Corinne Nielsen

Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis

Hedgehog signalling—an essential pathway during embryonic pancreatic development, the misregulation of which has been implicated in several forms of cancer—may also be an important mediator in human pancreatic carcinoma. Here we report that sonic hedgehog, a secreted hedgehog ligand, is abnormally expressed in pancreatic adenocarcinoma and its precursor lesions: pancreatic intraepithelial neoplasia (PanIN). Pancreata of Pdx–Shh mice (in which Shh is misexpressed in the pancreatic endoderm) develop abnormal tubular structures, a phenocopy of human PanIN-1 and -2. Moreover, these PanIN-like lesions also contain mutations in K-ras and overexpress HER-2/neu, which are genetic mutations found early in the progression of human pancreatic cancer. Furthermore, hedgehog signalling remains active in cell lines established from primary and metastatic pancreatic adenocarcinomas. Notably, inhibition of hedgehog signalling by cyclopamine induced apoptosis and blocked proliferation in a subset of the pancreatic cancer cell lines both in vitro and in vivo. These data suggest that this pathway may have an early and critical role in the genesis of this cancer, and that maintenance of hedgehog signalling is important for aberrant proliferation and tumorigenesis.

Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation

Wnt signaling defines the colonic epithelial progenitor cell phenotype, and mutations in the gene adenomatous polyposis coli (APC) that activate the Wnt pathway cause the familial adenomatous polyposis coli (FAP) syndrome and most sporadic colon cancers. The mechanisms that regulate the transition of epithelial precursor cells into their differentiated derivatives are poorly characterized. We report that Indian hedgehog (Ihh) is expressed by mature colonocytes and regulates their differentiation in vitro and in vivo. Hedgehog (Hh) signaling restricts the expression of Wnt targets to the base of the colonic crypt in vivo, and transfection of Ihh into colon cancer cells leads to a downregulation of both components of the nuclear TCF4–β-catenin complex and abrogates endogenous Wnt signaling in vitro. In turn, expression of Ihh is downregulated in polyps of individuals with FAP and expression of doxycycline-inducible dominant negative TCF4 (dnTCF4) restores Ihh expression in APC mutant DLD-1 colon cancer cells. These data identify a new Wnt-Hh axis in colonic epithelial renewal.

Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract

Background: Sonic hedgehog (Shh) is an important endodermal morphogenetic signal during the development of the vertebrate gut. It controls gastrointestinal patterning in general, and gastric gland formation in particular. We have previously shown that Shh regulates gastric gland proliferation in the adult but detailed analysis of its expression along the adult gastrointestinal tract has never been undertaken. We therefore studied Shh expression along the normal human and rodent adult gastrointestinal tract as well as in intestinal metaplasia of the stomach, gastric and intestinal metaplasia of the oesophagus, and gastric heterotopia in Meckel’s diverticulum. Methods: The studies were performed with in situ hybridisation and by immunohistochemistry using an antibody that recognises the Shh precursor form. Results: We found that in the normal gastrointestinal tract, high levels of Shh were expressed in the fundic glands of the stomach. Shh expression was also found in fundic gland metaplasia and heterotopia. However, Shh expression was lost in intestinal metaplasia of the stomach. Conclusion: We found a strong correlation between Shh expression and fundic gland differentiation. Our current study therefore provides evidence that in addition to its role in gastric epithelial development, Shh plays a unique role in gastric epithelial differentiation in adults.

Hh pathway expression in human gut tissues and in inflammatory gut diseases.

Sonic hedgehog (Shh) directs early gut patterning via epithelial–mesenchymal signaling and remains expressed in endoderm-derived tissues into the adult period. In human adult gut epithelium SHH/SHH expression is strongest in basal layers, which suggests that SHH may function in the maintenance of gut epithelial stem or progenitor cells. Recent publications suggest a role for aberrant SHH/SHH expression in gut epithelial neoplasias. We hypothesized that the regenerating gut epithelium in inflammatory gut disorders would show an upregulation of SHH/SHH signaling and this abnormal signal may explain the increased incidence of neoplasia in these diseases. Archived healthy gut and inflammatory gut diseased tissues were analyzed by RNA in situ hybridization and immunohistochemistry to describe location and levels of SHH signaling. We show that SHH/SHH and its receptor PTCH1/PTCH1 expression is restricted to the glandular epithelium of the gut, in an antiluminal pattern (strongest in basal layers and weak to absent in luminal epithelium). Inflammatory diseases of the gut show dramatic increases in epithelial SHH signaling. Expression increases in inflamed glandular epithelium (including metaplastic glandular epithelium), losing its radial (crypt-villous) polarity, and expression appears upregulated and present in all epithelial cells. We also describe strong SHH/SHH and PTCH1/PTCH1 expression in intraepithelial and mucosal inflammatory cells. We suggest that SHH signaling in inflammatory diseases of the gut acts to ensure stem cell restitution of damaged mucosal epithelium. However, such signaling may also present a risk for neoplastic transformation.

