglia, which are derivatives of the neural crest. Conclusions: In this paper, we show that the lethality has a biphasic pattern, and the data strongly suggests that mid-gestation lethality is due to cardiac defects and not the associated neural tube defects. This finding supports the idea that ‘conotruncal’ cardiac defects involving the ventricular outflow tracts develop as a result of failure of the ‘cardiac’ neural crest to colonise the developing heart in the mid-gestation embryo, and that the resulting heart defects are solely responsible for the observed mortality.

KEYWORDS Mouse mutant; Congenital heart disease; Neural tube defects; Pax; Splotch; Cardiac neural crest; Heart failure

	1 Introduction

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Congenital heart disease continues to be the most prevalent category of major birth defects in humans [1]. Although some cardiac defects can be diagnosed before birth, and later corrected by surgical intervention, there are no methods routinely available for preventing the development of these malformations. If we are to develop preventive measures, it is important to gain a detailed understanding of the embryonic mechanisms that underlie the abnormal formation of the heart. This goal is greatly facilitated by the availability of an increasing number of mutant mouse strains in which the causative genes are known, and in which heart defects develop resembling those seen in humans [2, 3]. Such mouse mutants provide model systems in which to probe the molecular and cellular basis of abnormal cardiac development.

Mutations at the splotch locus are known to result from disruption or deletion of the Pax3 transcription factor [4]. Homozygotes have long been known to develop neural tube defects, both exencephaly and spina bifida, and defects in neural crest-dependent structures such as the dorsal root ganglia and melanocytes [5]. Moreover, homozygotes for the Sp^1H allele have been described as developing defects within the ventricular outflow tract, specifically common arterial trunk (also known as persistence truncus arteriosus, PTA) [6]. In the avian embryo, a common trunk results when a specific sub-population of neural crest cells (the ‘cardiac neural crest’) fails to colonise the developing outflow tracts [7, 8]. Since splotch embryos are known to have neural crest-related defects, this provides a possible explanation for the development of this cardiac defect in the homozygous mutants.

One of the most intriguing features of the Sp, Sp^1H and Sp^2H mutant alleles is the mortality of homozygotes around day fourteen of gestation [5]. In previous studies, it was observed that all homozygotes die prenatally [5, 6], making it difficult to determine which aspect of the mutant phenotype is responsible for the lethal effect. However, we observed that only around 60% of Sp^2H homozygotes die at day 14, with the remainder surviving to term. This provided an opportunity for a detailed analysis of the developmental defects exhibited by both dying and surviving Sp^2H homozygous embryos, offering the potential to determine the cause of the prenatal mortality. The objective of the present study was to investigate the cause of lethality, and in this paper we show that there is no correlation between the type or severity of neural tube defect and death in utero of Sp^2H/Sp^2H embryos. On the other hand, there is a striking correlation between the presence of malformations within the ventricular outflow tract and mortality on days 13–14 of gestation, suggesting strongly that cardiac defects are the cause of prenatal death in the mutant embryos. Moreover, diminution in the size of the dorsal root ganglia, a marker of defective neural crest, is strongly associated with the presence of a common arterial trunk, providing support for a link between abnormal neural crest and cardiac malformations in splotch mice.

	2 Materials and methods

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
2.1 Mouse strains
Sp^2H is a radiation-induced allele at the splotch locus that arose on the C3H/101 background [9]. A heterozygous Sp^2H female, obtained from the MRC Radiobiology Unit, Harwell, UK, was mated with a CBA/Ca male and the offspring were mated inter se to found a randomly-bred colony. Sp^2H/+ heterozygotes, identified by the presence of a white belly spot, were mated to produce Sp^2H/Sp^2H embryos. Noon on the day of finding a copulation plug was designated 0.5 days of gestation. Pregnant females were killed by cervical dislocation and the embryos were explanted into Dulbecco's Modified Eagles Medium (DMEM, Flow Labs, UK) containing 10% fetal calf serum (FCS). The yolk sac and amnion were opened and the umbilical cord was cut. A pulsating stream of blood indicated ongoing contractions of the heart in viable embryos. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.2 Genotyping of embryos
Since splotch is a recessive lethal mutation, embryos must be generated by matings between Sp^2H/+ individuals. This yields three categories of offspring: Sp^2H/Sp^2H, Sp^2H/+ and +/+. In order to perform comparisons between mutant and normal embryos it is essential to be able to identify the various genotypes early in development, before the appearance of morphological abnormalities. Genotyping was performed using the polymerase chain reaction (PCR) to identify the 32 base pair deletion present in the Sp^2H mutant allele of Pax3 [4]. Genomic DNA was isolated from the yolk sac and/or a limb bud as described previously [10]. Oligonucleotide primers that flank the deletion [4]were used to amplify a DNA fragment of length 127 bp (wild type) or 95 bp (Sp^2H). PCR conditions were: initial denaturation, 95°C for 5 min; 30 cycles of amplification, 94°C for 30 s, 60°C for 1 min, 72°C for 30 s; final extension, 72°C for 10 min. Amplified Pax3 fragments were analysed by electrophoresis on a 2.5% agarose gel in TAE buffer: 0.04 M Tris-acetate, 1 mM EDTA (pH 8.0).

2.3 Histological examination
Embryos were fixed by immersion in Bouin's fluid overnight, dehydrated through a graded series of alcohols and stored in absolute ethanol. Embryos were then cleared in Histoclear and embedded in paraffin wax. Serial sections of 10 µm thickness were cut on a Leica microtome, stained with haematoxylin and eosin, and mounted in DPX. The diameter of structures including the dorsal root ganglia and ventricular septal defect were measured using an eyepiece graticule on a Zeiss Axiophot microscope.

