To investigate if the fully penetrant cardiovascular defects seen in mice on a congenic C57Bl/6 genetic background (B6-Pax9) were recapitulated on a different genetic background we examined Pax9-deficient mice (hereafter referred to as Pax9−/−) which had been backcrossed in excess of 20 generations on an outbred CD1 genetic background (CD1-Pax9). These congenic CD1-Pax9+/− mice were subsequently intercrossed to produce CD1-Pax9−/− embryos at E15.5 for analysis by MRI, µCT and histology (n = 25). A further 22 neonates were collected on the day of birth and analysed for aortic arch artery defects by dissection and direct visualisation. From a subset of these neonates (n = 12), the heart was removed and further examined for outflow tract and intracardiac defects by histology (Table 1). This analysis revealed that all CD1-Pax9−/− embryos and neonates presented with a cleft palate, a severely hypoplastic thymus absent from the normal position, and a pre-axial digit duplication (Fig. 1A-H) as previously reported [4]. Surprisingly, CD1-Pax9−/− mice had a significantly lower incidence of DORV compared to our published data for B6-Pax9−/− neonates and embryos [3] (n = 24; 16% versus 79%, p < 0.001; Fig. 2E; Table 1), although a very similar incidence of VSD and arch artery defects (IAA and A-RSA) was observed (Fig. 1I-Q; Fig. 2F, G; Table 1). Bicuspid aortic valve, however, was not observed. These data demonstrate that a change in genetic background affects the penetrance of the outflow tract defects in CD1-Pax9−/− mice, although the incidence of arch artery defects was consistent.
Table 1
Cardiovascular defects in Pax9 and Pax9;Msx1 mutant E15.5 embryos and neonates
Genetic background - genotype
|
n
|
VSD
|
DORV + IVC
|
cAo
|
IAA
± A-RSA
|
A-RSA
(w/o IAA)
|
Absent CC
|
B6-Pax9−/− a
|
24
|
3/19 (16%)
|
15/19 (79%)
|
0
|
22/24 (92%)
|
2/24
(8%)
|
17/24 (71%)
|
CD1-Pax9−/− b
|
47
|
10/37 (27%)
|
6/37
(16%) ***
|
2/47
(4%)
|
41/47
(87%)
|
6/47
(13%)
|
27/47
(57%)
|
CD1-Pax9−/−;
Msx1+/− c
|
38
|
2/29
(7%)
|
1/29
(3%)
|
9/38
(24%)*
|
11/38
(29%)***
|
19/38
(50%)***
|
9/38
(24%)*
|
CD1-Pax9−/−;
Msx1−/− d
|
7
|
0
|
1/7
(14%)
|
0
|
2/7
(29%)
|
0
|
0
|
a Data for Pax9−/− mice on a C57Bl/6J (B6) genetic background have been published [3]. Aortic arch artery defects for neonates (n = 5) and E14.5-15.5 embryos (n = 19) are pooled. VSD and DORV + IVC data from embryos only. A-RSA refers to a retro-oesophageal, cervical origin or isolated right subclavian artery. Absent common carotid artery (CC), resulting in the internal and external carotid arteries arising directly from the main aortic vessels, either unilaterally or bilaterally. All embryos had cleft palate and an absent thymus, and all embryos except Pax9−/−;Msx1−/− had a pre-axial digit duplication. b For CD1-Pax9−/− mice, aortic arch artery defects for neonates (n = 22) and E15.5 embryos (n = 25) are pooled. VSD and DORV + IVC data are from all embryos and n = 12 neonates (by histology). c For CD1-Pax9−/−;Msx1+/− mice, aortic arch artery defects for neonates (n = 20) and E15.5 embryos (n = 18) are pooled. VSD and DORV + IVC data from all embryos and n = 11 neonates (by histology). d CD1-Pax9−/−;Msx1−/− data for neonates (n = 1) and E15.5 embryos (n = 6) are pooled. All control B6-Pax9+/+ (n = 16) and CD1-Pax9+/+ (n = 9) embryos and neonates were normal. CD1-Pax9+/+;Msx1−/− (n = 7) and CD1-Pax9+/−;Msx1−/− (n = 14) embryos and neonates had normal heart, great arteries and thymus, but all had a cleft palate. ***p < 0.001; *p < 0.05 (Fisher’s exact test for associations). Abbreviations: Ao, aorta; A-RSA, aberrant right subclavian artery; DORV + IVC, double outlet right ventricle with interventricular communication; IAA, interrupted aortic arch; VSD, perimembranous ventricular septal defect.
