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.

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.

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.

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.

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.