Analysis of the airways

If motility of cilia in the airways is disturbed, mucus accumulates in the nasal cavities. Mucus can be visualized by PAS staining, which detects glycoproteins that are part of the mucus.

Three adult wild type and knock-out individuals were investigated for the presence of mucus in nasal cavities. Figure 2.27 shows exemplary images of each one 13 weeks old wild type and mutant mouse, in which mucus accumulations are indicated by arrowheads. In the wild type animals mucus was only present in small spots or not detected at all. In contrast in each of theCfap43knock-out mice mucus was accumulated in the nasal cavities.

From experiments, in which antibodies were evaluated using knock-out mouse tissues (figures 2.11, 2.24 and 2.28), it became apparent, that multiciliated cells are still present in the airways as well as in other tissues from mutant animals. More detailed analysis of multiciliated tracheal cells from mutant and wild type mice revealed no obvious alterations of

Figure 2.27: Mucus accumulation in Cfap43 knock-out mice. Mucus (arrowheads) present in the nasal cavities was visualized by PAS staining. While only few small spots of mucus could be found in wild type nasal cavities from 13 weeks old animals, big stretches of mucus pervade nasal cavities of homozygous knock-out littermates.

motile cilia, as determined by immunofluorescence staining of two cilia markers (acetylated tubulin and IFT88) and DIC imaging (figure 2.28 A-C). Thus, processes generally needed for ciliogenesis, such as basal body biogenesis and intraflagellar transport, are not suppressed by Cfap43 knock-out. In addition the ciliary ultrastructure from motile cilia of lungs was analyzed by transmission electron microscopy (TEM) by Dr. Jan Hegermann (Institute for functional and applied anatomy, Hannover medical school). High magnification images of individual cilia (figure 2.28 D) show the presence and regular arrangement of both, the nine outer microtubular doublets and the central pair of microtubuli. In addition electron-dense material next to the microtubule doublets was detected, which might represent inner and outer dynein arms (IDAs and ODAs; arrows indicate each one pair in wild type and mutant cilia). Pink asterisks mark places, where ODAs would be expected, but were not detected.

Analogous yellow asterisks mark "missing" IDAs. Since dynein arms are difficult to find in each section of the TEM images, it is not clear, whether all inner dynein arms are present.

However, comparing the depicted cross-sections it appears possible that some inner dynein arms might be absent in motile cilia ofCfap43knock-out airway cells. TEM images showing an overview over an array of motile cilia (figure 2.28 E) reveal that the central microtubule pairs of individual cilia are oriented in the same direction, as indicated by yellow bars in the left part of the images. Also basal feet, which are shown in the inlay for TEM overview of Cfap43-/-cilia, are uniformly oriented. Hence, rotational polarity of individual cilia appears not to be affected by CFAP43.

Given that motile cilia are present in airway cells of knock-out mice, defects in the ciliary ultrastructure could not unambiguously be shown, and the number of multciliated mTECs was reduced subsequent to lentiviral knock-down of CFAP43 in mTECs (see section 2.4.3), a lower number of multiciliated cells in the airways of knock-out mice could explain the reduced ability of mutant mice to perform mucociliary clearance. To control whether the results obtained in mTECs can be reproduced in the knock-out mouse, trachea sections and ALI cultured mTECs of wild type and knock-out mice were stained for FOXJ1 and IFT88.

As before, cells were counted, which expressed FOXJ1 and both FOXJ1 and IFT88 together (see table 2.4). Suprisingly, the reduction of cells, which completed multiple ciliation after initiation of the multiciliogenesis program (marked by FOXJ1 expression) was less severe in the knock-out situation as compared to the knock-down. Thus, the results observed before in the knock-down situation were not completely reproduced, neither in trachea sections nor in cell culture.

Taken together, mucociliary clearance is impaired inCfap43knock-out mice. Gross changes in ciliary structure, length and rotational polarity as well as a reduced number of multicili-ated cells in the airways are unlikely to cause this phenotype. TEM images suggested that

2.5 Analysis of murine Cfap43 in vivo

Figure 2.28: Appearance of multiple cilia of airways epithelia in wild type and knock-out mice.

ACilia (ac. tubulin, red) and basal bodies (CEP63, green) were indistinguishable in isolated mTECs from wild type and knock-out mice. BDIC images and immunofluorescence staining (ac. tubulin, red) of cilia in sections of PFA fixed tracheas revealed no differences between wild type and knock-out multiciliated cells. CIFT88 was distributed along the whole cilia in cultured mTECs from both wild type and mutant mice. D High magnification TEM images of individual cilia show regularly arranged 9+2 axonemes in wild type and knock-out cilia. Arrows point to electron-dense material, which represents dynein arms. Asterisks indicate non-detected inner (yellow) and outer (pink) dynein arms. E Overview TEM images over arrays of cilia reveal uniform orientation of individual cilia, as indicated by yellow bars representing orientation of the central microtubule pair.

Table 2.4:Ciliation of FOXJ1 expressing cells in wild type andCfap43knock-out mice Sample type Count FOXJ1 Count IFT88 Marker/FOXJ1 Normalized Wild type

Trachea sections 103 97 0.94 1

ALI cultured mTECs 306 292 0.95 1

Cfap43knock-out

Trachea sections 241 217 0.90 0.96

Trachea sections 257 217 0.84 0.90

ALI cultured mTECs 140 123 0.88 0.92

some inner dynein arms might be missing in the knock-out situation, which requires a more detailed analysis in future studies.