Autonomous and non-cell autonomous role of cilia in structural birth defects in mice.

Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140, an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140-deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.

Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.

Ion Torrent sequencing for conducting genome-wide scans for mutation mapping analysis.

Mutation mapping in mice can be readily accomplished by genome wide segregation analysis of polymorphic DNA markers. In this study, we showed the efficacy of Ion Torrent next generation sequencing for conducting genome-wide scans to map and identify a mutation causing congenital heart disease in a mouse mutant, Bishu, recovered from a mouse mutagenesis screen. The Bishu mutant line generated in a C57BL/6J (B6) background was intercrossed with another inbred strain, C57BL/10J (B10), and the resulting B6/B10 hybrid offspring were intercrossed to generate mutants used for the mapping analysis. For each mutant sample, a panel of 123 B6/B10 polymorphic SNPs distributed throughout the mouse genome was PCR amplified, bar coded, and then pooled to generate a single library used for Ion Torrent sequencing. Sequencing carried out using the 314 chip yielded >600,000 usable reads. These were aligned and mapped using a custom bioinformatics pipeline. Each SNP was sequenced to a depth >500×, allowing accurate automated calling of the B6/B10 genotypes. This analysis mapped the mutation in Bishu to an interval on the proximal region of mouse chromosome 4. This was confirmed by parallel capillary sequencing of the 123 polymorphic SNPs. Further analysis of genes in the map interval identified a splicing mutation in Dnaic1(c.204+1G>A), an intermediate chain dynein, as the disease causing mutation in Bishu. Overall, our experience shows Ion Torrent amplicon sequencing is high throughput and cost effective for conducting genome-wide mapping analysis and is easily scalable for other high volume genotyping analyses.

Imaging techniques for visualizing and phenotyping congenital heart defects in murine models.

Mouse model is ideal for investigating the genetic and developmental etiology of congenital heart disease. However, cardiovascular phenotyping for the precise diagnosis of structural heart defects in mice remain challenging. With rapid advances in imaging techniques, there are now high throughput phenotyping tools available for the diagnosis of structural heart defects. In this review, we discuss the efficacy of four different imaging modalities for congenital heart disease diagnosis in fetal/neonatal mice, including noninvasive fetal echocardiography, micro-computed tomography (micro-CT), micro-magnetic resonance imaging (micro-MRI), and episcopic fluorescence image capture (EFIC) histopathology. The experience we have gained in the use of these imaging modalities in a large-scale mouse mutagenesis screen have validated their efficacy for congenital heart defect diagnosis in the tiny hearts of fetal and newborn mice. These cutting edge phenotyping tools will be invaluable for furthering our understanding of the developmental etiology of congenital heart disease.

Congenital heart disease and the specification of left-right asymmetry.

Complex congenital heart disease (CHD) is often seen in conjunction with heterotaxy, the randomization of left-right visceral organ situs. However, the link between cardiovascular morphogenesis and left-right patterning is not well understood. To elucidate the role of left-right patterning in cardiovascular development, we examined situs anomalies and CHD in mice with a loss of function allele of Dnaic1, a dynein protein required for motile cilia function and left-right patterning. Dnaic1 mutants were found to have nodal cilia required for left-right patterning, but they were immotile. Half the mutants had concordant organ situs comprising situs solitus or mirror symmetric situs inversus. The remaining half had randomized organ situs or heterotaxy. Looping of the heart tube, the first anatomical lateralization, showed abnormal L-loop bias rather than the expected D-loop orientation in heterotaxy and nonheterotaxy mutants. Situs solitus/inversus mutants were viable with mild or no defects consisting of azygos continuation and/or ventricular septal defects, whereas all heterotaxy mutants had complex CHD. In heterotaxy mutants, but not situs solitus/inversus mutants, the morphological left ventricle was thin and often associated with a hypoplastic transverse aortic arch. Thus, in conclusion, Dnaic1 mutants can achieve situs solitus or inversus even with immotile nodal cilia. However, the finding of abnormal L-loop bias in heterotaxy and nonheterotaxy mutants would suggest motile cilia are required for normal heart looping. Based on these findings, we propose motile nodal cilia patterns heart looping but heart and visceral organ lateralization is driven by signaling not requiring nodal cilia motility.

Connexin 43 regulates epicardial cell polarity and migration in coronary vascular development.