SOX9 specifies the pyloric sphincter epithelium through mesenchymal-epithelial signals.

Gastrointestinal (GI) development is highly conserved across vertebrates. Although several transcription factors and morphogenic proteins are involved in the molecular controls of GI development, the interplay between these factors is not fully understood. We report herein the expression pattern of Sox9 during GI development, and provide evidence that it functions, in part, to define the pyloric sphincter epithelium. SOX9 is expressed in the endoderm of the GI tract (with the exclusion of the gizzard) and its derivate organs, the lung and pancreas. Moreover, SOX9 is also expressed at the mesoderm of the pyloric sphincter, a structure that demarcates the gizzard from the duodenum. Using retroviral misexpression technique, we show that Sox9 expression in the pyloric sphincter is under the control of the BMP signaling pathway, known to play a key role in the development of this structure. By misexpressing SOX9 in the mesoderm of the gizzard, we show that SOX9 is able to transdifferentiate the adjacent gizzard epithelium into pyloric sphincter-like epithelium through the control of mesodermal-epithelial signals mediated in part by Gremlin (a modulator of the BMP pathway). Our results suggest that SOX9 is necessary and sufficient to specify the pyloric sphincter epithelial properties.

Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development

The bone morphogenetic protein (BMP) signaling pathway plays an essential role during gastrointestinal (GI) tract development in vertebrates. In the present study, we use an antibody that recognizes the phosphorylated and activated form of Smad1, 5, and 8 to examine (by immunohistochemistry) the endogenous patterns of BMP signaling pathway activation in the developing GI tract. We show that the endogenous BMP signaling pathway is activated in the mesoderm, the endoderm, and the enteric nervous system (ENS) of the developing chick GI tract and is more widespread than BMP ligand expression patterns. Using an avian‐specific retroviral misexpression technique to activate or inhibit BMP signaling pathway activity in the mesoderm of the gut, we show that BMP activity is required for the pattern, the development, and the differentiation of all three tissue types of the gut: mesoderm (that forms the visceral smooth muscle), endoderm (that forms the epithelium), and ectoderm (that forms the ENS). These results demonstrate that BMP signaling is activated in all the tissue layers of the GI tract during the development and plays a role during interactions and reciprocal communications of these tissue layers. Developmental Dynamics 234:312–322, 2005.

Sonic hedgehog is required for vascular outgrowth in the hindbrain choroid plexus.

Critical to the exchange and metabolic functions served by tissues like brain choroid plexi and lung is the coherent development of an epithelial sheet of large surface area in tight apposition to an extensive vascular bed. Here, we present functional experiments in the mouse demonstrating that Sonic hedgehog (Shh) produced by hindbrain choroid plexus epithelium induces the extensive vascular outgrowths and vascular surface area fundamental to choroid plexus functions, but does not induce the more specialized endothelial cell features of fenestrations and bore size. Our findings indicate that these Shh-dependent vascular elaborations occur even in the presence of Vegf and other established angiogenic factors, suggesting either that the levels of these factors are inadequate in the absence of Shh or that a different set of factors may be more essential to choroid plexus outgrowth. Transducing the Shh signal is a perivascular cell-the pericyte-rather than the more integral vascular endothelial cell itself. Moreover, our findings suggest that hindbrain choroid plexus endothelial cells, as compared to other vascular endothelial cells, are more dependent upon pericytes for instruction. Thus, in addition to Shh acting on the progenitor pool for choroid plexus epithelial cells, as previously shown, it also acts on choroid plexus pericytes, and together serves the important role of coordinating the development of two disparate yet functionally dependent structures-the choroid plexus vasculature and its ensheathing epithelium.

Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract.

Background: Sonic hedgehog (Shh) is an important endodermal morphogenetic signal during the development of the vertebrate gut. It controls gastrointestinal patterning in general, and gastric gland formation in particular. We have previously shown that Shh regulates gastric gland proliferation in the adult but detailed analysis of its expression along the adult gastrointestinal tract has never been undertaken. We therefore studied Shh expression along the normal human and rodent adult gastrointestinal tract as well as in intestinal metaplasia of the stomach, gastric and intestinal metaplasia of the oesophagus, and gastric heterotopia in Meckel’s diverticulum. Methods: The studies were performed with in situ hybridisation and by immunohistochemistry using an antibody that recognises the Shh precursor form. Results: We found that in the normal gastrointestinal tract, high levels of Shh were expressed in the fundic glands of the stomach. Shh expression was also found in fundic gland metaplasia and heterotopia. However, Shh expression was lost in intestinal metaplasia of the stomach. Conclusion: We found a strong correlation between Shh expression and fundic gland differentiation. Our current study therefore provides evidence that in addition to its role in gastric epithelial development, Shh plays a unique role in gastric epithelial differentiation in adults.