2.4 Scanning electron microscopy (SEM)
Hearts were removed from embryos in cold PBS and immersed overnight in a fixative comprising 2% glutaraldehyde, 1% formaldehyde in 0.1 M cacodylate buffer (300 mosmol/l). Following fixation, hearts were washed overnight in 0.1 M cacodylate buffer, postfixed for one hour in 0.1 M cacodylate buffer containing 1% osmium tetroxide, rinsed twice in isotonic 0.1 M cacodylate buffer, and then stored in the same buffer. Microdissection of hearts was performed by the method of Pexieder [11]. The hearts were then dehydrated through a graded series of ethanols and critical point dried using liquid CO₂, at a temperature of 50°C and pressure of 1400 lb/m².

2.5 Wet and dry weight measurements on embryos
All measurements were made on a Precisa 80A-200M balance. After measuring wet weight, the pericardium of the embryo was opened and the morphology of the outflow tract was recorded. Embryos were then dehydrated through a graded alcohol series and placed on paper filter discs for one week, prior to determination of dry weight.

	3 Results

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Our preliminary observations suggested that some Sp^2H/Sp^2H embryos survive to birth, in contrast to previous reports that all Sp [5]and Sp^1H [6]homozygotes die in utero. We set out, therefore, to determine whether homozygosity for the Sp^2H allele is lethal, and at which stage of gestation.

3.1 Variable stage of lethality of Sp^2H homozygotes
We carried out a detailed study of the survival of embryos in litters from matings between Sp^2H/+ individuals. Litter size was found to be relatively constant, at an average of 9.2 embryos per litter, between 9.5 and 13.5 days of gestation, but from 14.5 days onwards there was a decrease in mean litter size and, between 16.5 and 19.5 days, litters contained an average of only 5.4 embryos (Fig. 1a). One way analysis of variance showed that the differences in the mean values among the 9.5 and 13.5 days of gestation group and the 16.5 and 19.5 days are greater than would be expected by chance, and are statistically significant (P = <0.001). These results indicated that a sub-population of embryos die in utero. Indeed, dying embryos (defined as lacking a pulsating stream of blood within the umbilical artery/vein and an absence of contractions of the heart) were detected between 11.5 and 15.5 days of gestation, comprising up to 20% of each litter (Fig. 1b), although dying embryos were not found prior to 11.5 days nor between 16.5 and 18.5 days. Dying fetuses could again be detected in litters at 19.5 days, just prior to birth. The number of dying embryos observed on any one day of gestation was smaller than the magnitude of the reduction in litter size. The cumulative effect of loss of embryos on several successive days of gestation can probably account entirely for the observed reduction in sizes of litters.

View larger version (49K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Histograms illustrating the loss of approximately 60% of Sp2H homozygotes around 14.5 days of gestation. (a) Mean number of embryos per litter is constant until 13.5 days when a steady decrease occurs until 16.5 days. Thereafter, litter size is relatively constant until birth. A total of 112 litters were analysed in this study. (b) Non-viable embryos are observed in Sp2H litters between 11.5 and 15.5 days, and further dying fetuses can be detected just prior to birth, at 19.5 days. A total of 59 non-viable embryos were found out of a total of 901 embryos studied. (c) Litters (viable embryos only) between 9.5 and 13.5 days contain wild type, heterozygous and homozygous mutant embryos in a 1:2:1 ratio. However, from 14.5 days onwards, there is a decline in the proportion of Sp2H/Sp2H embryos, suggesting a loss of approximately 60% of the homozygotes at this time. Total number of viable embryos studied: +/+=233, Sp2H/+=456, Sp2H/Sp2H = 153.

 
Pax3 genotyping revealed that all embryos dying between 11.5 and 15.5 days were Sp^2H homozygotes, never heterozygotes or wild type embryos. A striking finding, however, was that only around 60% of the Sp^2H homozygotes died during this period of gestation, whereas the remaining 40% survived to birth (Fig. 1c). Since living Sp^2H homozygotes have never been observed postnatally in our colony, we conclude that there is a second stage of mortality of Sp^2H/Sp^2H individuals, beginning just prior to birth (Fig. 1b) and continuing in the immediate postnatal period.

3.2 Mortality in utero of Sp^2H homozygotes is not caused by their neural tube defects
We found that the presence of a neural tube defect is invariable in Sp^2H/Sp^2H embryos, although the nature of the defect was variable (Table 1). Embryos can have either lumbosacral spina bifida, or exencephaly, or both (Fig. 2a,b). There was no correlation, between the type of neural tube defect and the in utero mortality of Sp^2H homozygotes. For instance, if a particular type of neural tube defect were lethal in utero, then one would expect to see a reduction in the frequency of embryos with this defect late in gestation. In contrast, both spina bifida and exencephaly were equally frequent in embryos at 11.5–12.5 days of gestation and in fetuses near term (Fig. 2c), making it seem very unlikely that neural tube defects are the cause of death of around 60% of Sp^2H/Sp^2H embryos between 13.5 and 15.5 days. On the other hand, neural tube defects were very likely the cause of death in homozygotes surviving to birth, as is seen in other mouse mutants with comparable defects [12].

