Distinct patterns of primary and motile cilia in Rathke's cleft cysts and craniopharyngioma subtypes

Abstract

Cilia are highly conserved organelles, which serve critical roles in development and physiology. Motile cilia are expressed in a limited range of tissues, where they principally regulate local extracellular fluid dynamics. In contrast, primary cilia are expressed by many vertebrate cell types during interphase, and are intimately involved in the cell cycle and signal transduction. Notably, primary cilia are essential for vertebrate hedgehog pathway activity. Improved detection of motile cilia may assist in the diagnosis of some pathologic entities such as Rathke's cleft cysts, whereas characterizing primary cilia in neoplastic tissues may implicate cilia-dependent signaling pathways as critical for tumorigenesis. We show that immunohistochemistry for the nuclear transcription factor FOXJ1, a master regulator of motile ciliogenesis, robustly labels the motile ciliated epithelium of Rathke's cleft cysts. FOXJ1 expression discriminates Rathke's cleft cysts from entities in the sellar/suprasellar region with overlapping histologic features such as craniopharyngiomas. Co-immunohistochemistry for FOXJ1 and markers that highlight motile cilia such as acetylated tubulin (TUBA4A) and the small GTPase ARL13B further enhance the ability to identify diagnostic epithelial cells. In addition to highlighting motile cilia, ARL13B immunohistochemistry also robustly highlights primary cilia in formalin-fixed paraffin-embedded sections. Primary cilia are present throughout the neoplastic epithelium of adamantinomatous craniopharyngioma, but are limited to basally oriented cells near the fibrovascular stroma in papillary craniopharyngioma. Consistent with this differing pattern of primary ciliation, adamantinomatous craniopharyngiomas express significantly higher levels of SHH, and downstream targets such as PTCH1 and GLI2, compared with papillary craniopharyngiomas. In conclusion, motile ciliated epithelium can be readily identified using immunohistochemistry for FOXJ1, TUBA4A, and ARL13B, facilitating the diagnosis of Rathke's cleft cysts. Primary cilia can be identified by ARL13B immunohistochemistry in routine pathology specimens. The widespread presence of primary cilia in adamantinomatous craniopharyngioma implicates cilia-dependent hedgehog signaling in the pathogenesis of adamantinomatous craniopharyngioma.

Figure 1

Hematoxylin and eosin (H&E) stained section of Rathke’s cleft cyst (a). FOXJ1 immunohistochemistry (brown) of Rathke’s cleft cyst (b and d) and fallopian tube (c). Panel d. shows immunohistochemistry on a Rathke’s cleft cyst that has significantly disrupted and fragmented cyst epithelium. Staining demonstrates robust nuclear expression of FOXJ1 in multi-ciliated epithelial cells. Scale bars, 50μm.

Figure 2

Immunohistochemistry for acetylated TUBA4A (brown) on fallopian tube (a) Rathke’s cleft cyst (b), Rathke’s cleft cyst with underlying clusters of normal anterior pituitary cells (c), and Rathke’s cleft cyst that has significantly disrupted and fragmented cyst epithelium (d). Immunohistochemistry for TUBA4A demonstrates robust staining of cilia; however, there is significant cytoplasmic background particularly in anterior pituitary cells. Scale bars, 50μm.

Figure 3

Co-immunohistochemistry for both FOXJ1 (brown) and acetylated TUBA4A (red) on fallopian tube (a), Rathke’s cleft cyst (b), Rathke’s cleft cyst that has significantly disrupted and fragmented cyst epithelium (c), Rathke’s cleft cyst with stratified squamous epithelium (d). Co-immunohistochemistry shows concordance between FOXJ1 and TUBA4A. Scale bars, 50μm.

Figure 4

Immunohistochemistry for ARL13B on fallopian tube (a) and Rathke’s cleft cyst (b). Co-immunohistochemistry for FOXJ1 (brown) and ARL13B (red) on fallopian tube (c) and Rathke’s cleft cyst (d). ARL13B demonstrates robust staining of motile cilia, and co-localization with FOXJ1 in motile ciliated cells. There is minimal background staining with ARL13B immunohistochemistry. Scale bars, 50μm.

Figure 5

Hematoxylin and eosin (H&E) stained section of adamantinomatous craniopharyngioma (a) and papillary craniopharyngioma (c). FOXJ1 immunohistochemistry (brown) for adamantinomatous craniopharyngioma (b) and papillary craniopharyngioma (d). Co-immunohistochemistry for FOXJ1 (brown) and ARL13B (red) on “ciliated papillary craniopharyngioma” (e and f). Adamantinomatous craniopharyngiomas do not express FOXJ1. The vast majority of papillary craniopharyngioma do not express FOXJ1; however, rare ciliated BRAF V600E positive papillary craniopharyngioma cases show FOXJ1 and ARL13B expression with apical multi-ciliated cells. Scale bars, either 50μm (a-d) or 20μm (e,f).

Figure 6

ARL13B immunohistochemistry (red) on adamantinomatous craniopharyngioma (a) and papillary craniopharyngioma (b). Adamantinomatous craniopharyngiomas diffusely express primary cilia, in both the palisading basal cells and non-palisading “apical” cells in 100% of cases. Papillary craniopharyngiomas express primary cilia in basally oriented cells near fibrovascular cores in 100% of cases, but show no expression of primary cilia in more “apical” cells. Statistical analysis of primary cilia in different lesions (c). Differences in expression between “apical” and “basal” cells (****) are statistically significant (p<0.05). Matched serial sections of an adamantinomatous craniopharyngioma with hematoxylin and eosin (H&E) (d), RNAscope in situ hybridization for SHH (e) or PTCH1 (g) mRNA, or immunoistochemistry for β-catenin (f). Regions of high SHH mRNA expression (e, arrows) show excellent correlation with regions of nuclear/cytoplasmic β-catenin translocation (f, arrows). Statistical analysis of PTCH1 and GLI2 expression in adamantinomatous craniopharyngioma and papillary craniopharyngioma (h). Differences in expression (****) are statistically significant (p<0.05). Scale bars, 20μm (a,b) or 50 μm (d,e,f,g).