Effects of genetic background on cardiovascular anomalies in the Ts16 mouse

Authors


Abstract

To investigate the genetic contribution to phenotypic variability in aneuploidy, we generated mice with trisomy 16 (Ts16) by mating [Rb(6.16)24Lub × Rb(16.17)7Bnr]F1 males with females from four inbred strains, BALB/cJ, C3H/HeJ, C57BL/6J, and DBA/2J. Among the four Ts16 strains that were generated, there were no significant differences in survival, weight, or length relative to euploid control littermates at either embryonic day (E) 14.5 or E17.5. All Ts16 fetuses at E14.5 had edema that ranged from mild to severe, increased amniotic fluid volume, and a thickened neck. At E17.5, Ts16 fetuses exhibited two distinct phenotypes, one with an edematous morphology and the other runt-like. None of these gross morphological abnormalities was strain-specific either in occurrence or frequency. At E10.5, there were pharyngeal arch artery (PAA) anomalies in all Ts16 embryos on the C3H/HeJ background, but none in trisomics on the other three backgrounds. However, at E17.5, there was in addition to ventricular and atrioventricular septal defects, a high frequency of aortic arch defects in Ts16 fetuses, irrespective of genetic background. Taken together, these findings indicate that there are at least two mechanistic responses to the presence of three copies of mouse chromosome 16 in the modeling of the cardiovascular system: one, development of PAA defects, is strongly influenced by genetic background; but the second, development of aortic arch anomalies in the absence of preexisting PAA anomalies, is not. Developmental Dynamics 232:131–139, 2005. © 2004 Wiley-Liss, Inc.

INTRODUCTION

Although the overall patterns of anomalies and minor variations in development that are characteristic of chromosomal disorders are quite distinct, these conditions are notable for the variability of their phenotypes. For example, in Down syndrome (DS), which results from trisomy 21, none of the typical physical features is present in all affected individuals, and it is very unlikely that two persons would be phenotypically identical. In the case of the major congenital malformations known to be associated with DS, congenital heart disease occurs only half of the time, and duodenal atresia or stenosis in only 2.5% (Epstein, 2001).

Several possible explanations for this phenotypic variability in aneuploidy have been suggested (Epstein, 1988). One is that allelic differences in the genes that are triplicated might have different effects when present in three copies. For example, heterotrisomy for a gene (or genes) within a 9.6 cM minimal region (D21S167) on human chromosome 21 has been postulated to be a contributing factor to the pathogenesis of ventricular septal defects (VSDs) in DS, either through the presence of three different specific alleles or through the presence of specific combinations of alleles (Baptista et al., 2000). Second, the overall genetic background of the individual in whom the aneuploidy occurs could affect the penetrance and expression of different phenotypic features. Precedents for such background effects, often attributable to the existence of modifier loci, exist for many genetic traits in mice (Nadeau, 2001). Third, environmental factors, whether internal or external, that may influence developmental processes could affect the development of different components of the phenotype. Fourth, developmental processes are subject to random or stochastic processes that could have a similar influence. Finally, the altered expression of genes on the aneuploid chromosome could have a more global effect on the regulation or homeostasis of developmental systems, which could lead to phenotypic variability (Shapiro, 1997).

At the present time, it is not possible to distinguish among these possibilities in human aneuploidies, although it is likely that insight into genetic background effects in the future could be derived from genome-wide analyses. However, mouse models that have been developed for the study of DS provide a useful tool for looking at the effect of background genotype on specific components of an aneuploid phenotype. Mouse trisomy 16 (Ts16) was one of the first such models to be developed (Gropp et al., 1975; Cox et al., 1984).

Although Ts16 is no longer used as a model for DS, its robust phenotype makes it an attractive subject for the investigation of the effects of genetic background on phenotype. Here, we explore the effects of four different genetic backgrounds on the phenotype of Ts16 embryos and fetuses. Particular attention has been paid to development of the cardiovascular system, which is known to be seriously affected in Ts16. Our data demonstrate the complete penetrance of pharyngeal arch artery (PAA) anomalies and subsequent defects in the patterning of the great vessels when the maternal contribution is from the C3H/HeJ strain, but not when other genetic backgrounds are present. The strain-specificity of this feature makes the genetic contribution responsible amenable to mapping. Our data also demonstrate that defects in the patterning of the great vessels can arise at a significant frequency even in the absence of preexisting PAA anomalies, suggesting that there are at least two mechanistic responses to the presence of three copies of MMU16 in the modeling of the cardiovascular system.

RESULTS

Frequency of Trisomy

Although previous studies have shown the prevalence of Ts16 fetuses to be greater on a C3H/HeJ female background (21%) compared with the C57BL/6J female strain (11%; Epstein and Vekemans, 1990), we did not find the incidence of Ts16 to vary significantly with the genetic background of the female parent at E14.5 (P > 0.24; Fig. 1). Discrepancies in the incidence of Ts16 fetuses may be explained by the use of different male strains heterozygous for the Robertsonian translocation chromosome, Rb(16.17). Of interest, we also report a frequency of 20–25% Ts16 at embryonic day (E) 14.5, whereas Epstein and Vekemans found Ts16 frequencies ranging from 10 to 21%, depending on the parental cross. At E17.5, the frequency of Ts16 fetuses was lower in litters with a C3H/HeJ maternal contribution compared with the C57BL/6J, BALB/cJ, and DBA/2J females, but the difference was not significant. The frequency of postimplantation death was determined by dividing the number of moles (deciduomata) and late deaths by the number of implantation sites. Again, no significant differences in the frequencies of moles and fetal deaths were detected at E14.5 (P > 0.05). However, there did appear to be a higher frequency of fetal loss at E17.5 in the C3H/HeJ females (62%) compared with the C57BL/6J (47%), BALB/cJ (41%), DBA/2J (31%) females; but again, these differences did not attain significance (P > 0.05; data not shown).

Figure 1.

Frequencies of live Ts16 mice among live embryos at embryonic day (E) 14.5 and E17.5. There are no significant differences among strains at either age. The following number of Ts16/2N embryos were examined: at E14.5, C57BL/6J (32/110), DBA/2J (19/103); BALB/cJ (24/121), C3H/HeJ (24/107); and at E17.5, C57BL/6J (17/95), DBA/2J (16/80), BALB/cJ (23/93), C3H/HeJ (16/164). Error bars indicate 95% confidence interval for each proportion.

Gross Morphology

Among the four strains of Ts16 that were generated, there were no significant differences in the ratios of trisomic/euploid fetal weights and lengths at E14.5 and E17.5 (Fig. 2). Although Ts16 weight was reduced by E17.5 compared with control littermates in all four strains, this reduction was not significant. There was, however, a significant difference in Ts16 placental weight relative to euploid controls among the four strains, with DBA/2J placentas weighing significantly less than C3H/HeJ and C57BL/6J (data not shown).

Figure 2.

Embryonic weights at embryonic day (E) 14.5 and E17.5. At E14.5, Ts16 embryonic weight approximates 2N embryonic weight, but by E17.5, Ts16 weight is reduced relative to 2N littermates in all four strains. However, the relative reduction in weight, demonstrated by the ratio of (Ts embryonic weight)/(2N littermate embryonic weight) is not significantly different among strains. Lines follow the ratio of (Ts embryonic weight)/(2N littermate embryonic weight) at each age: triangles for E14.5, squares for E17.5. Error bars indicate 95% confidence intervals for each ratio.

All Ts16 fetuses at E14.5 had mild to severe edema, an increased volume of amniotic fluid, and a thickened neck. By E17.5, the trisomic fetuses exhibited two distinct phenotypes, one with an edematous morphology and the other runt-like (Fig. 3A), but all had open eyelids and displayed placental defects similar to those found at E14.5. There was gross disorganization of the placenta with an increased area of spongiotrophoblast invading the labyrinth, an arrangement that could effectively reduce exchange between the maternal and fetal blood by as much as two thirds (manuscript in preparation). The majority of trisomic fetuses on all backgrounds had malformations of the spinal column and alterations of the vertebral arches that were often accompanied by rib anomalies. The bones of the limbs showed delayed ossification, but there were no malformations (Fig. 3B–D).

Figure 3.

Gross morphology at embryonic day (E) 17.5. A: Ts16 embryos exhibited two phenotypes: one edematous and the other runt-like. All four strains showed both phenotypes in approximately a 2:1 ratio. No significant differences between strains were found in the frequency or expression of these two phenotypes. B–D: Delayed development of the entire skeleton was striking in runt-like (C) and edematous (D) fetuses compared with euploid controls (B).

Analysis of PAAs at E10.5

We examined the development of the PAAs in a total of 213 E10.5 embryos, 45 (21%) of which were trisomic. At least eight trisomics of each strain were examined. The PAAs were visualized by intracardiac India ink injection and scored as “present” or “absent” (Table 1). All Ts16 embryos derived from C57BL/6J, BALB/cJ, and DBA/2J females, as well as euploid control littermates, displayed normal development of the PAAs (Fig. 4A). However, PAA anomalies were detected in all embryos on the C3H/HeJ maternal background and typically involved abnormal development of at least one of the fourth or sixth PAAs (Fig. 4B,C). Occasionally, there was severe disruption of the entire PAA structure, with absence of the third, fourth, and sixth PAAs and direct connection of the dorsal aorta to the aortic sac (Fig. 4D).

Table 1. Summary of PAA Anatomy in Ts16 Embryos on Different Genetic Backgrounds
StrainTs162N
NormalAbnormalAbsent 4thAbsent 6thAbsent PAAsNormalAbnormal
  • a

    PAA anomalies were observed on the left and/or right side of the embryo.

C57BL/6J120000340
BALB/cJ110000440
DBA/2J80000290
C3H/HeJa014795610
Figure 4.

Anatomy of pharyngeal arch arteries (PAAs) 3, 4, and 6 as visualized by intracardiac India ink injection at E10.5. A: PAAs are numbered, and the dorsal aorta and aortic sac are labeled (DAo and AS, respectively) in a typical euploid control embryo. B–D: The different PAA anomalies exhibited by embryos from the crossing with a C3H/HeJ female. B: The absence of the left fourth PAA. The fourth PAA on the contralateral side (arrow) appears normal. C: Both the left and right sixth PAAs are absent. D: None of the PAAs are recognizable in the embryo shown, and the dorsal aorta connects directly with the aortic sac.

Histology of the Heart at E17.5

Hearts from five Ts16 fetuses and five euploid controls on each of the four inbred genetic backgrounds were sectioned and examined for cardiac anomalies at E17.5. VSDs were present in all Ts16 fetuses, regardless of genetic background (Fig. 5B). Other defects observed were double outlet right ventricle (DORV), common atrioventricular (AV) canal, and, more rarely, transposition of the great arteries (TGA) and persistent truncus arteriosus (PTA; Table 2). These defects are similar to those described in previous reports (Miyabara et al., 1982; Miyabara, 1990; Buselmaier et al., 1991; Waller et al., 2000). Although variation in the incidence of distinct cardiovascular malformations in Ts16 mice with different genetic backgrounds has been described (Miyabara et al., 1984), we were unable to determine whether there were statistically significant differences in frequency among strains because of the small sample size. No cardiac anomalies were observed in any of the 20 euploid control fetuses (Fig. 5A).

Figure 5.

A–D: Long axis sections of hearts from control and Ts16 fetuses at embryonic day (E) 17.5 stained with hematoxylin and eosin (A,B), and frontal views of the anterior mediastinum of E17.5 euploid control and Ts16 fetuses (C,D). A: Normal ventricular septation in the control fetus, showing fusion of the ventricular septum with the proximal part of the outflow tract cushions. No differences were observed in the frequency (100%) or degree of severity in Ts16 among the different genetic backgrounds. B: A typical ventricular septal defect is shown (arrow). All Ts16 fetuses displayed thymic abnormalities. C: Variable degrees of hypoplasia and asymmetry of the lobes of the thymus (arrow) were observed irrespective of genetic background. D: A representative hypoplastic thymus (arrow) from a Ts16 fetus is shown. Original magnification, ×6. VSD, ventricular septal defect; LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium; Thy, thymus. Scale bar = 875 μm in A (applies to A,B), in C (applies to C,D).

Table 2. Histological Analysis of Cardiovascular Anomalies in Ts16 at E17.5
Type of anomalyaC57BL/6JBALB/cJDBA/2JC3H/HeJ
  • a

    Usually more than one anomaly was observed. AV, atrioventricular; DORV, double outlet right ventricle; E, embryonic day; PTA, persistent truncus arteriosus; TGA, transposition of the great arteries; VSD, ventricular septal defect.

VSD5555
DORV2112
PTA11  
Common AV canal2212
TGA  1 

Thymic dysplasia was observed in all fetuses with cardiac anomalies (Fig. 5C,D). In most cases the lobes were drop-shaped and occasionally asymmetric.

Analysis of Defects of the Great Vessels at E17.5

Corrosion casts of Ts16 and euploid control hearts at E17.5 were prepared. The size (relative to body size) and shape of the heart were normal in fetuses recognized to be trisomic, regardless of genetic background (data not shown). Because the abnormalities detected in the E10.5 trisomics were exclusively on the C3H/HeJ background, we first examined the E17.5 C3H/HeJ corrosion cast preparations and found that 100% (10/10) of Ts16 fetuses had abnormalities of the patterning of the great vessels that could be attributable to inappropriate persistence or regression of the fourth and sixth aortic arch arteries. Examination of the corrosion casts revealed that two fetuses had pulmonary atresia (Fig. 6B) with the pulmonary vasculature filling retrograde by means of the patent ductus arteriosus. Two other fetuses had interruption of the aortic arch (IAA), one each of type B and type C. IAA, type B, occurs between the left carotid artery and the left subclavian artery and is caused by interruption of the fourth aortic arch. With this type of IAA, the transverse arch of the aorta is not fully developed and is separated into two, unconnected parts where a single arch would normally be formed. With IAA type C, the interruption occurs between the innominate artery and the left common carotid artery (Fig. 6C).

Figure 6.

A–F: Examples of corrosion casts of euploid control (A) and Ts16 fetuses (B–F) with a maternal genetic contribution from the C3H/HeJ strain at embryonic day (E) 17.5. A: Representative euploid control. The innominate artery (IA) is the first branch to arise from the arch of the aorta (Ao) and divides into the right subclavian artery (RSA) and right common carotid artery (RCC). The next branch is the left common carotid artery (LCC) followed by the left subclavian artery (LSA). The proximal pulmonary artery (PA) gives rise to the left and right pulmonary arteries (LPA and RPA, respectively) and the ductus arteriosus (DA) joins the descending aorta just beyond the origin of the left subclavian artery. B: Ts16 fetus with pulmonary atresia. The proximal pulmonary artery has not formed. The position where the proximal PA should be present is marked by a black arrow. C: Ts16 fetus with interrupted aortic arch (IAA) type C. The interruption occurs between the innominate artery (IA) and the left carotid artery (LCC; arrowhead). D: Ts16 fetus with right-sided aortic arch and anomalous origin of LSA from a left-sided ductus arteriosus (posterior view). E: Ts16 fetus with severe coarctation of the aorta. Narrowing of the aorta is indicated by a black arrowhead. F: Ts16 fetus with truncus right aortic arch with RPA arising from trunk and no visualized LPA. The first branch of the ascending trunk is a common vessel leading to the innominate artery (IA) and LCC (arrowhead). The aortic arch courses posteriorly. The left subclavian artery (LSA) originates from the descending aorta.

Two fetuses had the right subclavian artery (RSA) originating from the descending aorta. Normally, the RSA arises from the innominate artery, which bifurcates into the right common carotid artery and the RSA. Another fetus displayed a right aortic arch and anomalous origin of the left subclavian artery (LSA) from a left-sided ductus arteriosus (Fig. 6D). Because the right-side aortic arch travels posterior to the esophagus and the trachea, a vascular ring was present with the potential for compressing these structures. Other defects included severe coarctation of the aorta (Fig. 6E), as well as a truncus with right aortic arch and absent ductus arteriosus (regression of the distal sixth arch; Fig. 6F).

Corrosion casts were prepared for each of the other maternal strains (n = 2). However, because the easy passage of methyl methacrylate across the ventricular septum through a VSD or common AV canal (see above) made the production of informative corrosion casts problematic, we also examined the patterning of the great vessels in additional fixed fetuses that were prepared for subsequent histological analysis. All euploid littermates displayed normal development of the heart, aortic arch, and associated vessels (Fig. 7A). A VSD and/or a common AV canal was present in all Ts16 fetuses. Moreover, in addition to being present in Ts16 fetuses with a C3H/HeJ background, aortic arch defects were also observed in trisomic fetuses with a maternal contribution from C57BL/6J (n = 5), BALB/cJ (n = 4), or DBA/2J (n = 5), which, unlike the C3H/HeJ trisomics, did not have preexisting PAA defects at E10.5.

Figure 7.

Morphology of great vessels of the heart in euploid control and Ts16 fixed fetuses at embryonic day (E) 17.5. A: Euploid control fetus. B: Ts16 fetus with interruption of the aortic arch, type B (indicated by the arrowhead). The interruption occurs between the left carotid artery (LCC) and the left subclavian artery (LSA). C: Ts16 fetus with right aortic arch with the expected inverted order of branches. For abbreviations, see legend to Figure 6.

Because the analysis of both the corrosion casts and fixed fetuses provided comparable data, the data on the vascular malformations associated with Ts16 at E17.5 observed by both methods have been combined in Table 3. All trisomic fetuses with a C3H/HeJ maternal contribution were abnormal (n = 13), as were 85% of those on the other three backgrounds. The most common anomaly was interruption of the aortic arch (type B; Fig. 7B). A right aortic arch with the expected inverted order of arch branches was also observed (Fig. 7C).

Table 3. Frequency of Cardiovascular Anomalies in Ts16a
Type of anomalyC57BL/6JBALB/cJDBA/2JC3H/HeJ
  • a

    RSA, right subclavian artery; IAA, interrupted aortic arch; DA, ductus arteriosis; PTA, persistent truncus arteriosus.

  • b

    Number of trisomics in the progeny selected for examination following fixation and production of corrosion casts.

Cases examinedb76713
Pulmonary atresia1 23
Abnormal RSA origin2112
IAA3214
Severe coarctation 1 1
Hypoplasia/aplasia of DA1112
PTA1 11
Right sided aorta   1
Left sided aorta   1
Normal111 

DISCUSSION

The relationship between genetic background and the incidence and/or variability of a specific phenotype is well documented (Houlston and Tomlinson, 1998; Nadeau, 2001, 2003; Zlotogora, 2003). Previous investigations by Miyabara and collaborators have examined the effect of genetic background on the frequency and nature of cardiovascular malformations in Ts16 mouse fetuses (Miyabara, 1990; Miyabara et al., 1982, 1984). These studies suggest a relationship between genetic background and differences in the frequency of cardiovascular anomalies; however, the interpretation of the results can be complicated by the use of different types of doubly heterozygous males in comparative studies, introducing sources of variability that preclude the assignment of a genetic component. In fact, when different doubly heterozygous males were crossed to an inbred female strain, C57BL/6, significant differences in the frequency of a variety of cardiovascular anomalies were observed, presumably attributable to a paternal genetic component (Miyabara, 1990).

In the studies reported here, a single strain of doubly heterozygous males was crossed with four different inbred strains of females. This strategy permitted the striking all-vs.-none difference that we observed in the presence of PAA defects at E10.5 to be attributed specifically to an allele or alleles present in the C3H/HeJ strain and not in C57BL/6J, BALB/cJ, or DBA/2J. The nature of the responsible gene(s) is unknown at this time, but it could be amenable to mapping and identified by techniques currently being used for the isolation of modifier loci.