View this table: [in this window] [in a new window]   Table 1 Correlation between defects of the heart, neural tube and dorsal root ganglia in viable 13.5 day Sp2H embryos

 

View larger version (71K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Neural tube defects in Sp2H embryos. (a) Wild type embryo obtained late on 13.5 days of gestation exhibiting normal morphology, and (b) Sp2H/Sp2H littermate with exencephaly (E), lumbosacral spina bifida (S) and a curled tail (T). Arrows indicate subcutaneous edema in the homozygous embryo. (c) Incidence of neural tube defects in Sp2H/Sp2H embryos throughout the second half of gestation. Every homozygous embryo has either exencephaly or spina bifida, or both, and the incidence of each defect shows no consistent variation with gestational age. This suggests that neither type of neural tube defect is specifically associated with the 60% of Sp2H homozygotes that are lost around 14.5 days of gestation. Scale bar in (a) and (b) represents 1 mm. A total of 136 Sp2H/Sp2H embryos were studied between 10.5 and 19.5 days of gestation.

 
3.3 A proportion of Sp^2H homozygotes have congenital cardiac defects
Since the presence of neural tube defects cannot easily explain the death of a proportion of Sp^2H homozygotes, we next considered the possibility that the presence of congenital heart malformations may be the critical factor. We performed a detailed study of cardiac morphology in 12.5 day and 13.5 day embryos obtained from litters of Sp^2H/+ mice. Observations made by SEM on whole hearts, and by serial histological sectioning, revealed three main types of cardiac morphology. Normal hearts were found in all wild-type and heterozygous embryos examined. Moreover, 42.3% of Sp^2H/Sp^2H embryos at 12.5 days, and 40.6% at 13.5 days (Table 1), also had hearts with normal morphology. The remaining Sp^2H/Sp^2H embryos exhibited conotruncal heart defects that comprised either a common arterial trunk arising from the right ventricle (PTA; 50.0% of homozygotes at 12.5 days and 53.1% at 13.5 days) or double outlet of aorta and pulmonary trunk from the right ventricle (DORV; 7.7% of homozygotes at 12.5 days and 6.3% at 13.5 days).

Fig. 3 illustrates the appearance of these cardiac phenotypes at 13.5 days in scanning electron micrographs, and Fig. 4 shows representative histological sections through the region of the ventricular outflow tracts. In the normal 13.5 day embryo, the aorta and pulmonary trunk are separate vessels (Fig. 3a and Fig. 4a,b) connecting, respectively, with the left and right ventricles. This morphology was seen in all wild-type and heterozygous hearts and in around 40% of Sp^2H/Sp^2H hearts. In contrast, in the majority of the Sp^2H/Sp^2H embryos the ventricular outflow tracts were completely undivided (Fig. 3b and Fig. 4c), with a common arterial trunk arising solely from the right ventricle via a single arterial valve. Communication of the arterial trunk with the left ventricle was never observed other than via a co-existing ventricular septal defect. The pulmonary arteries were observed arising as separate vessels taking origin directly from the common trunk (Fig. 4d), but these arteries were invariably of smaller diameter than the pulmonary arteries of the normal embryo. In a smaller proportion of Sp^2H/Sp^2H embryos, a less severe defect, double outlet right ventricle, was observed. In these mutant embryos the ventricular outflow tracts had divided to form the aorta and pulmonary trunk, but the vessels were abnormally positioned relative to each other, and both arose exclusively from the right ventricle via two separate arterial valves (Fig. 3c). In homozygotes with either common arterial trunk or double outlet right ventricle, the endocardium, myocardium and valves all appeared histologically normal, indicating the specific nature of the cardiac defects in Sp^2H homozygotes.

View larger version (148K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Scanning electron micrographs of whole hearts from embryos on 13.5 days of gestation. The atria have been removed in order to illustrate the arrangement of ventricular outflow tracts and the arterial trunks. (a) Normal wild-type heart demonstrating the appearance of the aorta (A) and pulmonary trunk (P) as clearly separate vessels. Serial sectioning confirms that the aorta connects solely with the left ventricle and the pulmonary trunk with the right ventricle. (b) Sp2H/Sp2H heart with common arterial trunk. The undivided arterial trunk arises exclusively from the right ventricle. (c) Sp2H/Sp2H heart with double outlet right ventricle. The aorta (small arrowhead) and pulmonary trunk (large arrowhead) are clearly demarcated, yet serial sectioning demonstrates that both trunks arise exclusively from the right ventricle. Scale bar represents 0.1 mm.

 

View larger version (170K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Transverse histological sections through the thoracic cavity, at the level of the cardiac outflow tract, of late 13.5 day embryos. (a) Wild type embryo to illustrate the normal morphology. The pulmonary trunk (P) and aorta (A) are separated by a thick cellular septum, with the pulmonary trunk arising solely from the right ventricle (R). The pulmonary trunk connects with the descending aorta (S) via the arterial duct (asterisk). Note the large size of the dorsal root ganglia (D). (b) Sp2H/Sp2H embryo with normal heart morphology. The pulmonary valve is visible in this section. Dorsal root ganglia are present but reduced in size compared with the wild-type embryo. (c), (d) Sections at slightly different levels through a Sp2H/Sp2H embryo with a common arterial trunk. An undivided arterial trunk (T) arises solely from the right ventricle and connects, in adjacent sections, with the descending aorta (S). Pulmonary arteries can be seen arising separately from the trunk (arrows in (d)). Note the large, over-expanded caval veins (V) and total absence of dorsal root ganglia. Other abbreviations: L, left atrial chamber; N, neural tube; large and small arrowheads in (a) indicate the trachea and oesophagus, respectively. Scale bar represents 0.2 mm.

