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Figure 1. Expression of Cfap161 in mouse and Xenopus. (A) Analysis of Cfap161 and Foxj1 expression by RT-PCR of RNA from wild type adult organs as indicated on top. Hprt was used as quality control. The full-size agarose gel is shown in Fig. S8. (B) WISH of E7.75 wild type murine embryo (a, a′) and SISH of E16.5 wild type embryos showing Cfap161 expression (arrowheads) in the node (a) and indicated tissues (b–e) that develop motile cilia. Red boxed area in (a) indicate the region shown in higher magnification in (a′). (C) SISH analysis of wild type adult tissues (indicated on top). Red boxed areas in (a–f) indicate the regions shown at higher magnification in (a′–f′). White arrowheads point to regions of expression in cells possessing motile cilia. Red arrowheads point to regions of cells with primary cilia. PRL photoreceptor layer, INL inner nuclear layer, GCL ganglion cell layer. (D) Xenopus WISH detected cfap161 transcripts in the left–right organiser (LRO; a, a′), floor plate (FP; a′), nephrostomes marked by white arrowheads (b), multiciliated cells (MCCs) of the skin (b), stomach (stom.; c, c′), branchial chamber (BC; c, c″) subcommissural organ (SCO; c, c‴) and in the zona limitans intrathalamica (ZLI; c, c‴). Transversal section planes are indicated with stippled lines and shown in (a′, c′, c″, c‴). Scale bars: (B, D) = 100 µm; (C) = 500 µm.
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Figure 2. Localisation of CFAP161 and the dependence on FOXJ1. (A) Schematic representation of the exon structure of Cfap161 and regions encoding the peptides (pI and pII) used for the generation of antibodies. (B) Western blot of overexpressed (CFAP161Flag in CHO cells) and endogenous CFAP161 (from mouse testis lysate) detected with monoclonal (pI) and polyclonal (pII) antibodies. The full-size Western blots are shown in Fig. S9A. (C) Induction of CFAP161 expression during cilia formation of air–liquid interface (ALI) cultures of mouse tracheal epithelial cells (mTEC). α-CFAP161 pII was used for visualising CFAP161, α-IFT88 to monitor ciliogenesis and α-β-Tubulin (β-TUB) as loading control. The full-size Western blot is shown in Fig. S9B. (D) Detection of endogenous CFAP161 by indirect immunohistochemistry of E17.5 wild type and Foxj1-mutant sections. Red boxed areas in a-h indicate the regions shown at higher magnification in a′–h′. Note that CFAP161 is absent in all analysed tissues of Foxj1lacZ/lacZ specimens. α-CFAP161 pI was used for the indirect DAB-immunostaining. (E) Expression of cfap161 was largely erased in foxj1-crispant embryos (b, b′) in comparison to wild types (a, a′). Red boxed areas in (a, b) indicate the regions shown at higher magnification in (a′, b′). (F) Unilateral foxj1 gain of function (foxj1-GOF) induced cfap161. Side of injection as indicated by asterisk. Scale bars: (D) = 500 µm; (E, F) = 100 µm.
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Figure 3. Subcellular localisation of CFAP161. (A) Indirect immunofluorenscence staining of murine adult fallopian tube sections (a, b), sections of adult respiratory epithelium of the nasal cavity (c, d), and seminiferous tubule of the adult testis (e, e′) showing localisation of CFAP161 to the ciliary axoneme and flagella, respectively. The distal region of cilia marked by white arrowhead lacks CFAP161. Boxed areas in a-d indicate the regions shown at higher magnification in a′–d′ and a″–d″, boxed areas in a′–d′ and a″–d″ indicate the regions shown enlarged in the respective insets. (B) Localisation of mouse GPF-CFAP161 in Xenopus MCCs co-expressing the basal body marker Cetn4-RFP (a) revealed accumulation of CFAP161 in anterior juxtaposition (a′) underneath Cetn4 (a″) as illustrated in (b). Boxed area in (a) indicate the region shown at higher magnification in (a′). Orthogonal section as indicated and shown in (a″). Scale bars: (A)a–d = 100 µm; (A)e, e′ = 25 µm; B = 100 µm.
