Probes for INS

Transdifferentiation of hypertrophic chondrocytes into bone-forming osteoblasts has been reported, yet the underlying molecular mechanism remains incompletely understood. SHP2 is an ubiquitously expressed cytoplasmic protein tyrosine phosphatase. SHP2 loss-of-function mutations in chondroid cells are linked to metachondromatosis in humans and mice, suggesting a crucial role for SHP2 in the skeleton. However, the specific role of SHP2 in skeletal cells has not been elucidated. To approach this question, we ablated SHP2 in collagen 2α1(Col2α1)-Cre- and collagen 10α1(Col10α1)-Cre-expressing cells, predominantly proliferating and hypertrophic chondrocytes, using "Cre-loxP"-mediated gene excision. Mice lacking SHP2 in Col2α1-Cre-expressing cells die at mid-gestation. Postnatal SHP2 ablation in the same cell population caused dwarfism, chondrodysplasia and exostoses. In contrast, mice in which SHP2 was ablated in the Col10α1-Cre-expressing cells appeared normal but were osteopenic. Further mechanistic studies revealed that SHP2 exerted its influence partly by regulating the abundance of SOX9 in chondrocytes. Elevated and sustained SOX9 in SHP2-deficient hypertrophic chondrocytes impaired their differentiation to osteoblasts and impaired endochondral ossification. Our study uncovered an important role of SHP2 in bone development and cartilage homeostasis by influencing the osteogenic differentiation of hypertrophic chondrocytes and provided insight into the pathogenesis and potential treatment of skeletal diseases, such as osteopenia and osteoporosis.

Guanylin (GUCA2A/Guca2a/GN) and uroguanylin (GUCA2B/Guca2b/UGN) are expressed in the gastrointestinal tract and have been implicated in ion and fluid homeostasis, satiety, abdominal pain, growth and intestinal barrier integrity. Their cellular sources are debated and include goblet cells, entero-/colonocytes, enteroendocrine (EE) cells and tuft cells. We therefore investigated the cellular sources of GN and UGN mRNAs in human and rat duodenal and colonic epithelium with in situ hybridization (ISH) to determine co-expression with Chromogranin A (CHGA/Chga/CgA; enterochromaffin [EC] cells), defensin alpha 6 (DEFA6/Defa6; Paneth cells), mucin 2 (MUC2/Muc2; goblet cells) and selected tuft cell markers. GUCA2A/Guca2a expression was localized to goblet cells and colonocytes in human and rat colon. In human duodenum, GUCA2A was expressed in Paneth cells and was scarce in villous epithelial cells. In rat duodenum, Guca2a was only localized to goblet cells. Guca2b was focally expressed in rat colon. In human and rat duodenum and in human colon, GUCA2B/Guca2b was expressed in dispersed solitary epithelial cells, some with a tuft cell-like appearance. Neither GUCA2A nor GUCA2B were co-expressed with CHGA in human duodenal cells. Consequently, EC cells are probably not the major source of human GN or UGN but other EE cells as a source of GN or UGN are not entirely excluded. No convincing overlap with tuft cell markers was found. For the first time, we demonstrate the cellular expression of GUCA2B in human duodenum. The specific cellular distribution of both GN and UGN differs between duodenum and colon and between human and rat intestines.

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.

Bilateral adrenalectomy forces the patient to undergo glucocorticoid replacement therapy and bear a lifetime risk of adrenal crisis. Adrenal autotransplantation is considered useful to avoid adrenal crisis and glucocorticoid replacement therapy. However, the basic process of regeneration in adrenal autografts is poorly understood. Here, we investigated the essential regeneration factors in rat adrenocortical autografts, with a focus on the factors involved in adrenal development and steroidogenesis, such as Hh signalling. A remarkable renewal in cell proliferation and increase in Cyp11b1, which encodes 11-beta-hydroxylase, occurred in adrenocortical autografts from 2-3 weeks after autotransplantation. Serum corticosterone and adrenocorticotropic hormone levels were almost recovered to sham level at 4 weeks after autotransplantation. The adrenocortical autografts showed increased Dhh expression at 3 weeks after autotransplantation, but not Shh, which is the only Hh family member to have been reported to be expressed in the adrenal gland. Increased Gli1 expression was also found in the regenerated capsule at 3 weeks after autotransplantation. Dhh and Gli1 might function in concert to regenerate adrenocortical autografts. This is the first report to clearly show Dhh expression and its elevation in the adrenal gland.

Sonic hedgehog (SHH) is an essential morphogenetic signal dictating cell fate decisions in several developing organs in mammals. In vitrodata suggest that SHH is required to specify LHX3+/LHX4+ Rathke's pouch (RP) progenitor identity. However, in vivo studies have failed to reveal such a function, supporting instead, a critical role for SHH in promoting proliferation of these RP progenitors and for differentiation of pituitary cell types. Here, we have used a genetic approach to demonstrate that activation of the SHH pathway is necessary to induce LHX3+/LHX4+ RP identity in mouse embryos. First, we show that conditional deletion of Shh in the anterior hypothalamus results in a fully penetrant phenotype characterised by a complete arrest of RP development, with lack of Lhx3/Lhx4 expression in RP epithelium at 9.0 dpc (days post coitum) and total loss of pituitary tissue by 12.5 dpc. Conversely, over-activation of the SHH pathway by conditional deletion of Ptch1 in RP progenitors leads to severe hyperplasia and enlargement of the Sox2+ve stem cell compartment by the end of gestation.