 
Sections through the ventricles of normal 13.5 day embryos revealed the ventricular septum to be intact (Fig. 5a). This was seen not only in +/+ and Sp^2H/+ embryos, but also in Sp^2H/Sp^2H embryos with normally septated outflow tracts. By contrast, all Sp^2H/Sp^2H embryos with abnormal ventriculo-arterial connections, either common arterial trunk or double outlet right ventricle, exhibited a co-existing ventricular septal defect of variable size (Fig. 5b–d) which was not seen to close, even in Sp^2H homozygotes that were dying at the time of dissection from the uterus. The interventricular communication between the left and right ventricles was directly adjacent to the membranous portion of the ventricular septum, the formation of the muscular outlet septum otherwise appears normal. To investigate in more detail the mechanics of closure of the ventricular septum, we measured the size of the interventricular communication at an earlier stage, 12.5 days, when the septum is open in both normal and malformed hearts (Table 2). Although there was no difference among the various embryo genotypes in diameter of the ventricles, the interventricular communication was approximately twice as large in Sp^2H/Sp^2H embryos with abnormal ventricular outflow tracts when compared to the embryos with normal outflow tract sepation, demonstrating abnormal formation of the ventricular outlet components the defective hearts.

View larger version (227K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Transverse histological sections through the heart, at the level of the interventricular septum, in late 13.5 day embryos. (a) Sp2H/+ heart showing normal morphology. The right (R) and left (L) ventricles are separated by a thick, muscular septum (S). (b) Sp2H/Sp2H heart with common arterial trunk. There is a wide communication between the ventricles in the apical part of the septum (arrowhead). (c) Another Sp2H/Sp2H heart with common arterial trunk in which the ventricular septal defect (arrowhead) is very small. Higher magnification (d) shows that the defect is patent, as blood cells can be seen passing between the ventricles. Scale bar represents 0.2 mm.

 

View this table: [in this window] [in a new window]   Table 2 Closure of the interventricular septum in normal and malformed splotch hearts at 12.5 days of gestation

 
3.4 Death in utero results from cardiac failure in embryos with cardiac defects
Observations on embryos at 13.5 and 14.5 days of gestation revealed a striking correlation between the presence of cardiac abnormalities and mortality in utero. Embryos were judged as dying if pulsatile release of blood from the umbilical vessels was absent when the embryos were dissected from the uterus. The circulation in the non-viable Sp^2H homozygotes appeared to ‘ebb and flow’, as in early development. Non-viable Sp^2H homozygotes all had common arterial trunk or double outlet right ventricle, whereas Sp^2H homozygotes with normal hearts appeared viable when removed from the uterus at this early fetal stage. Indeed, all the Sp^2H homozygotes surviving past 14.5 days of gestation did not have any heart defects. Histological analysis of the Sp^2H homozygotes with outflow tract defects revealed signs of cardiac failure: the superior caval veins were over-expanded (Fig. 4b) and the fetal liver was enlarged and engorged with blood. Moreover, measurement of wet and dry embryonic weights revealed that the wet/dry ratio was significantly higher in Sp^2H/Sp^2H embryos with cardiac defects (mean wet weight/dry weight in mg=117.9/6.2=19.0) (P<0.05) than in Sp^2H/Sp^2H embryos with normal hearts (134.5/8.2=16.4), in Sp^2H/+ embryos (138.1/8.9=15.5) or in +/+ embryos (137.5/9.2=15.0). This finding, taken together with the edematous appearance of Sp^2H homozygotes (Fig. 2b), suggests that Sp^2H/Sp^2H embryos with heart defects are fluid-overloaded and supports the idea that they die in utero from cardiac failure.

3.5 Sp^2H cardiac defects correlate with neural crest defects, but not neural tube defects
We examined the association between abnormal ventriculo-arterial connections, neural tube defects and size of the dorsal root ganglia in Sp^2H/Sp^2H embryos. Reduced size of dorsal root ganglia probably results from defective migration of the neural crest in splotch homozygotes [5]. There was no discernible correlation between the presence or type of cardiac defect and the type of neural tube defect: for instance, combined spina bifida and exencephaly was equally common in Sp^2H homozygotes with cardiac defects as in those with normal hearts (Table 1). In addition, around 4% of Sp^2H/+ embryos exhibited neural tube defects, although combined spina bifida and exencephaly was not seen in heterozygotes. In contrast to the lack of correlation between cardiac and neural tube defects, there was a strong association between cardiac and neural crest defects (Table 1). Wild-type and heterozygous embryos had closely similar sized dorsal root ganglia whereas Sp^2H/Sp^2H embryos had significantly smaller ganglia. Most strikingly, Sp^2H homozygotes with cardiac defects had very much smaller ganglia than homozygotes with normal hearts. Indeed, a number of homozygotes with abnormal ventriculo-arterial connections had no recognisable dorsal root ganglia at all, at the level of the anticipated contribution to the cardiac outflow tracts (Fig. 4b). These findings support the idea that the malformations of the ventricular outflow tracts result from failure of colonisation of the putative aorto-pulmonary septum by cells from the cardiac neural crest. It may be that severe diminution, or total absence, of the cardiac neural crest is necessary to yield a common arterial trunk or double outlet from the right ventricle, whereas persistence of sub-normalnormal numbers of neural crest cells is sufficient to promote normal septation of the outflow tract. Indeed, it was shown that neural crest ablations of only part of the ‘cardiac neural crest’ region gives rise to predominantly double outlet, whereas complete ablation gives rise to predominantly common arterial trunk [12].

	4 Discussion

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
We have examined litters of mouse embryos segregating the Sp^2H mutant allele in order to determine the cause of in utero mortality of splotch homozygotes. Our results demonstrate that although Sp^2H/Sp^2H embryos invariably have neural tube defects, there is no evidence that the nature or severity of the defect has any influence upon whether the embryo dies around 13.5–14.5 days of gestation. On the other hand, we find that presence of either a common arterial trunk or double outlet from the right ventricle, is strongly associated with in utero death of Sp^2H homozygotes.

