Figure 1.
Confocal analysis of Foxi1 and H+-ATPase subunits A1, B1, E2 and a4 expression in wt and Foxi1−/− endolymphatic sac (ES) epithelium.
Confocal images of inner ear sections from wt and Foxi1−/− mouse embryos (E16.5). Fluorescence images of wt ES tissue sections stained with a specific antibody against Foxi1 (green) and the H+-ATPase subunit A1 (red; A–B), subunit B1 (red; D–E), subunit E2 (red; G–H) and subunit a4 (red; J–K). The nuclei (blue) were visualized using To-Pro 3. Merged images reveal that cells with a strong staining of each of the H+-ATPase subunits also are positive for nuclear Foxi1 staining. In sections from Foxi1−/− ES no staining could be identified for A1 (C), B1 (F), E2 (I) or a4 (L). Scale bars 20 µm (A, D, G, J, C, F, I and L) and 10 µm (B, E, H and K) (L: lumen).
Figure 2.
Confocal analysis of Foxi1 and the H+-ATPase subunits A1, E2 and a4 expression in wt and Foxi1−/− distal nephrons.
Confocal images of wt and Foxi1−/− kidney sections. Double staining with Foxi1 and anti H+-ATPase antibodies showed exclusive expression of Foxi1 protein (green) in cells of distal nephron cells co-expressing subunits A1 (red; A–D), E2 (red; G–J) and a4 (red; M–P), in both cortex as well as medulla. Foxi1 is localized to the nuclei, which are stained blue (TOPRO 3), while the ATPase subunits are found apically, a4 (O–P) or both apically and in the cytosol, A1 (C–D) and E2 (I–J). No expression of ATPase subunits were detected in epithelial cells from Foxi1−/− kidneys, neither in medulla (E, K, Q) nor cortex (F, L, R). Scale bars 20 µm (L: lumen).
Figure 3.
Confocal analysis of Foxi1 and H+-ATPase subunits A1, E2 and a4 expression in wt and Foxi1−/− epididymides.
Confocal images of wt and Foxi1 −/− epididymal sections. In confocal images of wt epididymal sections, all three ATPase subunits (red) were exclusively found in Foxi1 (green) immunoreactive epididymal cells of caput (A, G, M), corpus (B, H, N) and cauda (C, I, O). Foxi1 (green) is localized to the nuclei (blue, TOPRO3), while the ATPase subunits are found on the apical/luminal (L) side of the cells. Staining of Foxi1−/− tissue sections showed no detectable A1 (D, E, F), E2 (J, K, L) nor a4 (P, Q, R) subunit expression in Foxi1 deficient epididymal tissue sections. Scale bars 20 µm (L: lumen).
Figure 4.
Foxi1 can activate an ATP6V0A4 promoter reporter construct.
Sequence analysis of the ATP6V0A4 upstream region reveals four putative FOXI1 sites located at −561/−547 (Fk1-3) and −358/−352 (Fk4) relative to start of transcription (NCBI: NC_000007.12, NM_130841). A −754 ATP6V0A4 promoter construct was transfected into COS7 cells using increasing amounts of Foxi1 expression plasmid. A significant step-wise induction is shown.
Figure 5.
Foxi1 interact and activate ATP6V0A4 promoter reporter construct at putative forkhead binding sites Fk1-3.
(A) Core TTT-triplets of potential forkhead sites were mutated to GGG according to schematic picture. (B) Mutations reduce Foxi1-depedent reporter activation. While Fk3 mutations seem to abolish this activation the effects on Fk1-2 are intermediate and Fk4 seems to contribute very little to Foxi1-dependent reporter gene activation. (C) EMSA using in vitro transcribed/translated Foxi1 demonstrates interactions with [32P] labeled oligonucleotides harboring the −561/−547 (Fk1-3) and −358/−352 (Fk4) regions. The comparatively weak interaction demonstrated for Fk4 correlates well with the modest effects on reporter gene activation seen in Fig. 5 B. (D–F) EMSA showing the −561/−547 interaction is competed for with either wt or mutated oligonucleotides specific for the Fk1, 2 and 3 interactions. As can be deduced, there is a higher sensitivity in EMSA competition experiments using wt probes as compared with mutated probes – typical of a sequence specific significant interaction.
Figure 6.
Chromatin Immuoprecipitation (ChIP) analysis.
Chromatin immunoprecipitation confirm presence of Foxi1- ATP6V0A4 promoter interaction at FK1-3. Transfected 3T3-L1 cells were fixed, and chromatin was prepared by sonication. After preclearance with Protein G Sepharose the chromatin was immunoprecipitated with or without an Anti-6X His tag antibody (Foxi1, No Ab). The purified precipitated DNA was used as template for PCR reactions with specific primers covering sequence with Foxi1 binding sites (Fk1-3) or unspecific primers (Random). ChIP analysis reveal a strong interaction of the ATP6V0A4 promoter with wt Foxi1 (Lane 1). The interaction is weakened using promoter with mutated FK1 (Lane2) and even more weakened with Fk2 and 3 (Lanes 3–4). Using the triple mutated promoter Fk1-3 (Lane 5) no interaction is detected.
Figure 7.
In silico identification of forkhead sites in genes encoding subunits A1 and E2.
Representation of potential forkhead binding sites in the upstream regions of genes encoding the A1 and E2 subunit. In the depicted 3 kb region 14 and 25 sites where identified for A1 and E2, respectively.
Figure 8.
mRNA in situ hybridization and immunohistochemistry on kidney sections from wt and Foxi1−/− mice.
Combined in situ hybridization and immunofluorescence. DIG labeled cRNA probes for A1 and E2 were hybridized to wt and Foxi1−/− kidney sections and the same sections were then subjected to immunofluorescent staining with an anti-CAII specific antibody (green) and the nuclear marker Topro3 (red). CAII expressing cells in wt kidney displayed positive hybridization signal for both A1 and E2 probes while in Foxi1−/− kidney sections, no hybridization signal could be detected. White arrows point to the same CAII positive cells in images from in situ hybridization and immunofluorescence. Scale bars 10 µm.
Figure 9.
Schematic illustration of the vacuolar H+-ATPase proton pump.
Subunits specifically expressed in endolymphatic epithelium of the inner ear, intercalated cells of kidney and narrow and clear cells of the epididymis in bold.