Primary cilia regulate Gli/Hedgehog activation in pancreas

Abstract

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. However, recent results describe a role of the pathway in pancreatic epithelium, both during development and in adult tissue during neoplastic transformation. To determine the consequences of epithelial Hh activation during pancreas development, we employed a transgenic mouse model in which an activated version of GLI2, a transcriptional mediator of the pathway, is overexpressed specifically in the pancreatic epithelium. Surprisingly, efficient Hh activation was not observed in these transgenic mice, indicating the presence of physiological mechanisms within pancreas epithelium that prevent full Hh activation. Additional studies revealed that primary cilia regulate the level of Hh activation, and that ablation of these cellular organelles is sufficient to cause 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 expansion of an undifferentiated cell population expressing progenitor markers. Thus, our findings suggest that Hh signaling plays a critical role in regulating pancreatic epithelial plasticity.

Fig. 1.

Primary cilia prevent full Hh activation in pancreas of myc-GLI2ΔN-overexpressing mice. (A) Analysis of β-gal activity in 3-week-old Pdx1-Cre;CLEG2;Ptch1^lacZ/+ mice revealed few cells within the pancreas displaying detectable activity. (B) Ablation of primary cilia through Kif3a inactivation results in a significant increase of both the area and intensity of β-gal staining of positive cells in the pancreata of Pdx1-Cre;CLEG2;Kif3a^lox/lox;Ptch1^lacZ/+ mice. (C) Myc-GLI2ΔN protein accumulates in a limited number of cells in Pdx1-Cre;CLEG2 pancreata (arrowheads). (D) In contrast, the number of pancreatic cells marked by strong expression of the myc-GLI2ΔN fusion protein significantly increases in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice. Note the increased stromal compartment (asterisks) and the presence of epithelial cell nests accumulating high levels of myc-GLI2ΔN (outlined). (E and F) Myc-GLI2ΔN fusion protein localizes to primary cilia in ductal cells of Pdx1-Cre;CLEG2 mice. Note that the areas of strong myc staining in the Pdx1-Cre;CLEG2 sample correspond to one of the few duct areas in which the transgene was active.

Fig. 2.

Full Hh activation results in perturbed pancreas morphology. (A–D) Hematoxylin/eosin-stained sections of 3-week-old mice reveal loss of pancreatic tissue and expansion of duct-like epithelium in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice. Note the increased stromal compartment in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice (asterisks in D). (E–H) Extensive acinar cell loss as shown by the decrease of amylase staining and increased dilation of duct-like structures marked by CK19 staining is observed in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice. (G) Mild ductal dilation is observed in Pdx1-Cre;Kif3a^lox/lox mice. (I–L) Expression of the ductal marker mucin-1 is lost in cells expressing high levels of myc-GLI2 in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice (arrowheads). (J) Myc-GLI2ΔN accumulation is observed in a subset of pancreatic cells in Pdx1-Cre;CLEG2 mice. Epithelial cell nests are outlined in D and H.

Fig. 3.

Activation of Hh signaling results in expression of progenitor markers in the ductal-like epithelium of Pdx1-Cre;CLEG2;Kif3a^lox/lox mice. (A and B) Expression of the embryonic pancreatic marker Sox9 in undifferentiated cells in 2- to 3-week-old Pdx1-Cre;CLEG2;Kif3a^lox/lox mice. E-cadherin expression confirms the epithelial nature of those cells. (C and D) FoxA2 is expressed in undifferentiated cells in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice. (E and F) Myc-GLI2ΔN-expressing cells in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice are highly proliferative as determined by staining with phospho-Histone H3. (G and H) Activation of Notch signaling in undifferentiated cells of Pdx1-Cre;CLEG2;Kif3a^lox/lox mice as determined by Hes1 expression. (I and J) Pdx-1 expression is excluded from undifferentiated cells expressing high levels of myc-GLI2ΔN in Pdx1-Cre;CLEG2;Kif3a^lox/lox pancreata. (K and L) Undifferentiated cells are first observed at P0 in Pdx1-Cre;CLEG2;Kif3a^lox/lox pancreata. (M and N) Abnormal mucin-1 expression in undifferentiated cells of P0 Pdx1-Cre;CLEG2;Kif3a^lox/lox pancreata. Mucin-1 expression is decreased and not restricted to the apical membrane in myc-GLI2ΔN-expressing cells of Pdx1-Cre;CLEG2;Kif3a^lox/lox mice (arrows). Mucin-1 is properly localized to the apical membrane in Pdx1-Cre; CLEG2 mice. Note the low number of myc-GLI2ΔN-expressing cells observed in Pdx1-Cre; CLEG2 mice. (O and P) Coexpression of exocrine (amylase) and ductal markers (Mucin-1) in myc-GLI2ΔN-expressing cells of P0 in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice. The area marked by arrowheads is shown at higher magnification in inset in P.

Fig. 4.

Increased Hh activity results in disruption of islet formation in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice. (A–D) Islets are reduced in number and their architecture is affected in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice at P5. Islet morphology is shown at higher magnification in insets. (E–H) Islet morphology appears unaffected in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice at P0. (I–K) Quantification of islet area in E15.5 embryos and P0 and P5 mice. Note the dramatic reduction of islet area in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice at P5. (L and M) Quantification of ngn3-positive cells and epithelial area in E15.5 embryos. Both epithelial area and neurogenin3 endocrine precursors are reduced in Pdx1-Cre;CLEG2 and Pdx1-Cre;CLEG2;Kif3a^lox/lox mice. (N and O) Quantification of endocrine cell proliferation based on phospho-Histone H3 expression at P0 and P5. (P and Q) Quantification of endocrine cell death based on cleaved caspase-3 expression at P0 and P5. Note the sustained 2- to 3-fold increased cell death rate in Pdx1-Cre;CLEG2;Kif3a^lox/lox mice. All values are relative to control littermates, whose averages were considered to be one. Sample numbers represent n ≥ 3. (See Table S1 for sample numbers.) Error bars represent SD. *P < 0.05 and **P < 0.005.