4.1 Why are abnormal ventriculo-arterial connections associated with mortality in splotch embryos?
Mortality in utero is not normally considered a feature of common arterial trunk or double outlet right ventricle either in humans [14]or in the chick following neural crest ablation [7]. However, in recent video-microscopy studies of 12.5 and 13.5 day Sp^2H/Sp^2H hearts, we found poor myocardial contractility compared with normal litter mates. In Sp^2H/Sp^2H hearts with outflow tract defects, the left ventricular area ejection fraction was less than 50% when compared with normal hearts. However, mutant hearts exhibited normal contractile force in detergent-skinned ventricular muscle strips but, when excitation-contraction coupling was examined, Ca²⁺ transients were found to be undetectable. Ca²⁺ currents were examined using the perforated patch clamp technique on isolated ventricular myocytes, and the magnitude of the Ca²⁺ current was found to be reduced in Sp^2H/Sp^2H hearts with common arterial trunk (Conway, Greene, Godt et al.; unpublished). These findings, together with our observations of edema, fluid-overload, expanded caval veins and liver engorgement, suggest that Sp^2H/Sp^2H embryos with abnormal ventriculo-arterial connections are dying of cardiac failure in utero.

Lethality in Sp^2H/Sp^2H embryos seems most likely to result from a functional haemodynamic defect of the cardiovascular system that shares a common developmental origin with the abnormal ventriculo-arterial connections. Altered hemodynamics could arise, for instance, from a restrictive ventricular septal defect, as occurs with small septal defects in humans [15], or from alteration in the pattern of aortic arch arteries, as has been described in splotch homozygotes [6]. An alternative mechanism is suggested by studies in the chick, where a reduction in myocardial contractility has been detected in neural crest-ablated embryos prior to the appearance of structural heart defects [16, 17], suggesting that the neural crest may exert an influence on the developing myocardium from an early stage in its development. A similar mechanism could operate in splotch embryos, although the nature of this putative interaction is currently unknown. What may be of significance is that all the embryos with abnormal hearts showed the arterial segment connected exclusively to the right ventricle. Clearly, further studies are required to determine the cause of cardiac failure in Sp^2H/Sp^2H embryos with abnormal hearts.

4.2 Requirement of the neural crest for normal development of the ventricular outflow tracts
The neural crest is an embryonic stem cell population that arises from the edges of the neural plate and migrates ventrally and laterally through the embryonic mesoderm, differentiating into a variety of cell types [18]. Labelling of pre-migratory neural crest cells, using the chick/quail cytological marker [19]has revealed that the aorto-pulmonary septum contains derivatives of the neural crest. Moreover, DiI labelling in the rat embryo [20]and tracing of neural crest migration using gene expression markers in the mouse [21]have both confirmed the existence of a ‘cardiac’ neural crest cell sub-population in mammals. Ablation of the chick pre-migratory cardiac neural crest leads to defective septation of the outflow tract in avian embryos [19]and it seems likely that defective neural crest migration in splotch embryos is similarly responsible for the presently observed cardiac defects. Indeed, we observed a marked reduction or absence of cardiac neural crest cells, as recognised by their expression of Pax3, Hoxa3, CrabpI, Prx1, Prx2 and c-met in 10.5 day Sp^2H/Sp^2H embryos [21]. In the present study, we found a close correlation between reduced size of dorsal root ganglia and presence of abnormal ventriculo-arterial connections, also pointing to a relationship between defective neural crest migration and failure of septation of the cardiac outflow tract. It should be noted, nonetheless, that the common feature of the abnormal hearts was origin of the arterial segment exclusively from the right ventricle rather than complete failure of septation, since the hearts with double outlet right ventricle had separate arterial valves and formed a muscular outlet septum. This fact, together with origin of the common trunks and double outlet vessels from the right ventricle, suggests that the Sp^2H/Sp^2H embryos fail to complete ventricular septation (which is partially present in those with double outlet right ventricle) because of the failure to transfer the arterial segment, in part, to the left ventricle. This is a most unusual finding within humans with this malformation, and points to more complex factors being involved. Similarly, within the chick neural crest-ablation system, the truncus arises predominantly over-riding the ventricular septum [16]. These differences may reflect different outflow tract architecture within the different species (Anderson, Kirby and Conway; unpublished observations).

The fact the that only approximately 60% of Sp^2H homozygotes die in utero at 13.5–14.5 days of gestation, and have cardiac malformations involving partial or complete failure of septation, suggests that there is sufficient neural crest colonisation of the outflow tract within those Sp^2H homozygotes that survive until birth. Thus, there appears to be a minimum threshold number of cardiac neural crest cells that are required for complete septation of the outflow tract.

4.3 Relationship between neural crest migration and neural tube closure
Homozygous Sp^2H embryos are characterised by defects of both closure of the neural tube and migration from the neural crest. This led Auerbach [5]to suggest that both types of developmental defect may result from an abnormality of dorsal neuroepithelial cells that comprise the presumptive neural crest and the site of fusion of the apposing neural folds during neurulation. However, there is no obligatory relationship between neural crest migration and neural tube closure. For instance, neural crest cells emigrate from the neuroepithelium prior to closure in the cranial region but following closure in the spinal region of avian and mammalian embryos. Moreover, neural tube defects have been observed in the presence of apparently normal dorsal root ganglia in the cranial region of splotch mice [22]and in the lumbosacral region of splotch/curly tail double mutant mice [23]. In the present study we show that there is no particular association between the type or severity of neural tube defect and the presence of neural crest-related heart defects. It seems most likely, therefore, that defects due to closure of the neural tube and migration from the neural crest are independent effects of a lack of the Pax3 transcription factor. Deficiency in the migration of somite-derived myoblast precursors to form the limb musculature is also observed in splotch homozygotes [24–26]and this may represent a third requirement for the Pax3 gene product. Recently, it has been shown that Pax3 is a key regulator of skeletal myogenesis, in that Pax3 expression is sufficient to induce expression of MyoD, Myf-5 and myogenin basic helix-loop-helix myogenic transcription factors [27]. There is no expression of MyoD, Myf-5 and myogenin in cardiac muscle, but as many of the genes that are regulated by them are expressed in both skeletal and cardiac muscle [3], this indicates that MyoD, Myf-5 and myogenin are not the targets of Pax3-regulation in the heart. Currently, it is not known if Pax3 also plays a role in cardiac myogenesis or even if it is expressed in the embryonic heart.

