Recovery of a novel Dnahc5 mutation.
As part of an ENU mutagenesis screen for recessive mutations causing congenital heart defects, we recovered a mouse mutant exhibiting situs anomalies. Homozygous animals exhibited situs inversus totalis, as well as heterotaxy (Figure ). Mutants with heterotaxy usually also exhibited complex structural heart defects and died before or shortly after birth. To map the mutation, heterozygous mutants in the C57BL/6J (B6) background were crossed to C3H mice to generate B6/C3H hybrid offspring, and these were further intercrossed to obtain embryos between E15.5 and birth. Genome scanning DNA obtained from these hybrid offspring using B6/C3H polymorphic microsatellite DNA markers mapped the mutation to chromosome 15 (18
). Further fine mapping using SNPs narrowed the interval to a 7.2-Mb interval between SNP rs13482485 and rs32364740. This region contains 26 genes, including Dnahc5
. As mutations in the human ortholog DNAH
5 are well described in patients with situs anomalies and PCD (19
), we analyzed Dnahc5
cDNA sequences and also the 79 exons of the Dnahc5
genomic DNA sequence from homozygous mutant embryos.
Situs anomalies in Dnahc5del593 mutants.
PCR amplification of cDNA and genomic DNA for Dnahc5 gave the expected products, except for primers targeting exons 7–17. PCR amplification of cDNA derived from these exons yielded no specific products, while amplification of genomic DNA with primers spanning exon 6 and 18 generated a 3.3-kb amplicon from the mutant embryo DNA (Figure A). This compares with the expected amplicon size of 30,688 bp. Sequencing of the central 2,765 bp showed that the mutated region consisted of a large deletion of 29,755 bp spanning exons 7–17, with the 5′ end containing sequences from intron 6, proceeding internally to base Chr15:28,178,819 (mm8), and the 3′ end containing sequences derived from intron 17, starting internally at base Chr15:28,208,575 (Figure C). Surprisingly, in this deleted interval, there was a 2,397-bp insertion composed of DNA derived from mouse chromosomes 2, 4, and X. This included both repetitive sequences and portions of exons of other genes, but these lacked known splice donor/acceptor sites (Figure D). In silico translation of the predicted mutant Dnahc5 mRNA showed that this novel Dnahc5 allele has an in-frame deletion of 1,779 nucleotides coding 593 amino acids from residues 267–859 at the N-terminal side of Dnahc5 — a region containing most of the heavy chain dynein interacting domain N1, but that does not include the motor domain (Figure C). The predicted transcript was experimentally verified by sequencing of cDNAs obtained from RT-PCR amplification of transcripts expressed in homozygous mutant embryos (Figure B). We refer to this mutant Dnahc5 allele as Dnahc5del593.
Dnahc5 mutation involves an in-frame DNA deletion.
Situs anomalies in homozygous Dnahc5del593 mutants.
With the identification of the mutation in Dnahc5
, we conducted a systematic analysis of genotype versus phenotype. This survey revealed that some of the Dnahc5del593
homozygous animals survived up to 2–4 weeks postnatally, exhibiting either normal situs (situs solitus; Figure A) or complete mirror reversal of body situs (situs inversus totalis; Figure B). The homozygous animals usually expired or had to be euthanized at 3–4 weeks of age, as they exhibited a dome-shaped head indicative of hydrocephaly. These findings were consistent with the known role of Dnahc5
in the ependymal cilia for moving cerebral spinal fluid and maintaining patency of the aqueduct (20
). A previous gene trap mouse model of PCD has also been found to have situs inversus totalis and hydrocephalus (21
Finding both situs solitus and situs inversus totalis in the postnatal homozygous animals suggested situs specification was randomized. However, the previous examination of embryos harvested preterm also showed heterotaxy (Figure C). To determine the frequency of different situs presentations, 123 embryos from 18 litters were harvested at E16.5–E18.5, which consisted of 30 wild-type (Dnahc5+/+
), 68 heterozygous (Dnahc5del593/+
), and 25 homozygous mutants (Dnahc5del593/del593
). This distribution did not differ significantly from the expected Mendelian ratio. All wild-type and heterozygous mutants showed situs solitus. Interestingly, of the 25 homozygous mutant embryos, 24% (6
) exhibited situs solitus, 36% (9
) situs inversus totalis, and 40% (10
) exhibited heterotaxy. We define heterotaxy as any deviation from situs solitus or situs inversus totalis, whether the changes were observed in the cardiac, pulmonary, or abdominal anatomy. These observations show this Dnahc5
mutation generates complete reversal of handedness as in situs inversus totalis at the same rate as the incidence of heterotaxy.