The one other mouse aneuploidy in which the effect of genetic background on phenotype have been investigated is the Df1 deletion in proximal mouse chromosome 16, which is a model for the 22q11 deletion that causes the DiGeorge and related syndromes in man. The Df1 deletion results in conotruncal and aortic arch anomalies similar to those found in Ts16, and their penetrance ranges from 16 to 50%, depending on background (Taddei et al., 2001). In contrast to our findings with Ts16, the genetic background did not affect the frequency of PAA defects, in this instance involving the 4th arches, that occurred in all E10.5 embryos regardless of genotype. Rather, the background appears to have affected the degree of “recovery” of the PAAs from these impairments in development (Lindsay and Baldini, 2001; Taddei et al., 2001). Therefore, quite different mechanisms appear to be at work in Ts16 and Df1.

The PAA system is transient and initially involves a symmetrical array of vessels that regress or are extensively remodeled into adult vascular structures, including the subclavian arteries and arch of the aorta. During gestation, the PAAs link the heart with paired dorsal aortae and develop symmetrically until approximately E11.5. The PAAs then undergo a dramatic remodeling process that requires the presence of neural crest cells to establish the embryonic asymmetric circulatory system (reviews Creazzo et al., 1998; Sucov, 1998; Epstein and Buck, 2000). The aberrant persistence and/or regression of different components of the PAA system is the presumed mechanism leading to specific congenital vascular malformations (Momma et al., 1999). For example, IAA, type B, is caused by the absence or regression of the 4th aortic arch.

In our studies at E10.5, we found that fully penetrant PAA abnormalities ranging from a reduction in size to the complete absence of the 4th and 6th PAAs, either unilaterally or bilaterally, occurred on a C3H/HeJ background. These findings can explain the defects in the patterning of the aortic arch and great vessels that subsequently occur at E17.5 (Momma et al., 1999). However, our data also demonstrate that cardiovascular anomalies can occur without preexisting defects of the PAAs, at least before E11, implicating additional mechanisms in the maldevelopment of the aortic arch and associated vessels. It has been suggested, for example, that VSDs may play a role in aortic arch defects by altering hemodynamic parameters and causing the aortic arch and pulmonary segments to compete for priority, with one segment being reabsorbed as a redundant feature. This phenomenon has been referred to as the “arch-rival” hypothesis (Buselmaier et al., 1991). However, although all fetuses with a female genetic component from C57BL/6J, BALB/cJ, or DBA/2J had a physiological anomaly, VSD and/or common AV canal, only 85% manifested aortic arch defects. Therefore, if this hypothesis is correct, other factors operative in development, such as stochastic events, may affect the ultimate phenotypic outcome.

Cardiac neural crest anomalies have been described in Ts16 (Webb et al., 1996; Waller et al., 2000) and other mouse mutants (Conway et al., 1997; Kochilas et al., 2002; Ohnemus et al., 2002) in association with outflow tract and aortic arch abnormalities. Immunohistochemical studies on serially sectioned hearts from normal and Ts16 mouse embryos, ranging from E8.5 to E14.5, strongly suggest that abnormal neural crest cell behavior is involved in the pathogenesis of the conotruncal malformations in the Ts16 mouse (Waller et al., 2000). Based on the PAA defects at E10.5 and the frequent involvement of the thymus gland, our results support the neural crest hypothesis. Although the significance of the similarities in cardiovascular defects between Df1 and Ts16 remains unclear, evidence suggests that the migration and distribution of the neural crest cells in early embryonic development are critical pathways that are sensitive to gene dosage effects.

EXPERIMENTAL PROCEDURES

Production of Ts16 Embryos and Fetuses

Rb(6.16)24Lub and Rb(16.17)7Bnr mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and crossed to generate [Rb(6.16)24Lub x Rb(16.17)7Bnr]F1 stocks heterozygous for the two Robertsonian translocation chromosomes, which share monobrachial homology for MMU16. F1 males were naturally mated with females from four inbred strains, BALB/cJ, C3H/HeJ, C57BL/6J, and DBA/2J, obtained from the Jackson Laboratory. We used the same four F1 males throughout the accumulation of embryos for PAA analysis. Females were checked daily for the presence of a vaginal plug, and the day on which it was found was designated 0.5 day of gestation. Females from each inbred strain were housed with the four F1 males for a 1-week period (plugged females were removed daily and replaced with a virgin female) for a period of 4 weeks (1 week/strain). This procedure was repeated for another 4-week cycle. Preliminary data indicating strain-specific PAA anomalies on the C3H/HeJ background prompted us to breed more C3H/HeJ females to accumulate embryos in excess of the other three inbred strains. Husbandry records demonstrated that each F1 male produced both Ts16 and 2N control embryos from each inbred female strain. Pregnant females were killed, and embryos/fetuses were collected at E10.5, E14.5, and E17.5. Karyotype analysis of cultured tail fibroblasts performed by conventional methods was used to determine the genotype of each animal. Whereas euploid embryos/fetuses have 38 acrocentric chromosomes and one metacentric (Robertsonian translocation) chromosome (total of 40 arms), Ts16 embryos/fetuses have 37 acrocentric and two metacentric chromosomes (total of 41 arms).

Histology

Fetuses were formalin-fixed and embedded in paraffin using standard protocols. Serial sections were cut at 5 μm and stained with hematoxylin and eosin for light microscopy. Comparative morphometric analyses were performed on equivalent sections through the heart and thymus. Digital images were captured and analyzed by using Spot Software version 2.1 (Diagnostic Instruments, Inc.).

Ink Injection and Corrosion Casts

For India ink injections, embryos collected at E10.5 were injected intracardially with India ink (diluted 1:20 in H2O), using a finely drawn glass pipette, and were then immediately fixed in 95% ethanol:1% acetic acid:1% chloroform. The embryos were dehydrated overnight and cleared in 1:1 benzyl benzoate:methyl salicylate. The aortic arches were scored as present or absent and, if present, for relative thickness.

To identify vascular patterning defects, pregnant females were anesthetized and the fetuses removed at E17.5. The fetal hearts were exposed by a thoracic incision and rib removal. Methyl methacrylate (LADD Research Industries, Inc.) was injected into the right and left ventricles until the entire fetal vasculature was filled. After allowing the resin to harden overnight, the fetuses were placed in maceration solution (Polysciences, Inc.) for 2–3 days to dissolve the soft tissues, leaving a mold of the vasculature. Photographs were digitally captured and processed with Adobe Photoshop software for Mac.

Because of the difficulties involved in producing corrosion casts in fetuses with VSDs, we also examined the heart and associated great vessels in fixed fetuses at E17.5. The thymus was also inspected. Subsequently, the fetuses were serial sectioned and stained with hematoxylin and eosin to expose VSDs.

Skeletal Preparations

Fetuses were collected at E17.5 and fixed in 80% ethanol for a minimum of 24 hr before removal of the skin and viscera. The carcasses were dehydrated and stained with Alcian blue for cartilage and with Alizarin red for calcified tissue according to standard procedures (McLeod, 1980). Euploid and Ts16 skeletal preparations in 100% glycerin were viewed in uncovered 60-mm Petri dishes using a Zeiss dissecting microscope. Digital images were captured by using Spot Software v2.1 and processed with Photoshop software.

Statistics

The frequency of live trisomy fetuses was recorded at E14.5 and E17.5. The weight and head-to-rump length of Ts16 embryos were compared with their euploid littermates using analysis of variance for parametric data and the Kruskal–Wallis test for nonparametric data, with the litter as the unit of comparison. Ninety-five percent confidence intervals for nonparametric data were calculated by using Vassarstat's Web site for statistical computation at http://faculty.vassar.edu/lowry/VassarStats.html. Strains were compared pairwise by using the Tukey t-test for multiple parametric comparisons and the Mann–Whitney test for multiple nonparametric comparisons.

Acknowledgements

We thank R. Gacayan and M. Doan for excellent animal care. Intracardiac ink injection and corrosion cast protocols were kindly provided by Anne Moon.

Ancillary