4.4 Developmental role of the Pax3 transcription factor
The Sp^2H mutation involves a 32 base pair deletion in the homeodomain, one of the two main DNA-binding regions, of the Pax3 gene [4]. This deletion leads to formation of a stop codon so that the C-terminal portion of the homeodomain is missing from the mature protein. DNA-binding studies in vitro have shown that an expressed protein with structure similar to the predicted truncated Pax3 protein in Sp^2H has reduced DNA-binding activity with an altered specificity for target sequences [28]. The other splotch alleles disrupt the Pax3 gene in varying ways: Sp^r and Sp^4H involve deletion of the entire gene, Sp involves a point mutation at a splice acceptor site that leads to aberrantly spliced transcripts and Sp^d has a point mutation in the paired box region. Sp^1H is probably an identical mutation to Sp^2H, since both mutations were isolated from the same mutagenised male [9]. It seems likely that each mutation results in loss of some or all of the functions of the Pax3 gene in controlling downstream gene expression. Until the downstream targets of Pax3 are determined, it will not be possible to explain the variation in phenotype, for instance, between Sp^2H in which homozygotes develop exencephaly, spina bifida and heart defects, and Sp^d in which only spinal neural tube defects are seen in homozygotes.

Pax3 is expressed in the dorsal part of the neural tube, throughout the early somite, in the dermomyotome, in muscle precursors that migrate from the ventrolateral portion of the somite to colonise the limb, and in the cardiac and other early migrating neural crest [21, 25, 26, 29]. Recent studies have suggested several likely targets for Pax3 gene regulation at these sites, including N-cadherin, c-met, Msx2 and NCAM. For instance, N-cadherin expression co-localises with Pax3 in the developing somite and dermomyotome [30], while c-met expression is absent from the dermomyotome and limb muscle precursors in splotch mutant embryos [30, 31]. Similarly, in the neural tube, Msx2 exhibits a closely similar pattern of expression to Pax3 [32], whereas NCAM expression is altered in splotch mutants [33]. It is difficult to determine from gene expression studies of this type whether the putative regulatory interactions are direct or mediated via intermediate gene products. Further insight is likely to emerge from cell transfection experiments in which the action of Pax3 in regulating target gene expression can be tested directly.

There is an ever increasing list of different gene mutations that show similar embryonic heart and neural tube defects (e.g., RXR alpha knockout mice [34]) which also exhibiting different combinations of neural crest-related heart defects [2, 3], so that it should be possible in future to gain insight into the mechanisms underlying human cardiovascular development and congenital heart defects.

Time for primary review 21 days.

	Acknowledgements

 
This work was supported by the British Heart Foundation and the Wellcome Trust.