Connexin 43 knockout (Cx43 KO) mice exhibit conotruncal malformations and coronary artery defects. We observed epicardial blisters in the Cx43 KO hearts that suggest defects in epicardial epithelial-mesenchymal transformation (EMT), a process that generates coronary vascular progenitors. Analysis using a three-dimensional collagen gel invasion assay showed that Cx43 KO epicardial cells are less invasive and that, unlike wild-type epicardial cells, they fail to organize into thin vessel-like projections. Examination of Cx43 KO hearts using Wt1 as an epicardial marker revealed a disorganized pattern of epicardial cell infiltration. Time-lapse imaging and motion analysis using epicardial explants showed a defect in directional cell migration. This was associated with changes in the actin/tubulin cytoskeleton. A defect in cell polarity was indicated by a failure of the microtubule-organizing center to align with the direction of cell migration. Forced expression of Cx43 constructs in epicardial explants showed the Cx43 tubulin-binding domain is required for Cx43 modulation of cell polarity and cell motility. Pecam staining revealed early defects in remodeling of the primitive coronary vascular plexuses in the Cx43 KO heart. Together, these findings suggest an early defect in coronary vascular development arising from a global perturbation of the cytoarchitecture of the cell. Consistent with this, we found aberrant myocardialization of the outflow tract, a process also known to be EMT dependent. Together, these findings suggest cardiac defects in the Cx43 KO mice arise from the disruption of cell polarity, a process that may be dependent on Cx43-tubulin interactions.

Heterotaxy and complex structural heart defects in a mutant mouse model of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder associated with ciliary defects and situs inversus totalis, the complete mirror image reversal of internal organ situs (positioning). A variable incidence of heterotaxy, or irregular organ situs, also has been reported in PCD patients, but it is not known whether this is elicited by the PCD-causing genetic lesion. We studied a mouse model of PCD with a recessive mutation in Dnahc5, a dynein gene commonly mutated in PCD. Analysis of homozygous mutant embryos from 18 litters yielded 25% with normal organ situs, 35% with situs inversus totalis, and 40% with heterotaxy. Embryos with heterotaxy had complex structural heart defects that included discordant atrioventricular and ventricular outflow situs and atrial/pulmonary isomerisms. Variable combinations of a distinct set of cardiovascular anomalies were observed, including superior-inferior ventricles, great artery alignment defects, and interrupted inferior vena cava with azygos continuation. The surprisingly high incidence of heterotaxy led us to evaluate the diagnosis of PCD. PCD was confirmed by EM, which revealed missing outer dynein arms in the respiratory cilia. Ciliary dyskinesia was observed by videomicroscopy. These findings show that Dnahc5 is required for the specification of left-right asymmetry and suggest that the PCD-causing Dnahc5 mutation may also be associated with heterotaxy.

Automated identification of abnormal respiratory ciliary motion in nasal biopsies.

Motile cilia lining the nasal and bronchial passages beat synchronously to clear mucus and foreign matter from the respiratory tract. This mucociliary defense mechanism is essential for pulmonary health, because respiratory ciliary motion defects, such as those in patients with primary ciliary dyskinesia (PCD) or congenital heart disease, can cause severe sinopulmonary disease necessitating organ transplant. The visual examination of nasal or bronchial biopsies is critical for the diagnosis of ciliary motion defects, but these analyses are highly subjective and error-prone. Although ciliary beat frequency can be computed, this metric cannot sensitively characterize ciliary motion defects. Furthermore, PCD can present without any ultrastructural defects, limiting the use of other detection methods, such as electron microscopy. Therefore, an unbiased, computational method for analyzing ciliary motion is clinically compelling. We present a computational pipeline using algorithms from computer vision and machine learning to decompose ciliary motion into quantitative elemental components. Using this framework, we constructed digital signatures for ciliary motion recognition and quantified specific properties of the ciliary motion that allowed high-throughput classification of ciliary motion as normal or abnormal. We achieved >90% classification accuracy in two independent data cohorts composed of patients with congenital heart disease, PCD, or heterotaxy, as well as healthy controls. Clinicians without specialized knowledge in machine learning or computer vision can operate this pipeline as a "black box" toolkit to evaluate ciliary motion.

Decellularized tracheal extracellular matrix supports epithelial migration, differentiation, and function.

Tracheal loss is a source of significant morbidity for affected patients with no acceptable solution. Interest in engineering tracheal transplants has created a demand for small animal models of orthotopic tracheal transplantation. Here, we examine the use of a decellularized graft in a murine model of tracheal replacement. Fresh or decellularized tracheas harvested from age-matched female donor C57BL/6 mice were transplanted into syngeneic recipients. Tracheas were decellularized using repeated washes of water, 3% Triton X-100, and 3 M NaCl under cyclic pressure changes, followed by disinfection with 0.1% peracetic acid/4% ethanol, and terminal sterilization by gamma irradiation. Tracheas were explanted for immunolabeling at 1, 4, and 8 weeks following surgery. Video microscopy and computed tomography were performed to assess function and structure. Decellularized grafts supported complete reepithelialization by 8 weeks and motile cilia were observed. Cartilaginous portions of the trachea were maintained in mice receiving fresh transplants, but repopulation of the cartilage was not seen in mice receiving decellularized transplants. We observed superior postsurgical survival, weight gain, and ciliary function in mice receiving fresh transplants compared with those receiving decellularized transplants. The murine orthotopic tracheal transplant provides an appropriate model to assess the repopulation and functional regeneration of decellularized tracheal grafts.