Pax9 and Msx1 are known to interact in craniofacial development [14]. To explore if Pax9 and Msx1 also interacted in cardiovascular development, Pax9+/− and Msx1+/− mice, with both lines congenic on a CD1 background, were crossed to produce compound mutant embryos and neonates for analysis. Double heterozygous mice (i.e. Pax9+/−;Msx1+/−) were viable, fertile, and phenotypically normal except for the previously recognised absence of the lower teeth [14]. Pax9+/−;Msx1+/− mice were intercrossed to produce all possible Pax9;Msx1 genotypes. We confirmed that Msx1−/− mice on a CD1 background have cleft palate but no cardiovascular defects as previously reported [12, 19], and this was also found for the Pax9+/−;Msx1−/− genotype (Additional Table 1). For the Pax9−/−;Msx1+/− genotype, 18 embryos at E15.5 and 20 neonates were collected and examined, revealing a highly significant reduction in IAA-B, with or without A-RSA (p < 0.001) when compared with CD1-Pax9−/− mice (Fig. 2A-D, F; Table 1). There was also a significant reduction in the incidence of absent common carotid arteries, a hallmark of the B6- Pax9−/− cardiovascular phenotype (p < 0.05) [3] (Table 1), and a very low incidence of DORV (one case in 29 mutants examined; Fig. 2E). In our initial analysis, A-RSA referred to the right subclavian artery being retro-esophageal, isolated, or of a cervical origin. When this data was further analysed the incidence of retro-esophageal right subclavian artery (RE-RSA) was seen to be significantly reduced in Pax9−/−;Msx1+/− embryos (p < 0.01) (Fig. 2G) and cervical origin of the right subclavian artery (cRSA) was found to be significantly increased (p < 0.05) (Fig. 2H). Cervical origin of the aorta (cAo) was also increased significantly (p < 0.05) (Fig. 2I). Wild type (n = 2), Pax9−/− (n = 4) and Pax9−/−;Msx1+/− (n = 6) embryos at E12.5 were analysed by µCT to assess the remodelling of the aortic arch arteries. This revealed that the 3rd and 4th PAAs were absent or aberrant in all Pax9−/− embryos as expected [3], whereas in Pax9−/−;Msx1+/− embryos the 3rd and 4th PAAs were maintained in 67% (8/12) and 58% (7/12) of cases, respectively (p < 0.05; Fig. 2J-M; Table 2).
It therefore appears that mice deficient for Pax9 and heterozygous for Msx1 (i.e. Pax9−/−;Msx1+/−) present with alternative arch artery defects when compared to Pax9−/− mice. These compound mutant mice, however, did show the other Pax9−/− associated developmental defects affecting the palate, thymus and digits (as shown in Fig. 1) [4]. Overall, lack of one Msx1 allele in the context of Pax9 deficiency appeared to rescue the cardiovascular phenotype to a degree, with the incidence of fatal lesions, such as IAA-B, reduced and replaced with the potentially non-lethal defect of cervical origin of the aortic arch.
Table 2
Cardiovascular defects in Pax9:Msx1 mutant embryos at E12.5
Genotype
|
n
|
Absent 3rd PAA
|
Absent 4th PAA
|
Control
|
2
|
0/4
|
0/4
|
CD1-Pax9−/−
|
4
|
7/8
|
8/8
|
CD1-Pax9−/−;Msx1+/−
|
6a
|
4/12*
|
5/12*
|
Embryos were assessed by µCT and the 3rd and 4th PAAs (i.e. two of each per embryo) scored for being absent in each genotype. PAA defects in CD1-Pax9−/−;Msx1+/− embryos were significantly reduced when compared to CD1-Pax9−/− embryos. *p < 0.05 (Fisher’s exact test for associations). a Two embryos were normal with no PAA defects. Abbreviation: PAA, pharyngeal arch artery.