	References

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 

1. Edmonds L.D, James L.M. Temporal trends in the birth prevalence of selected congenital malforamtions in the Birth Defects Monitoring Progam/Commission on Professional and Hospital Activities, 1979–1989. Teratology (1993) 48:647–649.[CrossRef][Web of Science][Medline]
2. Rossant J. Mouse mutants and cardiac development — New molecular insights into cardiogenesis. Circ Res (1996) 78:349–353.[Abstract/Free Full Text]
3. Olson E.N, Srivastava D. Molecular pathways controlling heart development. Science (1996) 272:671–676.[Abstract]
4. Epstein D.J, Vekemans M, Gros P. Splotch (Sp^2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3. Cell (1991) 67:767–774.[CrossRef][Web of Science][Medline]
5. Auerbach R. Analysis of the developmental effects of a lethal mutation in the house mouse. J Exp Zool (1954) 127:305–329.[CrossRef][Web of Science]
6. Franz T. Persistent truncus arteriosus in the Splotch mutant mouse. Anat Embryol (1989) 180:457–464.[CrossRef][Medline]
7. Kirby M.L, Waldo K.L. Role of neural crest in congenital heart disease. Circulation (1990) 82:332–340.[Free Full Text]
8. Kirby M.L, Waldo K.L. Neural crest and cardiovascular patterning. Circ Res (1995) 77:211–215.[Free Full Text]
9. Beechey C.V, Searle A.G. Mutations at the Sp locus. Mouse News Lett (1986) 75:28.
10. Estibeiro JP, Copp AJ, Cockroft DL, Brown NA, Clarke DO. Extraction of macromolecules from embryonic material, in Copp AJ, Cockroft DL, editors. Postimplantation mammalian embryos: a practical approach. Oxford: IRL Press, 1990, pp. 173–204.
11. Vuillemin M, Pexieder T. Normal stages of cardiac organogenesis in the mouse: I. Development of the external shape of the heart. Am J Anat (1989) 184:101–113.[CrossRef][Web of Science][Medline]
12. Nishibatake M.D, Kirby M.L, Van Mierop M.D. Pathogenesis of persistent truncus arteriosus and dextroposed aorta in the chick embryo after neural crest ablation. Circulation (1987) 75:255–264.[Abstract/Free Full Text]
14. Mair DD, Edwards WD, Fuster V, Seward JB, Danielson GK. Truncus arteriosus and aortopulmonary window, in Anderson RH, Mccartney FJ, Shinebourne EA, Tynan M, editors. Paediatric cardiology, vol. 2. Edinburgh: Churchill Livingstone, 1992, pp. 913–929.
15. Anderson RH, Macartney FJ, Shinebourne EA, Tynan M. Paediatric cardiology. Edinburgh: Churchill Livingstone, 1987.
16. Tomita H, Connuck D.M, Leatherbury L, Kirby M.L. Relation of early hemodynamic changes to final cardiac phenotype and survival after neural crest ablation in chick embryos. Circulation (1991) 84:1289–1295.[Abstract/Free Full Text]
17. Leatherbury L, Connuck D.M, Kirby M.L. Neural crest ablation versus sham surgical effects in a chick embryo model of defective cardiovascular development. Pediatr Res (1993) 33:628–631.[Web of Science][Medline]
18. Weston J.A. The migration and differentiation of neural crest cells. Adv Morphog (1970) 8:41–114.[Medline]
19. Kirby M.L, Gale T.F, Stewart D.E. Neural crest cells contribute to normal aorticopulmonary septation. Science (1983) 220:1059–1061.[Abstract/Free Full Text]
20. Fukiishi Y, Morriss-Kay G.M. Migration of cranial neural crest cells to the pharyngeal arches and heart in rat embryos. Cell Tissue Res (1992) 268:1–8.[CrossRef][Web of Science][Medline]
21. Conway S.J, Henderson D.J, Copp A.J. Pax3 is required for cardiac neural crest migration in the mouse: evidence from the splotch (Sp^2H) mutant. Development (1997) 124:505–514.[Abstract]
22. Franz T. Neural tube defects without neural crest defects in Splotch mice. Teratology (1992) 46:599–604.[CrossRef][Web of Science][Medline]
23. Estibeiro J.P, Brook F.A, Copp A.J. Interaction between splotch (Sp) and curly tail (ct) mouse mutants in the embryonic development of neural tube defects. Development (1993) 119:113–121.[Abstract]
24. Franz T, Kothary R, Surani M.A.H, Halata Z, Grim M. The Splotch mutation interferes with muscle development in the limbs. Anat Embryol (1993) 187:153–160.[Medline]
25. Bober E, Franz T, Arnold H.-H, Gruss P, Tremblay P. Pax-3 is required for the development of limb muscles: A possible role for the migration of dermomyotomal muscle progenitor cells. Development (1994) 120:603–612.[Abstract]
26. Goulding M, Lumsden A, Paquette A.J. Regulation of Pax-3 expression in the dermomyotome and its role in muscle development. Development (1994) 120:957–971.[Abstract]
27. Rawls A, Olson E.N. MyoD meets its maker. Cell (1997) 89:5–8.[CrossRef][Web of Science][Medline]
28. Chalepakis G, Goulding M, Read A, Strachan T, Gruss P. Molecular basis of splotch and Waardenburg Pax-3 mutations. Proc Natl Acad Sci USA (1994) 91:3685–3689.[Abstract/Free Full Text]
29. Goulding M.D, Chalepakis G, Deutsch U, Erselius J.R, Gruss P. Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J (1991) 10:1135–1147.[Web of Science][Medline]
30. Daston G, Lamar E, Olivier M, Goulding M. Pax-3 is necessary for migration but not differentiation of limb muscle precursors in the mouse. Development (1996) 122:1017–1027.[Abstract]
31. Yang X.-M, Vogan K, Gros P, Park M. Expression of the met receptor tyrosine kinase in muscle progenitor cells in somites and limbs is absent in Splotch mice. Development (1996) 122:2163–2171.[Abstract]
32. Takahashi Y. Organization of the spina bifida neural tube in Splotch (Pax-3 defective) mouse embryos. Dev Growth Diff (1996) 38:23–31.[CrossRef][Web of Science]
33. Moase C.E, Trasler D.G. N-CAM alterations in splotch neural tube defect mouse embryos. Development (1991) 113:1049–1058.[Abstract]
34. Sucov H.M, Dyson E, Gumeringer C.L, Price J, Chien K.R, Evans R.M. RXR mutant mice establish a genetic basis for vitaminA signaling in heart morphogenesis. Genes Dev (1994) 8:1007–1018.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				E. Goldmuntz, S. Woyciechowski, D. Renstrom, P. J. Lupo, and L. E. Mitchell Variants of Folate Metabolism Genes and the Risk of Conotruncal Cardiac Defects Circ Cardiovasc Genet, December 1, 2008; 1(2): 126 - 132. [Abstract] [Full Text] [PDF]

				C.-P. Chang, K. Stankunas, C. Shang, S.-C. Kao, K. Y. Twu, and M. L. Cleary Pbx1 functions in distinct regulatory networks to pattern the great arteries and cardiac outflow tract Development, November 1, 2008; 135(21): 3577 - 3586. [Abstract] [Full Text] [PDF]

				D Obler, A L Juraszek, L B Smoot, and M R Natowicz Double outlet right ventricle: aetiologies and associations J. Med. Genet., August 1, 2008; 45(8): 481 - 497. [Abstract] [Full Text] [PDF]

				M. Ream, A. M. Ray, R. Chandra, and D. M. Chikaraishi Early fetal hypoxia leads to growth restriction and myocardial thinning Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R583 - R595. [Abstract] [Full Text] [PDF]

				F. Bajolle, S. Zaffran, R. G. Kelly, J. Hadchouel, D. Bonnet, N. A. Brown, and M. E. Buckingham Rotation of the Myocardial Wall of the Outflow Tract Is Implicated in the Normal Positioning of the Great Arteries Circ. Res., February 17, 2006; 98(3): 421 - 428. [Abstract] [Full Text] [PDF]