ANKS6 is the critical activator of NEK8 kinase in embryonic situs determination and organ patterning.

The ciliary kinase NEK8 plays a critical role in situs determination and cystic kidney disease, yet its exact function remains unknown. In this study, we identify ANKS6 as a target and activator of NEK8. ANKS6 requires NEK8 for localizing to the ciliary inversin compartment (IC) and activates NEK8 by binding to its kinase domain. Here we demonstrate the functional importance of this interaction through the analysis of two novel mouse mutations, Anks6(Streaker) and Nek8(Roc). Both display heterotaxy, cardiopulmonary malformations and cystic kidneys, a syndrome also characteristic of mutations in Invs and Nphp3, the other known components of the IC. The Anks6(Strkr) mutation decreases ANKS6 interaction with NEK8, precluding NEK8 activation. The Nek8(Roc) mutation inactivates NEK8 kinase function while preserving ANKS6 localization to the IC. Together, these data reveal the crucial role of NEK8 kinase activation within the IC, promoting proper left-right patterning, cardiopulmonary development and renal morphogenesis.

Changes in activin and activin receptor subunit expression in rat liver during the development of CCl4-induced cirrhosis.

Amounts of betaA-activin, betaC-activin, activin receptor subunits ActRIIA and ActRIIB mRNA, and betaA- and betaC-activin subunit protein immunoreactivity were investigated in male Lewis rats, either untreated or after 5 or 10 weeks of CCl(4) treatment to induce cirrhosis. Apoptosis was assessed histologically and with an in situ cell death detection kit (TUNEL). Reverse transcription and polymerase chain reaction were used to evaluate mRNA levels. Activin betaA- and betaC-subunit immunoreactivity was studied by immunohistochemistry using specific monoclonal antibodies. Hepatocellular apoptosis (P<0.001), increased betaA- and betaC-activin mRNAs (three- to fourfold; P<0.01) and increased betaA- and betaC-activin tissue immunoreactivity were evident, whereas ActRIIA mRNA concentrations fell (30%; P<0.01) after 5 weeks of CCl(4) treatment. The mRNA concentrations at 10 weeks were not significantly different from controls, despite extensive hepatic nodule formation. We conclude that the increased activin subunit expression is associated with apoptosis, rather than hepatic fibrosis and nodule formation.

Airway ciliary dysfunction and sinopulmonary symptoms in patients with congenital heart disease.

RATIONALE: Patients with congenital heart disease with heterotaxy exhibit a high prevalence of abnormal airway ciliary motion and low nasal nitric oxide, characteristics associated with primary ciliary dyskinesia, a reflection of the role of motile cilia in airway clearance and left-right patterning. OBJECTIVES: To assess the potential broader clinical significance of airway ciliary dysfunction in congenital heart disease, we assessed the prevalence of ciliary dysfunction versus respiratory symptoms in patients with congenital heart disease with or without heterotaxy. METHODS: Patients with a broad spectrum of congenital heart disease were recruited (n = 218), 39 with heterotaxy. Nasal nitric oxide measurements and nasal biopsies for ciliary motion video microscopy were conducted. Sinopulmonary symptoms were reviewed by questionnaire. MEASUREMENTS AND MAIN RESULTS: A high prevalence of ciliary motion defects (51.8%) and low or borderline low nasal nitric oxide levels (35.5%) were observed in patients with congenital heart disease with or without heterotaxy. Patients with ciliary motion defects or low nasal nitric oxide showed increased sinopulmonary symptoms, with most respiratory symptoms seen in those with both abnormal ciliary motion and low nitric oxide. Multivariate analysis showed that abnormal ciliary motion and low nasal nitric oxide were more important in determining risk of sinopulmonary symptoms than heterotaxy status. CONCLUSIONS: Patients with congenital heart disease without heterotaxy exhibit a high prevalence of abnormal ciliary motion and low nasal nitric oxide. This was associated with more sinopulmonary symptoms. These findings suggest that patients with a broad spectrum of congenital heart disease and respiratory symptoms may benefit from screening for ciliary dysfunction and implementation of medical interventions to reduce sinopulmonary morbidities.

Initiation and maturation of cilia-generated flow in newborn and postnatal mouse airway.

Mucociliary clearance in the adult trachea is well characterized, but there are limited data in newborns. Cilia-generated flow was quantified across longitudinal sections of mouse trachea from birth through postnatal day (PND) 28 by tracking fluorescent microsphere speed and directionality. The percentage of ciliated tracheal epithelial cells, as determined by immunohistochemistry, was shown to increase linearly between PND 0 and PND 21 (R(2) = 0.94). While directionality measurements detected patches of flow starting at PND 3, uniform flow across the epithelia was not observed until PND 7 at a approximately 35% ciliated cell density. Flow became established at a maximal rate at PND 9 and beyond. A linear correlation was observed between the percentage of ciliated cells versus flow speed (R(2) = 0.495) and directionality (R(2) = 0.975) between PND 0 and PND 9. Cilia beat frequency (CBF) was higher at PND 0 than at all subsequent time points, but cilia beat waveform was not noticeably different. Tracheal epithelia from a mouse model of primary ciliary dyskinesia (PCD) harboring a Mdnah5 mutation showed that ciliated cell density was unaffected, but no cilia-generated flow was detected. Cilia in mutant airways were either immotile or with slow dyssynchronous beat and abnormal ciliary waveform. Overall, our studies showed that the initiation of cilia-generated flow is directly correlated with an increase in epithelial ciliation, with the measurement of directionality being more sensitive than speed for detecting flow. The higher CBF observed in newborn epithelia suggests unique physiology in the newborn trachea, indicating possible clinical relevance to the pathophysiology of respiratory distress seen in newborn PCD patients.

Primordial germ cell deficiency in the connexin 43 knockout mouse arises from apoptosis associated with abnormal p53 activation.

Connexin 43 knockout (Cx43alpha1KO) mice exhibit germ cell deficiency, but the underlying cause for the germ cell defect was unknown. Using an Oct4-GFP reporter transgene, we tracked the distribution and migration of primordial germ cells (PGCs) in the Cx43alpha1KO mouse embryo. Analysis with dye injections showed PGCs are gap-junction-communication competent, with dye coupling being markedly reduced in Cx43alpha1-deficient PGCs. Time-lapse videomicroscopy and motion analysis showed that the directionality and speed of cell motility were reduced in the Cx43alpha1KO PGCs. This was observed both in E8.5 and E11.5 embryos. By contrast, PGC abundance did not differ between wild-type and heterozygous/homozygous Cx43alpha1KO embryos until E11.5, when a marked reduction in PGC abundance was detected in the homozygous Cx43alpha1KO embryos. This was accompanied by increased PGC apoptosis and increased expression of activated p53. Injection of alpha-pifithrin, a p53 antagonist, inhibited PGC apoptosis and prevented the loss of PGC. Analysis using a cell adhesion assay indicated a reduction in beta1-integrin function in the Cx43alpha1KO PGCs. Together with the abnormal activation of p53, these findings suggest the possibility of anoikis-mediated apoptosis. Overall, these findings show Cx43alpha1 is essential for PGC survival, with abnormal p53 activation playing a crucial role in the apoptotic loss of PGCs in the Cx43alpha1KO mouse embryos.

Endothelin receptor A blockade ameliorates hypothermic ischemia-reperfusion-related microhemodynamic disturbances during liver transplantation in the rat.

BACKGROUND: The objective of this study was to investigate the effect of graft treatment with specific endothelin receptor antagonists (ET(A) and ET(B)) on the microhemodynamic disturbances which occur following ischemia/reperfusion injury during orthotopic liver transplantation (OLT) in the rat. MATERIALS AND METHODS: OLT was performed in male Sprague-Dawley rats. An ET(A) receptor antagonist (BQ-610; 0.3 mg/kg) or ET(B) receptor antagonist IRL-1038 (20 nmol/kg) was administered intraportally into liver grafts in vitro at the beginning of 2- and 6-h cold storage (4 degrees C) using physiological saline. Sham-operated animals served as controls (Cont). Seven groups were studied: Cont; vehicle-2 h (saline treated); ET(B) antagonist-2 h; ET(A) antagonist-2 h; vehicle-6 h; ET(A) antagonist-6 h; and ET(B) antagonist-6 h. At 1 h after graft implantation, the liver microcirculation was investigated by intravital fluorescence microscopy. RESULTS: In vehicle-treated livers, the hepatic microcirculation was markedly impaired compared with the Cont as manifested by a reduced lobular perfusion index, increased incidence of sinusoidal nonperfusion, elevated leukocyte adhesion in sinusoids and terminal hepatic venules, and increased hepatic venous resistance (23-fold; 6-h group). In addition, plasma liver enzymes were significantly elevated in the vehicle treated groups. Alterations to all these parameters were markedly reduced in the ET(A) receptor antagonist-treated liver grafts although there was still evidence of hepatic injury. The ET(B) receptor antagonist had little effect on the I/R-induced changes to the hepatic microcirculation. CONCLUSIONS: Our results indicate that the ET(A) antagonism ameliorates hypothermic I/R-related microhemodynamic disturbances during OLT in the rat, suggesting that application of an ET(A) antagonist to liver grafts may have therapeutic potential in human liver transplantation.