To further examine the effect of genetic background on the Pax9−/− cardiovascular phenotype, and to also analyse the effect of Msx1 heterozygosity, intra-cardiac ink injections were performed on CD1-Pax9−/− and CD1-Pax9−/−;Msx1+/− embryos at E10.5 to visualise the patency of the developing PAAs (Fig. 2N). Data for CD1-Pax9−/− embryos (n = 9) was first compared to our published ink injected B6-Pax9−/− embryos (n = 20) [3]. This analysis revealed that, like B6-Pax9−/− embryos, the 4th PAAs in CD1-Pax9−/− embryos were bilaterally non-patent to ink and therefore considered to be absent at this stage. The 3rd PAAs in CD1-Pax9−/− embryos were also similarly affected as B6-Pax9−/− embryos in the majority of cases (Fig. 2O; Table 3). Approximately half of B6-Pax9−/− embryos also had aberrantly persisting 1st and/or 2nd PAAs patent to ink, and this increased to 78% (p = 0.41) and 89% (p < 0.05) respectively in CD1-Pax9−/− embryos. CD1-Pax9−/−;Msx1+/− embryos (n = 16) also displayed bilateral defects of the 4th PAAs although 37.5% (6/16) embryos had at least one vessel that was hypoplastic rather than absent, which is significantly different to CD1-Pax9−/− embryos (p < 0.05; Fig. 2P, Q; Table 3; Additional file 1). There was a significant reduction in the incidence of 3rd PAA defects seen in CD1-Pax9−/−;Msx1+/− embryos compared with CD1-Pax9−/− embryos (25% versus 89%; p < 0.005). The reduction in persistent 1st and 2nd PAAs observed, however, was not significant.
Table 3
Pharyngeal arch artery defects in mutant E10.5 embryos
|
Bilateral defects
|
Genetic background
|
n
|
PAA
|
Abnormal
|
Unilateral
|
Bilateral
|
Present
|
Hypo/Int/Abs
|
Absent
|
B6-Pax9−/− a
|
20
|
1
|
11 (55%)
|
1
|
10
|
9
|
1
|
0
|
2
|
8 (40%)
|
3
|
5
|
4
|
1
|
0
|
3
|
15 (75%)
|
3
|
12
|
-
|
8
|
4
|
4
|
20 (100%)
|
1
|
19
|
-
|
3
|
16
|
CD1-Pax9−/−
|
9
|
1
|
7 (78%)
|
1
|
6
|
6
|
0
|
0
|
2
|
8 (89%) *
|
1
|
7
|
7
|
0
|
0
|
3
|
8 (89%)
|
0
|
8
|
-
|
3
|
5
|
4
|
9 (100%)
|
0
|
9
|
-
|
0
|
9
|
CD1-Pax9−/−;
Msx1+/−
|
16
|
1
|
8 (50%)
|
0
|
8
|
8
|
0
|
0
|
2
|
8 (50%)
|
0
|
8
|
8
|
0
|
0
|
3
|
4 (25%) **
|
0
|
4
|
-
|
3
|
1
|
4
|
16 (100%)
|
0
|
16
|
-
|
6 *
|
10
|
Embryos were collected at E10.5 and assessed for pharyngeal arch artery (PAA) defects by intracardiac ink injection. a Data for Pax9−/− embryos on a C57Bl/6J (B6) genetic background have been published [3]. For Pax9−/− embryos, each left and right PAA 1–4 was scored as having a unilateral or bilateral defect, and the bilateral defects categorised as either present, a combination of hypoplastic, interrupted and/or absent (Hypo/Int/Abs), and bilaterally absent. All control B6-Pax9+/+ (n = 18) and CD1-Pax9+/+ (n = 12) embryos were normal. CD1-Pax9+/+;Msx1−/− (n = 6) and CD1-Pax9+/−;Msx1−/− (n = 7) embryos were normal. The increase in abnormal 2nd PAAs in CD1-Pax9−/− compared with B6-Pax9−/− embryos is significant. The decrease in 3rd PAA defects, and the increase in hypoplastic 4th PAA defects, in CD1-Pax9−/−;Msx1+/− compared with CD1-Pax9−/− embryos is significant. **p < 0.005, *p < 0.05 (Fisher’s exact test for associations).