				Y. Wang, E. Vachon, J. Zhang, V. Cherepanov, J. Kruger, J. Li, K. Saito, P. Shannon, N. Bottini, H. Huynh, et al. Tyrosine phosphatase MEG2 modulates murine development and platelet and lymphocyte activation through secretory vesicle function J. Exp. Med., December 5, 2005; 202(11): 1587 - 1597. [Abstract] [Full Text] [PDF]

				P. R.J. Bois, K. Izeradjene, P. J. Houghton, J. L. Cleveland, J. A. Houghton, and G. C. Grosveld FOXO1a acts as a selective tumor suppressor in alveolar rhabdomyosarcoma J. Cell Biol., September 12, 2005; 170(6): 903 - 912. [Abstract] [Full Text] [PDF]

				C. A. Nichols and T. L. Creazzo L-type Ca2+ channel function in the avian embryonic heart after cardiac neural crest ablation Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1173 - H1178. [Abstract] [Full Text] [PDF]

				T. L. Macatee, B. P. Hammond, B. R. Arenkiel, L. Francis, D. U. Frank, and A. M. Moon Ablation of specific expression domains reveals discrete functions of ectoderm- and endoderm-derived FGF8 during cardiovascular and pharyngeal development Development, December 22, 2003; 130(25): 6361 - 6374. [Abstract] [Full Text] [PDF]

				S. J. Kwang, S. M. Brugger, A. Lazik, A. E. Merrill, L.-Y. Wu, Y.-H. Liu, M. Ishii, F. O. Sangiorgi, M. Rauchman, H. M. Sucov, et al. Msx2 is an immediate downstream effector of Pax3 in the development of the murine cardiac neural crest Development, March 3, 2003; 129(2): 527 - 538. [Abstract] [Full Text] [PDF]

				Z. Yin, J. Haynie, X. Yang, B. Han, S. Kiatchoosakun, J. Restivo, S. Yuan, N. R. Prabhakar, K. Herrup, R. A. Conlon, et al. The essential role of Cited2, a negative regulator for HIF-1alpha , in heart development and neurulation PNAS, August 6, 2002; 99(16): 10488 - 10493. [Abstract] [Full Text] [PDF]

				D. U. Frank, L. K. Fotheringham, J. A. Brewer, L. J. Muglia, M. Tristani-Firouzi, M. R. Capecchi, and A. M. Moon An Fgf8 mouse mutant phenocopies human 22q11 deletion syndrome Development, January 10, 2002; 129(19): 4591 - 4603. [Abstract] [Full Text] [PDF]

				A. F. Machado, E. F. Zimmerman, D. N. Hovland Jr., R. Weiss, and M. D. Collins Diabetic Embryopathy in C57BL/6J Mice: Altered Fetal Sex Ratio and Impact of the Splotch Allele Diabetes, May 1, 2001; 50(5): 1193 - 1199. [Abstract] [Full Text]

				S. Ausoni and S. Sartore Cell Lineages and Tissue Boundaries in Cardiac Arterial and Venous Poles : Developmental Patterns, Animal Models, and Implications for Congenital Vascular Diseases Arterioscler Thromb Vasc Biol, March 1, 2001; 21(3): 312 - 320. [Abstract] [Full Text] [PDF]

				M. J. Anderson, G. D. Shelton, W. K. Cavenee, and K. C. Arden Embryonic expression of the tumor-associated PAX3-FKHR fusion protein interferes with the developmental functions of Pax3 PNAS, February 13, 2001; 98(4): 1589 - 1594. [Abstract] [Full Text] [PDF]

				P. J. Scambler The 22q11 deletion syndromes Hum. Mol. Genet., October 1, 2000; 9(16): 2421 - 2426. [Abstract] [Full Text] [PDF]

				M. J.B. van den Hoff and A. F.M. Moorman Cardiac neural crest: the holy grail of cardiac abnormalities? Cardiovasc Res, August 1, 2000; 47(2): 212 - 216. [Full Text] [PDF]

				S. J. Conway, J. Bundy, J. Chen, E. Dickman, R. Rogers, and B. M. Will Decreased neural crest stem cell expansion is responsible for the conotruncal heart defects within the Splotch (Sp2H)/Pax3 mouse mutant Cardiovasc Res, August 1, 2000; 47(2): 314 - 328. [Abstract] [Full Text] [PDF]

				R. Eferl, M. Sibilia, F. Hilberg, A. Fuchsbichler, I. Kufferath, B. Guertl, R. Zenz, E. F. Wagner, and K. Zatloukal Functions of c-Jun in Liver and Heart Development J. Cell Biol., May 31, 1999; 145(5): 1049 - 1061. [Abstract] [Full Text] [PDF]

				T. Clark, S. Conway, I. Scott, P. Labosky, G Winnier, J Bundy, B. Hogan, and D. Greenspan The mammalian Tolloid-like 1 gene, Tll1, is necessary for normal septation and positioning of the heart Development, January 6, 1999; 126(12): 2631 - 2642. [Abstract] [PDF]

				D. J. Henderson, S. J. Conway, N. D. E. Greene, D. Gerrelli, J. N. Murdoch, R. H. Anderson, and A. J. Copp Cardiovascular Defects Associated With Abnormalities in Midline Development in the Loop-tail Mouse Mutant Circ. Res., July 6, 2001; 89(1): 6 - 12. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Conway, S. J Articles by Copp, A. J Search for Related Content PubMed PubMed Citation Articles by Conway, S. J Articles by Copp, A. J Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics