K14-cre–Driven Apc Loss Results in Hypoplastic/Athymic Mice
Thymus is an organ that is also known to have K14 expression [16]. It represents the primary lymphoid organ for thymocyte development and selection. Distinct population of TECs of cortex and medulla mediates both of these critical functions. Cortical and medullary TEC subsets are characterized by differential expression of four keratin species: K8, K18, K5, and K14.
The normal thymus is a lobulated lymphoid organ, each lobule clearly showing the two distinct TEC compartments, an outer cortex and an inner medulla (Figure 6). There were no major differences in the histology of thymus between the ages P3 to P17 in phenotypically normal littermates. As shown in the H&E staining of thymus, the cortex was formed of dense lymphoid tissue that lacks nodules (Figure 6A). Since the stroma of the medulla is less heavily infiltrated with lymphocytes than the cortex, the medulla stained more lightly than the cortex. In normal mice, the thymus retains its size until the young adult age and regresses thereafter by atrophy. In the normal young mice we examined (P3–P17), it is evident that thymocytes were mitotically active in the cortex as determined by BrdU immunostaining (Figure 6B). Immunohistochemistry of normal thymus from P3 to P17 mice showed a similar staining pattern for K14 in that its expression was restricted to a small population of TECs in the inner medullary region and in the keratinocytes in Hassall's corpuscles (Figure 6D). Diffuse cytoplasmic staining for β-catenin was also detected in the medullary epithelial cells (Figure 6C). In contrast to K14 expression, diffuse staining for K8 was observed in epithelial cells both in the medulla and cortex (Figure S3). K1 staining was not detected in young mice at P3 but in older mice it was detected in differentiated keratinocytes in some of Hassall's corpuscles (Figure S3).
Figure 6  Histological and Immunochemical Examination of Thymus
(A–D) P3 normal thymus. (E–G) Mild P3 mutant thymus. (H–K) Severe P3 mutant thymus. (L–O) P13 mutant thymus. Stained with H&E for histology (A, E, H, L), BrdU (B, I, M), β-catenin (C, F, J, N), and K14 (D, G, K, O). (B) Actively dividing thymocytes are visible at the superficial edge of cortex of normal P3 thymus. Note the progression of histological abnormalities in the mutant thymus from mild P3, severe P3 to P13 (A, E, H, L). Scale bars, 20 μm. The histological abnormalities of thymus were evident as early as P3 in KA mutants (Figure 6E and 6H). The thymus was made of two lobules as in the normal mice but the mutant thymus was significantly smaller in size than that of the age-matched controls (Figure 2G). Interestingly, variations in the phenotypic severity of the mutant pups at P3 were prominently reflected in the extent of histological abnormalities of thymus. A P3 KA mutant pup that showed milder phenotype with a comparable body weight to its normal littermates (Figure 2A, M2) showed milder thymus abnormalities (Figure 6E) compared to its more severe mutant littermate (Figure 2A, M1; Figure 6H). The milder P3 mutant thymus was already much smaller in size compared to those of normal littermates (data not shown) but two epithelial compartments of thymus were histologically still distinguishable, with colonization of thymocytes evident in the cortex. However, there were small populations of lightly stained cells by H&E extending from the edge of the outer cortex towards inner medulla (Figure 6E), and these cells showed intense nuclear β-catenin staining whereas the rest of the medullary cells showed diffuse β-catenin staining pattern similar to that of the control (Figure 6F). Localization of K14 was limited to a few cells in the medulla and some overlapped with K8 localization (Figure 6G).
In the other P3 mutant thymus the distinct thymic epithelial compartments have been lost completely, and only a few lymphocytes were remaining at the edges and some in the middle (Figure 6H). Proliferative activities were no longer observed in thymocytes as prominently as in the normal thymus, but the epithelial cells seemed to be forming concentric structures (Figure 6I). Unlike in the normal or mild mutant thymi, the severe P3 mutant thymus showed extensive K14 expression that overlapped with K8 expression (Figure 6K). These cells were more like basal cells of the skin than TECs and were adjacent to the most immature looking cells that were showing strong nuclear and cytoplasmic β-catenin staining (Figure 6J). The nuclear staining of β-catenin was not observed in the normal age-matched thymus (Figure 6C). Most notably, the K14 and β-catenin staining patterns were mutually exclusive (Figure 6J and 6K).
At P10–P13, the mutant thymus consisted of numerous enlarged Hassall's corpuscle–like structures, made of arrays of K14- and K8-expressing keratinizing epithelial cells surrounding large keratin deposits (Figure 6L and 6O). There were numerous neutrophils and macrophages infiltrating the thymus in response to these keratins; hence, these structures could be called pyogranuloma. Varying degrees of differentiation-specific markers depending on the age of mice, in this case K1 and involucrin that are normally present only in Hassall's corpuscles, were also detected in mutant thymi (Figure S3). In these mice no thymocytes were detectable. BrdU incorporation was only observed in very few keratinizing epithelial cells, looking somewhat similar to the pattern of mature skin (Figure 6M). The diffuse expression of β-catenin was also present in these epithelial cells, and at this age fewer cells were positive for nuclear β-catenin staining (Figure 6N). As in the younger mutant mice, however, nuclear localization of β-catenin was only observed in K14-negative cells that looked like undifferentiated basal cells. In older P17 mice, the histopathology and keratin expression pattern of the mutant thymus was similar to that of P13 except for the fact that β-catenin expression became increasingly diffuse and appeared to colocalize with K8/K14 expression (data not shown). This coincided with fewer immature cells in the older mutant thymus.
Collectively, these results suggest that loss of Apc and consequent stabilization of β-catenin in K14-expressing TECs lead to their aberrant proliferation and differentiation to keratinocytes, causing massive squamous metaplasia, rather than to form either medullary or cortical TECs. Loss of proper TEC compartments consequently resulted in loss of thymocytes for maturation and the mice to be “athymic.”