Breeding double heterozygous mice also produced double null (i.e. Pax9−/−;Msx1−/−) embryos (n = 6 at E15.5, n = 1 at E12.5, n = 2 at E10.5), and one neonate, for analysis (Additional file 2; Table 1). From the mutant neonates and embryos at E15.5, two presented with IAA-B and A-RSA (Additional file 2H), and one of these also had DORV. Interestingly, Pax9−/−;Msx1−/− mice did not have the pre-axial digit duplication seen in Pax9−/− mice (Additional file 2G). Analysis of the three Pax9−/−;Msx1−/− embryos at E12.5 and E10.5 by HREM and ink injection showed that the PAA defects were similar to Pax9−/− embryos at these stages (Additional file 2L-N). The incidence of IAA-B in Pax9−/−;Msx1−/− neonates and E15.5 embryos was the same as seen in Pax9−/−;Msx1+/− neonates and E15.5 embryos (Table 1) but the majority of double nulls (n = 5/7; 71%) had a normal cardiovascular system at these stages.
To investigate if cell fate within the pharyngeal arches was affected in CD1-Pax9;Msx1 mutant embryos, cell death and proliferation were assessed. There was no significant difference in the levels of apoptosis and proliferation between the cells of the different pharyngeal tissue layers in control, CD1-Pax9−/− and CD1-Pax9−/−;Msx1+/− embryos at either E9.5 or E10.5 (n≥3 per genotype and stage) (Additional file 3). In B6-Pax9−/− embryos a significantly reduced number of NCC observed in the 3rd and 4th pharyngeal arches at E10.5 has been described [3]. We firstly counted the number of cells in the pharyngeal arch mesenchyme of CD1 congenic mutant mice as this is predominantly comprised of NCC (n≥3 per genotype and stage; Fig. 3A). This revealed that at E9.5 there was a significant reduction in cell number in the 3rd pharyngeal arch mesenchyme in Pax9−/− and Pax9−/−;Msx1+/− embryos compared to controls (p < 0.05). At E10.5 there was also a significant reduction in the number of mesenchymal cells in the 3rd pharyngeal arch of Pax9−/− embryos (p < 0.001), but the reduction in cell number in Pax9−/−;Msx1+/− embryos was not significantly different to controls in this tissue. In the 4th pharyngeal arch, however, there was a significant reduction in cell number in both Pax9−/− and Pax9−/−;Msx1+/− embryos (p < 0.001; Fig. 3A). This data, therefore, suggests that there is a reduction in NCC number in the 4th pharyngeal arch of Pax9−/− and Pax9−/−;Msx1+/− embryos, but the reduction in cell number in the 3rd pharyngeal arch of Pax9−/−;Msx1+/− embryos at E10.5 is not significantly reduced when compared to controls. To validate this observation specifically for NCC, we immunostained coronal sections of E10.5 embryos (n = 3 per genotype) with an anti-AP-2α antibody which labels NCC (Fig. 3B). This confirmed our mesenchymal cell counting data and demonstrated that the number of NCC in the 3rd pharyngeal arch of Pax9−/−;Msx1+/− embryos at E10.5 was significantly increased when compared with Pax9−/− embryos (p < 0.001). The number of NCC in the 4th pharyngeal arch was significantly reduced in both Pax9−/− and Pax9−/−;Msx1+/− embryos when compared to controls (p < 0.0001; Fig. 3C).
In B6-Pax9−/− embryos the reduction in SMC surrounding the 3rd PAAs was linked to the failure of this vessel to be maintained resulting in an absent common carotid artery at the foetal stage [3]. To investigate this in CD1-Pax9−/− and Pax9−/−;Msx1+/− embryos, control and mutants at E10.5 were immunostained using antibodies raised against ERG1 and smooth muscle actin for endothelium and SMC respectively (n = 6 of each genotype examined; Fig. 3D-F). This staining revealed that, like B6-Pax9−/− embryos, SMC recruitment to the 3rd PAA was greatly reduced or absent in CD1-Pax9−/− embryos (Fig. 3E). In Pax9−/−;Msx1+/− embryos, however, SMC were observed surrounding the 3rd PAAs (Fig. 3F).
