Conditional deletion of dnaic1 in a murine model of primary ciliary dyskinesia causes chronic rhinosinusitis

Abstract

Studies of primary ciliary dyskinesia (PCD) have been hampered by the lack of a suitable animal model because disruption of essential ciliary genes in mice results in a high incidence of lethal hydrocephalus. To develop a viable mouse model for long-term studies of PCD, we have generated a transgenic mouse line in which two conserved exons of the mouse intermediate dynein chain gene, Dnaic1, are flanked by loxP sites (Dnaic1(flox/flox)). Dnaic1 is the murine homolog of human DNAI1, which is mutated in approximately 10% of human PCD cases. These mice have been crossed with mice expressing a tamoxifen-inducible Cre recombinase (CreER). Treatment of adult Dnaic1(flox/flox)/CreER(+/-) mice with tamoxifen results in an almost complete deletion of Dnaic1 with no evidence of hydrocephalus. Treated animals have reduced levels of full-length Dnaic1 mRNA, and electron micrographs of cilia demonstrate a loss of outer dynein arm structures. In treated Dnaic1(flox/flox)/CreER(+/-) animals, mucociliary clearance (MCC) was reduced over time. After approximately 3 months, no MCC was observed in the nasopharynx, whereas in the trachea, MCC was observed for up to 6 months, likely reflecting a difference in the turnover of ciliated cells in these tissues. All treated animals developed severe rhinosinusitis, demonstrating the importance of MCC to the health of the upper airways. However, no evidence of lung disease was observed up to 11 months after Dnaic1 deletion, suggesting that other mechanisms are able to compensate for the lack of MCC in the lower airways of mice. This model will be useful for the study of the pathogenesis and treatment of PCD.

Figure 1.

Expression of Dnaic1 mRNA in various mouse tissues. RT-PCR was performed on RNA isolated from the indicated tissues using primers specific for Dnaic1 mRNA. A strong positive signal was obtained from tissues with cilia (trachea, nasal) and flagella (testis); other tissues were negative.

Figure 2.

Schematic diagram of the Dnaic1 protein (top) showing the approximate location of the WD repeats. Diagram of the targeted genomic region of Dnaic1 (middle) showing the loxP sites flanking exons 17 and 18. These exons include one of the WD repeat regions. Activation of CreER with tamoxifen causes the removal of exons 17 and 18 (bottom).

Figure 3.

(A) Genomic DNA was isolated from untreated cultures of homozygous loxP/Dnaic1 (control) or from cultures treated for 6 days with tamoxifen and amplified with primers specific to exons 17 and 18. The tamoxifen-treated cultures demonstrate an almost complete absence of full-length Dnaic1. In the tamoxifen-treated culture, the absence of a specific target results in the generation of some nonspecific products. (B) Western blot analysis of Dnaic1 expression in mouse tracheal cells (left panel). Total cell lysates from differentiated cultures of wild-type (WT) (lane 1), Dnaic1^flox/Wt (lane 2), Dnaic1^flox/flox (lane 3), and undifferentiated WT (lane 4) mouse tracheal epithelial cells were separated by gel electrophoresis and probed with a monoclonal antibody against Dnaic1. Cultures from WT mice express only the full length Dnaic1 protein; cultures of Dnaic1^flox/flox cells express only the shorter, deleted protein. Heterozygote cultures (Dnaic1^flox/Wt) express both products; undifferentiated cells express neither. Cilia isolated from cultures of WT (right panel, lane 1) and Dnaic1^flox/Wt (lane 2) cultures show clear incorporation of the full-length protein, whereas cilia isolated from Dnaic1^flox/flox cells show no incorporation of the deleted Dnaic1 protein (lane 3). (C) Ciliary activity in cultures of WT and Dnaic1^flox/flox mouse tracheal epithelial cells treated with tamoxifen or vehicle control. All cultures were positive for the CreER transgene. Cultures were treated for the first 6 days of culture, and ciliary activity was measured on Day 20. Control cultures averaged greater than 30% ciliated surface area, whereas the PCD cultures averaged less than 1%. Data shown are from one representative experiment with three cultures in each group and nine measurements per culture (n = 27 for each condition). Error bars indicate SEM. (D) Hematoxylin and eosin–stained paraffin section of Dnaic1^flox/flox mouse tracheal epithelial cells cultured in the absence (top) or presence (bottom) of 1 μM tamoxifen. Fully differentiated ciliated cells are abundant in both cultures. Images are representative of two independent experiments.

Figure 4.

(A) Genomic DNA isolated from lung tissue of tamoxifen treated mice was amplified by PCR using primers that span the loxP targeted region of Dnaic1. DNA from WT mice (Dnaci1^+/+) produced only the expected 1.4 kb full-length product, DNA from the homozygous loxP/Dnaic1 mouse (Dnaic1^−/−) produced essentially only the deleted 564 bp product, and samples from the heterozygous mice (Dnaic1^+/−) produced both. (B) RNA isolated from the same tissue as in A was analyzed by RT-PCR. Note the almost complete absence of intact Dnaic1 mRNA in the homozygous treated animal (Dnaic1^−/−).

Figure 5.

Transmission electron micrographs of tracheal cilia from (A) a control mouse (Dnaic1^wt/flox) and (B) a mouse with primary ciliary dyskinesia (PCD) (Dnaic1^flox/flox) 7 months after tamoxifen treatment. Outer dynein arms (ODAs) are clearly visible in cilia from the control animal but are absent in cilia from the PCD mouse (arrows). Computer-assisted image averaging of individual outer doublets more clearly illustrates the presence of the ODA in control axonemes (asterisk in C) and the absence of ODA in PCD axonemes (asterisk in D). Images shown are the average of 17 and 27 doublets, respectively.

Figure 6.

Mucociliary clearance (A) in the nasopharynx and (B) in the trachea of tamoxifen-treated Dnaic1^flox/flox mice. Groups of animals were treated with tamoxifen, and mucociliary clearance (MCC) was measured at the times indicated and expressed as a percentage of the rate of MCC in a parallel group of control animals. Complete inhibition of MCC requires approximately 2 months in the nasopharynx and approximately 6 months in the trachea after the deletion of Dnaic1. x axis = time after tamoxifen treatment in months. Each symbol represents a single animal.

Figure 7.

Rhinosinusitis in PCD mice. (A) Paraffin section of a control mouse (11 mo) showing the nasal septum (1), the ethmoturbinates (2), and the maxillary sinus (3). Nasal cavity of representative PCD mice 2 (B) and 11 (C) months after Dnaic1 deletion. Higher magnification images of ciliated epithelium from the maxillary sinus (D, E) and olfactory epithelium (F, G) of a control (D, F) and of the PCD mouse (E, G) shown in B. Note the accumulation of mucus and inflammatory neutrophils and the degeneration of the epithelium in the PCD animal. (H and I) Low-magnification images of the ethmoturbinates from the animals shown in A and C, showing the attenuation of epithelium and the underlying bone structure in the PCD mouse. A, B, and C were stained with alcian blue–periodic acid Schiff; D, E, H, and I were stained with hematoxylin and eosin; F and G were stained with alcian blue–toluidine yellow. Scale bars for D, E, F, and G = 10 μm; scale bars for H and I = 40 μm.