DeltaNp63 regulates thymic development through enhanced expression of FgfR2 and Jag2

Abstract

p63, a homologue of the tumor suppressor p53, is pivotal for epithelial development, because its loss causes severe epithelial dysgenesis, although no information is so far available on the role of p63 in the thymus. We identified the expression of all p63 isoforms in the developing thymus. The p63(-/-) thymi show severe abnormalities in size and cellularity, even though the organ expresses normal levels of keratins 5 and 8, indicating a p63-independent differentiation of thymic epithelial cells (TEC). TEC were sufficiently developed to allow a significant degree of education to produce CD4/CD8 single- and double-positive T cells. To study the selective contribution of transactivation-active p63 (TAp63) and amino-deleted p63 (DeltaNp63) isoforms to the function of the TEC, we genetically complemented p63(-/-) mice by crossing p63(+/-) mice with transgenic mice expressing either TAp63alpha or DeltaNp63alpha under the control of the keratin 5 promoter. Thymic morphology and cellularity were partially restored by complementation with DeltaNp63, but not TAp63, one downstream effector being fibroblast growth factor receptor 2-IIIb (FgfR2-IIIb). Indeed, FgfR2-IIIb is regulated directly by p63, via its interaction with apobec-1-binding protein-1, and its knockout shows thymic defects similar to those observed in p63(-/-) thymi. In addition, expression of Jag2, a component of the Notch signaling pathway known to be required for thymic development, was enhanced by p63 in vivo genetic complementation. Like Jag2(-/-) thymi, p63(-/-) thymi also show reduced gammadelta cell formation. Therefore, p63, and particularly the DeltaNp63 isoform, is essential for thymic development via enhanced expression of FgfR2 and Jag2. The action of DeltaNp63 is not due to a direct regulation of TEC differentiation, but it is compatible with maintenance of their "stemness," the thymic abnormalities resulting from epithelial failure due to loss of stem cells.

Fig. 1.

Fetal thymic development in p63^−/− and genetically complemented mice. (A) Relative size of thymi (E19.5 embryos), dissected with the heart attached to provide a frame of reference. Thymi from p63^−/− animals are reduced in size and exhibit severe structural anomalies. (B) Thymic histology (E19.5 sections stained with hematoxylin–eosin) in p63^−/− mice before and after the reintroduction of TAp63α and/or ΔNp63α. (Scale bars, 300 μm.) (C) Histology of thymic cortical area in p63^−/− mice before and after the reintroduction of TAp63α and/or ΔNp63α at bigger magnification. (Scale bars, 10 μm.) (D) The thymus/heart (E19.5 embryos) ratio of p63^−/−, and p63^−/−;TA mice was markedly reduced compared with wild type (wt), and increased in p63^−/−;ΔN and p63^−/−;ΔN;TA mice (n = 11; P value is indicated). (E) Thymic cell number (mechanical extraction from thymus from E19.5 embryos) in the different genotypes. Bar indicates standard deviation (n = 11; statistical significance is indicated).

Fig. 2.

Histochemistry of keratins, cell death detection, and T cell counts in complemented mice. (A) Confocal staining of fetal thymus at E19.5 in wild-type (wt); p63^−/− mice; and complemented p63^−/−;ΔN, p63^−/−;TA, and p63^−/−;ΔN;TA mice. (Top and Middle) Tissue was stained for K5 (red) (Top) or K8 (green) (Middle). (Bottom) Merged fluorescence with DAPI (blue). The expression of K5 and K8 has a similar pattern in wild-type, p63^−/−, and genetically complemented mice. (Scale bars, 60 μm.) (B and C) Staining for CD8 and CD4 in cells extracted from the thymi obtained from seven different mice for each genotype, wild type or p63^−/−. Differences between control (wt) and p63^−/− (knockout) genotypes for each particular subset were not statistically significant; bar indicates standard deviation. (D) Cell death was evaluated by TUNEL positivity (red); samples are as in A. In the thymi of p63^−/− mice, a higher incidence of cell death is detected. The reintroduction of TAp63α results in an increase of cell death. The reintroduction of ΔNp63α results in a slight improvement of both parameters. The experiment was performed three times, and a representative experiment is shown. (Scale bars, 60 μm.)

Fig. 3.

Expression of FgfR2 and Jag2 in complemented mice. (A) Quantitative real-time PCR indicates that the p63^−/− reduced FgfR2-IIIb expression was restored upon reintroduction of ΔNp63α, but not TAp63α, whereas Jag2 expression was restored upon reintroduction of TAp63α and ΔNp63α [∗, 0.001 < P < 0.01 between wild-type (wt) and genetically complemented mice; ○, 0.01 < P < 0.05 between wt and genetically complemented mice; n = 7]. (B) (Upper) Semiquantitative RT-PCR for FgfR2 splicing variants IIIc and IIIb and for Jag1 and Jag2. The results indicate that only the expression of the IIIb isoform is down-regulated in the p63^−/− thymus. Among the Notch ligands, the expression of Jag2 is more markedly reduced in comparison with Jag1. (Lower) Quantification of the semiquantitative RT-PCR (n = 5).

Fig. 4.

Expression of FgfR2, Jag1, and Jag2 in TAp63 and ΔNp63 Saos-2 Tet-on inducible cell lines. (A) Semiquantitative RT-PCR in Saos-2 Tet-on inducible cell lines (Dx, 2 μg/ml) overexpressing TAp63α or ΔNp63α isoforms. Actin is shown in the bottom image as a control. FgfR2, Jag1, and Jag2 expression were normalized to actin by densitometry, and results are reported as fold change over zero time (see graphics below actins). We performed three independent experiments, and a representative one is shown. Results obtained from the densitometry are shown as mean from three independent experiments. (B) Scheme of the role of ΔNp63 in “stemness” of thymic development. ∗, Jag2 is also induced by TAp63.