Collectively, these data show that the reduced number of NCC in the 3rd pharyngeal arch in Pax9−/− embryos is rescued to some extent in Pax9−/−;Msx1+/− embryos. Along with the concomitant recruitment of SMC to the 3rd PAA, this suggests a developmental mechanism to explain the reduced aortic arch artery severity in mice with Pax9 deficiency coupled with Msx1 heterozygosity. Pax9−/− mice die in the neonatal period, presumably from arterial duct-dependent defects such as IAA-B caused by failure of the left 4th PAA to form, as well as absent common carotid arteries caused by the collapse of the 3rd PAAs [3]. Pax9−/−;Msx1+/− mice also died after birth but with a much lower incidence of IAA-B and absent common carotid arteries, and a higher occurrence of other arch artery defects such as cervical origins of the aortic arch and right subclavian artery were observed. Analysis at mid-embryogenesis showed that, although morphogenesis of the 4th PAAs was affected in all Pax9−/−;Msx1+/− embryos, a proportion of embryos had a mildly affected 4th PAA. Moreover, there were fewer embryos with 3rd PAA defects and this was linked to the maintenance of the 3rd PAAs which were invested with SMC. We therefore hypothesised that Msx1 heterozygosity, rather than rescuing arch artery defects, altered the type of defect to one that is apparently compatible with a functioning systemic circulation. For example, a 3rd PAA in combination with an absent left 4th PAA and a persistent carotid duct may remodel to a cervical aortic arch. Pax9−/−;Msx1+/− mice with an apparently intact systemic circulation, however, still died soon after birth. Pax9−/− and Pax9−/−;Msx1+/− mice also have a cleft secondary palate, and we speculated that this could theoretically compromise postnatal survival independent of the cardiovascular defects [4, 7]. We therefore further hypothesised that mutant mice with a normal palate and an intact systemic cardiovascular system could survive the neonatal period following closure of the arterial duct. To engineer this configuration we used Isl1Cre mice [31] in conjunction with a Pax9-floxed allele [32]. Isl1Cre causes recombination in the second heart field (SHF), encompassing the pharyngeal endoderm, and the developing limb, two domains where Pax9 is also expressed [31, 33]. We expected that the palate would not be affected in these mice as the cleft palate observed with Pax9 deficiency is caused by a lack of expression in NCC [3, 32], a cell type with only minimal activity of Isl1Cre [34]. The Pax9−/flox;Isl1Cre mutant mice, hereafter referred to as Pax9ΔSHF, should therefore develop the typical Pax9−/− cardiovascular defects but without a cleft palate. Firstly, to demonstrate the efficacy of deleting Pax9 from the Is1Cre domain, we generated Pax9ΔSHF mutant mice on a C57Bl/6J genetic background. From nine Pax9ΔSHF embryos at E15.5 examined by MRI, all presented with the typical Pax9−/− phenotype, affecting the cardiovascular system, thymus and digit formation, although the palate was normal as predicted (Fig. 4; Table 4). There was no significant difference from the cardiovascular defects observed in B6-Pax9−/− mice.
Table 4
Cardiovascular defects in second heart field mutant embryos and neonates
Genetic background – genotype & stage
|
n
|
VSD
|
DORV + IVC
|
cAo
|
IAA-B ±
A-RSA
|
A-RSA
|
Absent CC
|
B6-Pax9ΔSHF
E15.5
|
9
|
1/9
(11%)
|
5/9
(56%)
|
1/9
(11%)
|
8/9
(89%)
|
1/9
(11%)
|
9/9
(100%)
|
CD1-Pax9ΔSHF
Neonate
|
1
|
N/A
|
N/A
|
0
|
1
|
0
|
1
|
CD1-Pax9ΔSHF;Msx1+/−
Neonate
|
8
|
0
|
0
|
2/8
(25%)
|
0
|
2/8
(25%)
|
0
|
All mice with Pax9 conditionally inactivated from the second heart field with Isl1Cre (Pax9ΔSHF) had pre-axial digit duplication, absent thymus and normal palate. N/A, not assessed. Abbreviations: A-RSA, aberrant right subclavian artery; cAo, cervical aorta; CC, common carotid artery; DORV + IVC, double outlet right ventricle with interventricular communication; IAA,-B interrupted aortic arch type B; SHF, second heart field; VSD, perimembranous ventricular septal defect.
