Flattop regulates basal body docking and positioning in mono- and multiciliated cells
- Helmholtz Center Munich, Germany
- Université libre de Bruxelles, Belgium
- University of Edinburgh, United Kingdom
- Univeristy of Edinburgh, United Kingdom
Figures
Figure 1 with 2 supplements
FLTP has active FOXA2 binding sites in its promoter and is conserved among species.
(A) A high amount of the endodermal transcription factor FOXA2 binds the human FLTP promoter in pancreatic progenitors and in adult islets, indicating that FLTP is a direct target of FOXA2 and …
-
Figure 1—source data 1
Mendelian ratio of Fltp intercrosses on different backgrounds.
(A–C) Fltp animals are born roughly at the expected Mendelian ratio in C57Bl6/6NCrl, 129S6/SvEvTac, or CD1 background. Note: FltpZV/ZV animals are slightly underrepresented on the C57Bl6 and 129S6 background.
- https://doi.org/10.7554/eLife.03842.004
Figure 1—figure supplement 1
Fltp antibody specificity.
(A) Western blot of Strep Flag (SF) Tag, Fltp SF-tagged transiently transfected HEK293T cells and testis lysate of Fltp+/+, FltpZV/+, and FltpZV/ZV animals incubated with Fltp116-1 antibody. Note, …
Figure 1—figure supplement 2
Fltp construct and confirmation of the knock-out strategy.
(A) The FltpZV targeting strategy deletes the entire open reading frame (ORF) from exon 2 to 6 (exons: black and orange boxes; orange boxes: ORF, primers for genotyping: 418, 420, 565, 566; for …
Figure 2 with 1 supplement
Fltp reporter and protein is detectable in mono- and multiciliated tissues.
(A and B) mRNA (Fltp in situ hybridization) (A) and lacZ reporter expression (B) are restricted to the node (n) at E7.5. (C–E)Whole-mount lacZ stained and benzyl alcohol/benzyl benzoate (BABB) …
Figure 2—figure supplement 1
Additional Fltp reporter expression in mono- and multiciliated tissues.
(A–H) Whole-mount lacZ stained and benzyl alcohol/benzyl benzoate (BABB) cleared (D, F, H) FltpZV/+ embryos and organs. β-gal activity is restricted to the node (n) (A). (B) Cross-section of the …
Figure 3
Loss of Fltp leads to constricted distal airways and cilia formation defects in the lung.
(A–C) Whole-mount lacZ stained and BABB cleared distal airways of left lung lobes at P60. Red lines show how diameters were measured. (A) FltpT2AiCre/+; R26R/+is used as control (Ctrl). (B and C) Flt…
Figure 4 with 1 supplement
Fltp is expressed and necessary during BB docking.
(A) Scheme of multiciliated cell (MCC) maturation. A MCC progenitor projects a primary cilium at the apical surface (stage I). Centrosome amplification is the first sign of differentiation (stage …
Figure 4—figure supplement 1
ALI differentiation efficiency and quantification of BB docking defect.
(A) Graph showing the percentage of cells in stage II–IV for WT and FltpZV/ZV (HOM) ALIs at day zero (before the switch to ALI condition), one, two, and four measured by GFP and pericentrin (HOM) …
Figure 5
Fltp is required for kinocilium positioning and stereocilia bundle formation.
(A) Whole-mount LSM of an FltpZV/+ organ of Corti (OC) showing its different regions. Fltp reporter expression is restricted to all sensory HCs (IHC and OHC) of the OC (red dashed line). (B–M) …
Figure 6
Fltp is a potential downstream mediator of PCP signaling.
(A–B) LSM of an E18.5 Fltp+/+; Celsr1crsh/crsh (WT) organ of Corti (OC) (A) revealed rotated outer (OHC) and inner hair cells (IHC). FltpZV/ZV; Celsr1crsh/crsh (HOM) OC (B) shows more severely …
Figure 7
Fltp is located at the interface of apical actin and Dlg3 in IE hair cells.
(A, B, C–D′, F–H) Single section LSM of outer HCs of an Fltp+/+; Dlg3-Venus animal at E18.5 reveals that Dlg3-Venus is located at the lateral membrane and at the medial membrane (or the lateral …
Figure 8 with 1 supplement
Loss of Fltp or Dlg3 leads to BB mispositioning in IE hair cells.
(A–D) LSM of Fltp+/+; Dlg3-Venus (WT) IE HCs at E18.5 reveals BBs in the middle of the lateral Dlg3-Venus crescent. (B′–D′) IMARIS surface rendering pictures of WT HCs. (E–H) LSM of FltpZV/ZV; …
Figure 8—figure supplement 1
BB mispositioning in FltpZV; Dlg3-Venus and Dlg3 animals.
(A) In FltpZV/ZV; Dlg3-Venus (HOM) IE significantly more hair cells across all rows show BB mispositioning over 8° (red) compared to controls Fltp+/+; Dlg3-Venus (WT). (B) The same analysis as in (A)…
Figure 9
Fltp interacts with proteins associated with the TJ complex (Dlg3), the BB (γ-Tubulin) and with the core PCP protein Dvl2.
(A) Dlg3 and Fltp constructs used for interaction domain mapping. (B) N-terminus of Fltp is essential for interaction with Dlg3. HEK293T cells were transfected with SF Dlg3 variants and with …
Author response image 1
Author response image 2
Author response image 3
(A-B) Show cryosections of a WT lung stained with acetylated tubulin (red) and pericentrin (green) and DAPI (blue). BBs are docked and cilia formed. (C-D) Cryosections of a Dlg3 KO animal show …
Author response image 4
(A-A’’) WT cultures at day 0, ALI day 2 and ALI day 4. In day 0 cultures MCCs are already present (A). Day 2 and day 4 ALIs show mostly 100% docked BBs (red, pericentrin; white, cilia) (A’,A’’). …