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Figure 4. No cilia related phenotypes in Xenopus cfap161-crispants. (A) Representative wild type (a) and crispant (b) tadpoles at stage 45. (B) Evaluation of organ situs, embryonic cysts (C) and hydrocephalus (D) in 297 crispants obtained in 5 independent experiments. Statistical calculation of organ situs defects, cyst analysis or hydrocephalus formation was performed using chi square (http://www.physics.csbsju.edu/stats/contingency_NROW_NCOLUMN_form.html). (E) Ciliary beat frequency (CBF) in wild type and crispants. (a) Statistical evaluation of CBF. Results from 3 independent experiments with each 5 embryos and 5 analysed MCCs per embryo. Graph displays respective values with mean and s.d. Raw data are shown in Table S3. (b) Kymographs of ciliary motility of single MCCs of wild type and crispant embryos. (F) Cilia generated flow (CGF) (a) Statistical evaluation of CGF, velocities of bead transport in wild type and crispant embryos. Results from 3 independent experiments with 8 analysed specimens each. Graph displays respective values with mean and s.d. Raw data are shown in Table S4. (b) Maximum intensity projection of bead transport of single wild type and crispant embryo. Compared to wild type specimens no significant deviations were recorded in any experiment. ns P > 0.05. p-values were calculated via Wilcoxon-Match-Pair test in RStudio. Scale bar: (A) = 500 µm.
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Figure 5. Generation of a murine Cfap161-null allele Cfap161∆ex2,3. (A) Schematic drawing showing the structure of the targeted Cfap161 locus and mutated alleles (Cfap161loxP and Cfap161∆ex2,3). (B) Western blots of testis and epididymis lysates with monoclonal pI and polyclonal pII antibody show absence of CFAP161 in the mutant (Cfap161∆ex2,3/∆ex2,3). α-IFT88 was used as loading control. The full-size Western blots are shown in Fig. S9C. (C) Indirect immunofluorescence of wild type (a, a′, c, c′) and Cfap161∆ex2,3 mutant (b, b′, d, d′) testis sections showing absence of CFAP161 from flagella of mutant sperm. White arrowheads: flagella; asterisk: non-specific staining around seminiferous tubules. Scale bar: (C) = 50 µm.
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Figure 6. Absence of phenotypes in Cfap161∆ex2,3 mutants. (A) Schematic drawing of the brain indicating the planes of the sections shown in (Ca–d). (B) Schematic drawing of the face skull indicating the planes of the sections shown in (Cg–j). (C) (a–d) Sections of wild type (a, c) and Cfap161∆ex2,3 mutant (b, d) brains at the two horizontal levels indicated in (A). Red boxed areas indicate the regions shown at higher magnification in a′, b′, c′, c″, d′, and d″. Asterisks marks the choroid plexus in the ventricle lumen. (e, f) Representative sections of wild type (e) and Cfap161∆ex2,3 mutant (f) lungs. Red boxed areas indicate the regions shown at higher magnification in e′, and f′. Arrows point to PAS-positive (purple stained) cells, representing the mucus producing cells. (g–j) Sections of wild type (g, i) and Cfap161∆ex2,3 mutant (h, j) nasal cavities at the two horizontal levels indicated in (B). Red boxed areas indicate the regions shown at higher magnification in g′, and h′. Scale bars: (C)a–d: 1 mm; (C)e–j = 500 µm.
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cfap161 (cilia and flagella associated protein 161) gene expression in dorsal explant from a X. laevis embryo, assayed via in situ hybridization at NF stage 16.
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cfap161 (cilia and flagella associated protein 161) gene expression in a X. laevis embryo, assayed via in situ hybridization NF stage 32, lateral view, anterior left, dorsal up.
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Fig. S4. Subcellular localisation of Xenopus Cfap161
N- or C-terminally HA-tagged Cfap161 was expressed in Xenopus
skin MCCs in combination with GFP-Cetn4 to label basal bodies.
Cfap161 signals were found throughout the cells but did not
specifically accumulate in cilia. Apical and orthogonal views as
indicated. Scales bars = 100 µm.
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Fig. S6. Genome editing of the cfap161 locus in Xenopus. (A) Sequence of the
cfap161 L-allele of genome edited specimens injected with sgRNA1, which targets
exon 1. (B) Sequence of the cfap161 L-allele of genome edited specimens injected
with sgRNA2, which targets exon 3. (C) cfap161 transcripts were reduced in
cfap161-crispants (b) at stage 30, compared to wt (a) with a focus on the skin. Red
boxed areas in (a,b) indicate the regions shown at higher magnification in (a‘, b‘).
Scale bar: C = 150 µm.
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