We next generated Pax9ΔSHF and Pax9ΔSHF;Msx1+/− neonates for analysis on a CD1 genetic background. Due to the complex mating scheme required to generate mutant mice we only collected neonates so as to conserve the dams for subsequent breeding. All neonates that were found dead soon after birth were collected and analysed for arch artery defects, cleft palate and pre-axial digit duplication. All surviving neonates were culled five days after birth and examined the same way. Genotyping revealed that all neonates found dead on the day of birth had a Pax9−/− genotype. All control genotypes analysed (n = 5) had a normal palate and arch arteries (Fig. 5A-C). All Pax9−/− neonates (n = 5) presented with IAA-B and A-RSA as well as a cleft palate and pre-axial digit duplication (Fig. 5D-F). From two Pax9−/−;Msx1+/− neonates recovered from this cross, both had a cleft palate and pre-axial digit duplication, one had a cervical right subclavian artery, and the other a cervical aortic arch. Only one Pax9ΔSHF neonate was recovered, but showed the expected arch artery defects (IAA-B and A-RSA) and pre-axial digit duplication seen in Pax9−/− mice, although the palate was normal (Fig. 5G-I; Table 4). Eight neonates with the Pax9ΔSHF;Msx1+/− genotype (i.e. Pax9 deleted from the SHF in conjunction with Msx1 heterozygosity) were recovered, all found dead on the day of birth. The pre-axial digit duplication was seen, but the palate was unaffected, and the aorta and right subclavian artery were either normal (n = 6) or of cervical origin (n = 2; Fig. 5J-L; Table 4). Histology confirmed that the outflow tract, arterial valves and ventricular septum were normal. The cardiovascular system of Pax9ΔSHF;Msx1+/− neonates was therefore normal or had a phenotype theoretically compatible with a functioning systemic circulation, and an unaffected palate, yet these mice died on the day of birth.
As Pax9 deficiency has been shown to cause bone malformations such as pre-axial digit duplication and cleft palate [4] we investigated the skeletons of control, Pax9−/− and Pax9ΔSHF;Msx1+/− neonates to see if any phenotype here could explain the neonatal death. Neonatal skeletons were stained for cartilage and bone using alcian blue and alizarin red. All control neonates (n = 5) had a normal skeleton (Fig. 6A). The Pax9−/− (n = 4) and Pax9ΔSHF;Msx1+/− (n = 6) skeletons all presented with a pre-axial digit duplication on the hind and forelimbs (Fig. 6B,C) as expected. The ulna length for each line was measured and showed there was little difference in neonate size between the genotypes (Fig. 6G). In control neonates the normal hyoid bone, which connects to various ligaments and muscles such as the thyrohyoid and stylohyoid ligaments [35], had a horseshoe-shape, with an elongated and flat body, and two pairs of greater and lesser horns which projected posteriorly and anteriorly, respectively, from the outer borders of the body (Fig. 6D). In the Pax9−/− and Pax9ΔSHF;Msx1+/− mutants, the ossification centre of the body of the hyoid bone was significantly shorter compared to controls (p < 0.0001; Fig. 6E, F, H), the lesser horn extended laterally and the greater horn was significantly reduced in length (Fig. E, F I). The angle between the greater and lesser horns was significantly reduced in Pax9−/− and Pax9ΔSHF;Msx1+/− neonates (p < 0.0001) when compared to controls (Fig. 6E, F, J). Pax9 deficiency also causes thyroid cartilage deformities, where the thyroid cartilage is broader and lacks the lateral processes normally connecting the thyroid and cricoid cartilages [4]. Control neonates showed a normal thyroid cartilage with two laminae that fused together anteriorly (Fig. 6K, L). The posterior border of each lamina was free and created the superior and inferior horn projections. In Pax9−/− and Pax9ΔSHF;Msx1+/− neonates the inferior horn of the thyroid cartilage was significantly shorter compared to controls, and the superior horn was reduced to a stump (Fig. M-Q). Fused tracheal rings were also observed in Pax9−/− neonates (Fig. 6M, N). The hyoid bone and thyroid cartilage are both derived from NCC [36, 37] but neonates with a conditional inactivation of Pax9 from NCC, however, did not have any hyoid bone or thyroid cartilage defects (n = 11; Fig. 6R, S).