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Figure 3


Fig. 3. T1alpha is a direct in vivo target of Foxp2 and Foxp1 in the lung. (A-H) Immunohistochemical detection of the expression of SP-C (A,B), T1alpha (C,D), SP-B (E,F) and CC10 (G,H) in wild-type (A,C,E,G) and Foxp2 mutant (B,D,F,H) mouse lungs at E18.5, showing elevated T1alpha expression in the Foxp2 mutant. Arrows, AEC-2 cells; arrowheads, lymphatic vessel. (I-K) Co-staining for expression of Foxp2 (I) and T1alpha (J) reveals that both proteins are co-expressed in the same cells within the distal lung at E18.5 (K). Note that the dim fluorescence displayed by cells located between the airways in I is non-specific background. (L,M) Expression of T1alpha in lymphatic endothelium at E18.5 (arrows) does not differ between wild-type and Foxp2-/- lungs in contrast to the upregulation of T1alpha expression in alveoli (asterisk) of the mutant. (N) Q-PCR to assess expression of the genes encoding the indicated lung cell markers at E18.5 confirming specific upregulation of T1alpha (T1{alpha}) expression in Foxp2-/- lungs. Values are relative to wild-type expression levels, which are set at 1.0 for each gene. aqua-5, aquaporin 5. Q-PCR results are the average of three lung samples performed in triplicate±s.e.m.; *, P<0.001. (O) Luciferase reporter assays were performed in NIH-3T3 cells using the rat 1.3 kb T1alpha (T1{alpha}) promoter. Fox DNA-binding sites located in the T1alpha promoter are indicated by either green ovals (conserved between mouse and rat) or white ovals (present in the rat promoter but not conserved across species). Comparison between the rat sequence (upper sequence) and the mouse sequence (lower sequence) in the two conserved sites in the T1alpha promoter. Bar chart showing that co-expression of either Foxp1 or Foxp2 leads to a fivefold repression of the T1alpha promoter relative to pCMV. (P) ChIP assays performed using mouse lung chromatin and the previously characterized Foxp1 and Foxp2 polyclonal antibodies show that Foxp1 and Foxp2 are both found associated with the region containing the conserved Fox DNA-binding sites in the mouse T1alpha promoter in vivo. Scale bars: 50 µm in A-M.