Previous studies have suggested that defects in pancreatic epithelium caused by

Previous studies have suggested that defects in pancreatic epithelium caused by activation of the Hedgehog (Hh) signaling pathway are secondary to changes in the differentiation state of the surrounding mesenchyme. significant up-regulation of the Hh pathway in pancreata of mice overexpressing GLI2. As a consequence of overt Hh activation we observe profound morphological changes in both the exocrine and endocrine pancreas. Increased Hh activity also induced the growth of an undifferentiated cell populace expressing progenitor markers. Thus our findings suggest that Hh signaling plays a critical role in regulating pancreatic epithelial plasticity. mice results in the formation of undifferentiated tumors (7). INK 128 These findings suggest an additional cell-autonomous role of activated Hh signaling within the mature pancreas epithelium. To determine whether activation of Hh signaling in the pancreatic epithelium also affects pancreas formation we have analyzed pancreas organogenesis in mice. Surprisingly we find that ectopic expression of GLI2ΔN fails to efficiently up-regulate Hh pathway within the pancreas epithelium. This observation suggests that mechanisms exist in pancreatic epithelial cells that block inappropriate activation of the pathway. Recent studies have shown that main cilia cellular organelles are crucial regulators of the Hh pathway during embryonic development organ function and in malignancy (11-15). Specifically cilia ablation increases Hh activation mediated by GLI2ΔN during medulloblastoma and basal cell carcinoma (BCC) formation (11 15 Our findings show that concomitant removal of cilia in the presence of GLI2ΔN in mice results in overt Hh activation in pancreatic epithelium and consequently impaired pancreas formation. These pancreata display a significant loss of both exocrine and endocrine tissue accompanied by the appearance of undifferentiated epithelial cells expressing pancreatic progenitor cell markers. Thus INK 128 our study discloses a role for main cilia in regulating Hh signaling during pancreas formation and demonstrates that excessive Hh activation results in unique phenotypes in the pancreas underscoring a potential role for Hh signaling in modulating the differentiated state of pancreatic cells. Results Main Cilia Prevent Full Hh Activation upon GLI2ΔN Overexpression. INK 128 We have recently shown that in transgenic mice GLI2ΔN accumulation is usually observed in a mosaic fashion within the pancreatic epithelium. The activated GLI2ΔN expressed in CLEG2 mice is usually fused to a myc-tag in its N terminus thus allowing for immunodetection by an anti-myc antibody (myc-GLI2ΔN hereafter) (7). The restricted expression pattern of myc-GLI2ΔN is usually surprising because the transgene should be transcribed in all pancreatic cells due to the efficient elimination of the preceding cassette that places the transgene under direct control of the strong ubiquitous CMV early enhancer/chicken β-actin (CAG) promoter (7). To determine whether expression of the transgene in the pancreas indeed prospects to activation of the Hh signaling pathway we crossed mice with mice. is usually a direct transcriptional target of Hh signaling and mice transporting the β-galactosidase (β-gal) gene (locus serve as accurate reporters of Hh pathway activity (16). Analysis of β-gal activity in 3-week-old mice revealed few cells within the pancreas displaying detectable activity (Fig. 1mice. Fig. 1. Main cilia prevent full Hh activation in pancreas of myc-GLI2ΔN-overexpressing mice. (mice revealed few cells within the pancreas displaying detectable ARF3 activity. … Main cilia regulate the level of Hh signaling during mouse development in different organs and tissues (17 18 and therefore could also potentially regulate Hh signaling in the pancreas. Importantly cilia have been recently shown to repress Hh activation mediated by myc-GLI2ΔN during medulloblastoma and BCC formation (11 15 To address the role of cilia in pancreatic epithelial Hh signaling we generated compound mice characterized by ectopic expression of myc-GLI2ΔN (gene (as a marker for Hh INK 128 activity (compared with mice during postnatal (Fig. 1mice. Of notice cilia ablation in mice resulted in decreased β-gal activity during embryonic stages compared with controls (Fig. S1) thus suggesting a role for main cilia in regulating endogenous Hh activity. Importantly β-gal assay conditions used at embryonic stages were more sensitive than those used in 3-week-old mice (and expression was marginally increased in tissue.

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