In situs solitus, the heart apex points leftward (levocardia) and the aortic arch is leftward (Figure A). The aorta is posterior and right of the pulmonary artery at the base of the heart. Both superior vena cavae (SVCs) return to the right atrium — the left SVC via the coronary sinus and the inferior vena cava (IVC) returns to the right atrium. There are 4 left and 1 right lung lobes, and the stomach, pancreas, and spleen are on the left. Only the left liver lobe is visible posterior to the medial lobe, which straddles both sides of the body. In situs inversus totalis, there is complete mirror-image reversal of organ situs in the thoracic and abdominal cavities. For example, the heart apex is pointed to the right (dextrocardia), with a right-sided aortic arch, and the stomach, spleen, and pancreas are on the right (Figure B). In embryos with heterotaxy, we observed a wide range of situs anomalies (Table ). Some had 1 lung lobe on each side of the chest cavity, indicative of left pulmonary isomerism. There were also examples of symmetrical bilobed liver, midline stomach and pancreas, as well as azygos continuation of IVC (Figure , C and D, and Table ). Abnormal heart situs and complex structural heart defects were also observed in many of the mutants exhibiting heterotaxy (Table ). Light blue in Table indicates normal situs; pink, inverted situs; gray, right-left isomerism; and green, midline position or otherwise anomalous position/connection. It is interesting to note that isomerism (gray) and the more anomalous pathologies (green) were more frequently associated with levocardia.
Cardiovascular, pulmonary, and abdominal phenotypes associated with heterotaxy
Complex structural heart defects in heterotaxic embryos.
To evaluate the structural heart defects associated with heterotaxy, the hearts were further analyzed using serial section histology carried out by episcopic fluorescence image capture (EFIC) followed by 3D reconstruction. To broaden the analysis for structural heart defects, an additional 8 homozygous mutant embryos exhibiting heterotaxy were included in this histological analysis. Of the 18 hearts examined, 6 showed levocardia, 10 had dextrocardia, and 2 exhibited midline position or mesocardia (Table ). To delineate the complex structural heart defects, we adapted Van Praagh’s segmental analysis (22
), in which the heart is subdivided into atrial, ventricular, and arterial segments, with each segment containing 2 components — the right versus left atrial chambers, the right versus left ventricular chambers, and the aortic versus pulmonary trunks (Table ). In addition, we examined the atrioventricular (AV) and ventriculoarterial (VA) connections, venous drainage, aortic arch connections, and septal defects.
Summary of heart, lung, stomach, situs associated with heterotaxy
As mouse atria do not exhibit hallmark features seen in human left versus right atrial appendages, atrial situs was classified according to the pattern of venous return and the presence of a coronary sinus. If the ipsilateral SVC of an atrium returned directly to it, it was designated a morphologic right atrium. If its ipsilateral SVC returned via a coronary sinus to the contralateral atrium, it was designated a morphologic left atrium. In left atrial isomerism, there is often a common atrium to which both SVCs return. In right atrial isomerism, each SVC returns directly to its ipsilateral atrium, and in addition, each atrium has an inferior venous connection as well. Designating the atrial situs as such, we were able to classify all atrial pairs, yielding 4 situs solitus, 9 situs inversus, 4 with left atrial isomerism (Figure C) and 1 with right atrial isomerism (Figure ). All cases of atrial isomerism occurred in hearts with levocardia, and both hearts with mesocardia had situs solitus atrial arrangement (Table ).
Histological analysis of cardiac anomalies.
Images of anomalous venous return.
