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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
) 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) 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.
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