The ventricular morphology was determined by a combination of hallmarks: the morphologic left ventricle is characterized by the attachment of papillary muscles to the free wall. The roof of the right ventricle typically has a band of muscle known as the trabecula septomarginalis. The lack of conal tissue between the ventricular inflow and outflow valve is typical of a mitral and aortic valve, respectively. In contrast, conal tissue is typically seen between the tricuspid and pulmonic valve. Using these criteria, 14 of the hearts had ventricles that could be classified (Table ). The loop direction was concordant with the orientation of the apex in all but 1 of these hearts. However, the latter heart exhibited AV concordance, as indicated by dextrocardia presenting with atrial situs solitus and ventricular D-loop. The remaining 4 hearts had a superior-inferior, rather than left-right, arrangement of ventricles (Figure , A–D, and Figure B). In all 4 hearts, the morphologic right ventricle was positioned superiorly and was the larger of the 2 ventricular chambers (Figure , C and D). In 2 cases, the left inferior ventricle was displaced leftward and hence designated “D superior-inferior,” and in the other 2, the left ventricle was rightward, hence “L superior-inferior” (Table ). Independent of left-right or superior-inferior positioning of ventricles, the 18 hearts were almost equally divided between D- and L-loops (Table ).
Superior-inferior ventricles and AV canal defect.
Aorta–pulmonary artery positioning.
The position of the aorta is right and posterior in relation to the pulmonary artery in normal hearts, or left and posterior in hearts with mirror-image reversal of situs. D-malposition occurs when the aorta is displaced toward the right and anterior relative to the pulmonary artery, and L-malposition when the aorta is displaced toward the left and anterior in a heart with inverted situs. Ten of the 18 hearts had malpositioned aortas, which were equally divided between D-malpositioning (Figure , A and C) and L-malpositioning (Figure B; Tables and ).
Necropsies showing outflow tract defects in Dnahc5del593 mutants.
Variations from the normal AV connection comprising 2 AV valves included a single AV valve and a common AV canal that was either balanced or unbalanced, the latter being committed either to the morphologic left or right ventricles. The 18 hearts were equally divided between those that had normal AV connections and those that did not (Table ). Hearts with atrial or ventricular segments that deviated from normal situs or a simple situs inversion, such as with atrial isomerism or superior-inferior positioning of ventricles, invariably had AV canal defects (Figure D and Figure , E and F). Two of the 4 hearts with superior-inferior ventricles had a single AV valve of mitral valve morphology connecting a common atrium (left atrial isomerism) or 2 atria with primum and secundum atrial septal defects (ASDs) (right atrial isomerism) to the inferiorly positioned morphologic left ventricle. In hearts with normal or simply inverted atria and ventricles, AV canal defects were accompanied by VA malformations, e.g., transposition of great arteries (TGA).
VA alignment is said to be concordant if the morphologic left ventricle connects to the aorta and the morphologic right ventricle to the pulmonary artery. Ten of the 18 hearts had discordant VA alignment, of which double outlet right ventricle (DORV) was the most common (Figure B, Figure D, and Table ). Three of the 4 hearts with superior-inferior positioning of ventricles had DORV accompanied by ventricular septal defects (VSDs) (Figure C). All 6 D-looped hearts had abnormalities in VA alignment: 2 were DORV (Figure B), 2 were D-TGA (Figure , A and C, and Figure ), 1 was L-TGA (Figure B), and 1 was tetralogy of Fallot (TOF) (Figure A and Table ). Of the 8 L-looped hearts, 6 had concordant VA alignment. Of these, 1 had an L-malposition of the great arteries, and 1 had TOF. Only 2 of the L-looped hearts showed discordance in VA alignment, 1 having D-TGA and the other DORV (Figure D).
EFIC imaging of heart exhibiting mesocardia with inlet VSD and D-TGA.
The direction of the aortic arch was concordant with the ventricular loop in 15 of the 18 hearts (Table ), in that a D-looped ventricle had a left aortic arch or an L-looped ventricle had a right aortic arch. There was 1 example each of a D-looped ventricle discordant with a right aortic arch and an L-looped ventricle discordant with a left aortic arch (Table , nos. 7, 15). There was 1 case of a type B interrupted aortic arch (Table , no. 2). Other aortic anomalies were infrequent, with 1 case of hypoplastic transverse arch (Table , no. 11) and 1 aberrant right subclavian artery off the descending aorta (Table , no. 4).
By far the most common venous anomaly was azygos continuation of interrupted IVC (Figure D), seen in 8 mutants (Table ). Duplicated IVC was seen twice, giving a total of 10 hearts with venous anomalies. Eight of the 10 were associated with either pulmonary or abdominal situs abnormality, e.g., left pulmonary isomerism, malpositioned pancreas, or symmetric liver (Table ). Only in 1 case was the venous anomaly found as an isolated defect (Table , no. 16).
Cardiac-pulmonary and cardiac-abdominal situs.
Analysis of the bronchial tree patterning showed substantial correlation with atrial patterning (Table ). Nine of the 10 heterotaxic embryos with either situs solitus or situs inversus bronchial tree patterning had the corresponding atrial arrangement. All of the embryos that had left atrial isomerism also had left isomerism of the bronchial tree. However, the converse was not true, as 3 of the embryos with left isomerism of the bronchial tree exhibited atrial arrangement that was either situs solitus or situs inversus. In 4 of the embryos, we observed discordance between the direction of heart loop and the situs of stomach, spleen, and pancreas. Thus, a D-looped heart was associated with a right stomach, spleen, and pancreas, and the converse was found for an L-looped heart. These embryos were invariably associated with pulmonary isomerism (Tables and ).
Analysis of ciliary ultrastructure, orientation, and function.
The finding of 40% heterotaxy associated with this Dnahc5del593
mutation is striking and somewhat unexpected. This compares with only a 6.3% incidence of heterotaxy in a recent clinical study of 337 PCD patients (16
). To ascertain whether the Dnahc5del593
mutation is indeed a bona fide model of PCD, we analyzed ciliary structure and function in the respiratory airway. For this analysis, trachea were isolated from homozygous animals 2–4 weeks old and processed for scanning sand transmission electron microscopy (SEM and TEM).
SEM showed an orderly arrangement of the cilia in wild-type and heterozygous animals such that all of the cilia were oriented in a similar direction, presumably aligned with the direction of mucus flow in the airway (Figure , A and B). In contrast, among homozygous mutants, a wide variation was observed, ranging from marked disorganization of cilia with no obvious alignment (Figure C) to organized cilia similar to those seen in wild-type animals (compare with Figure A; and data not shown). SEM analysis of embryos at E7.75–E8.0 revealed that cells at the embryonic node are monociliated in both wild-type and homozygous mutant embryos (Figure , J and K)
SEMs and TEMs of tracheal epithelial cilia.
TEM analysis of the wild-type tracheal cilia showed the expected 9 plus 2 arrangement of microtubules (Figure , D–I), with outer and inner dynein arms decorating the peripheral doublets (Figure G). PCD patients with mutations in DNAH5
typically have cilia with missing ODAs (3
). In our mutants, the number of ODAs was markedly reduced (Figure I), with 7.5 ± 2.1 ODAs per cilium seen in wild-type animals and 0.29 ± 0.56 and 5.7 ± 2.5 ODAs per cilium in homozygous and heterozygous mutants, respectively. A frequency distribution plot of the number of ODAs per cilium showed a significant difference between all 3 genotypes (Figure ), indicating that the Dnahc5del593
allele causes ODA deficiency, as DNAH5
mutations do in PCD patients. We also examined the variability in ciliary orientation by drawing a line through the central doublet in a cross-section view of the cilia and calculating the angular variance relative to the reference line (Table ; see Methods), a measurement often used in the clinical diagnosis of PCD (23
). As expected, the cilia of homozygous mutants showed significantly greater variation in orientation than wild-type cilia, in agreement with the qualitative SEM analysis (Table ).
Graph depicting distribution of ODAs per cilium.
Quantitation of ciliary ultrastructure, orientation and function
To examine ciliary function in the airway epithelia, we used another standard assay for PCD involving motion analysis of the tracheal epithelia by videomicroscopy. Wild-type tracheal epithelia exhibited synchronous movement of the cilia, while the airway epithelia of homozygous mutants were immotile, or slow and dyskinetic. We quantitatively assessed the effectiveness of ciliary beating using fluorescent beads deposited above the epithelia. Motion analysis showed a marked reduction in both the velocity and directionality of bead displacement by the homozygous mutant tracheal epithelia, indicating a marked reduction in net fluid flow (Table ). However, the airway epithelia of wild-type and heterozygous animals showed no significant difference in bead displacement, consistent with the absence of any situs defects in such animals (Table ). Together these findings confirm that the Dnahc5del593 allele is a recessive mutation that models PCD.