Combined activities of hedgehog signaling inhibitors regulate pancreas development

Hedgehog signaling is known to regulate tissue morphogenesis and cell differentiation in a dose-dependent manner. Loss of Indian hedgehog (Ihh) results in reduction in pancreas size, indicating a requirement for hedgehog signaling during pancreas development. By contrast, ectopic expression of sonic hedgehog (Shh) inhibits pancreatic marker expression and results in transformation of pancreatic mesenchyme into duodenal mesoderm. These observations suggest that hedgehog signaling activity has to be regulated tightly to ensure proper pancreas development. We have analyzed the function of two hedgehog inhibitors, Hhip and patched 1 (Ptch), during pancreas formation. Our results indicated that loss of Hhip results in increased hedgehog signaling within the pancreas anlage. Pancreas morphogenesis, islet formation and endocrine cell proliferation is impaired in Hhip mutant embryos. Additional loss of one Ptch allele in Hhip-/-Ptch+/- embryos further impairs pancreatic growth and endodermal cell differentiation. These results demonstrate combined requirements for Hhip and Ptch during pancreas development and point to a dose-dependent response to hedgehog signaling within pancreatic tissue. Reduction of Fgf10 expression in Hhip homozygous mutants suggests that at least some of the observed phenotypes result from hedgehog-mediated inhibition of Fgf signaling at early stages.     

Hedgehog signaling regulates expansion of pancreatic epithelial cells

Embryonic Hedgehog signaling is essential for proper tissue morphogenesis and organ formation along the developing gastrointestinal tract. Hedgehog ligands are expressed throughout the endodermal epithelium at early embryonic stages but excluded from the region that will form the pancreas. Ectopic activation of Hedgehog signaling at the onset of pancreas development has been shown to inhibit organ morphogenesis. In contrast, Hedgehog signaling components are found within pancreatic tissue during subsequent stages of development as well as in the mature organ, indicating that a certain level of pathway activation is required for normal organ development and function. Here, we ectopically activate the Hedgehog pathway midway through pancreas development via expression of either Sonic (Shh) or Indian Hedgehog (Ihh) under control of the human Pax4-promoter. Similar pancreatic defects are observed in both Pax4-Shh and Pax4-Ihh transgenic lines, suggesting that regulation of the overall level of Hedgehog activity is critical for proper pancreas development. We also show that Hedgehog signaling controls mesenchymal vs. epithelial tissue differentiation and that pathway activation impairs formation of epithelial progenitors. Thus, tight control of Hedgehog pathway activity throughout embryonic development ensures proper pancreas organogenesis.     

Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.     

Glypican-6 stimulates intestinal elongation by simultaneously regulating Hedgehog and non-canonical Wnt signaling

We report here that Glypican-6 (GPC6)-null mice display at birth small intestines that are 75% shorter than those of normal littermates. Notably, we demonstrate that the role of GPC6 in intestinal elongation is mediated by both Hedgehog (Hh) and non-canonical Wnt signaling. Based on results from in vitro experiments, we had previously proposed that GPC6 stimulates Hh signaling by interacting with Hh and Patched1 (Ptc1), and facilitating/stabilizing their interaction. Here we provide strong support to this hypothesis by showing that GPC6 binds to Ptc1 in the mesenchymal layer of embryonic intestines. This study also provides experimental evidence that strongly suggests that GPC6 inhibits the activity of Wnt5a on the intestinal epithelium by binding to this growth factor, and reducing its release from the surrounding mesenchymal cells. Finally, we show that whereas the mesenchymal layer of GPC6-null intestines displays reduced cell proliferation and a thinner smooth muscle layer, epithelial cell differentiation is not altered in the mutant gut.     

Identification of osteogenic purmorphamine derivatives

During embryonic and cancer development, the Hedgehog family of proteins, including Sonic Hedgehog, play an important role by relieving the inhibition of Smo by Ptc, thus activating the Smo signaling cascade. Recently, a purine compound, purmorphamine, has been reported to target the Hedgehog signaling pathway by interacting with Smo. Interestingly, both Sonic Hedgehog and purmorphamine were found to promote the osteogenic differentiation of mouse chondroprogenitor cells. However, there is insufficient information as to how the activation of this seemingly unrelated signaling pathway, either by Sonic Hedgehog or purmorphamine, contributes to osteogenesis. Using alkaline phosphatase assays, we screened 125 purmorphamine derivatives from the Korea Chemical Bank for effects on the differentiation of preosteoblast C2C12 cells. Here, we report that two purine derivatives modulate ALP activity as well as the expression of genes whose expression is known or suggested to be involved in osteogenesis.     

Proliferation of Murine Midbrain Neural Stem Cells Depends upon an Endogenous Sonic Hedgehog (Shh) Source

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.     

Ptc1 and Ptc2 transcripts provide distinct readouts of Hedgehog signaling activity during chick embryogenesis.

Hedgehog (Hh) signaling in vertebrates controls patterning and differentiation of a broad range of tissues during development. The Hh receptor Patched (Ptc) is a critical regulator of signaling, maintaining active repression of the pathway in the absence of stimulation, limiting excess diffusion of ligand, and providing an efficient negative feedback mechanism for fine-tuning the responsiveness of receiving cells. Two distinct Ptc genes have been isolated from several vertebrates. Here, we describe the cloning of a second Ptc gene from chick (Ptc2). We show that Ptc1 and Ptc2 are both upregulated at sites of active Hh signaling but that the expression patterns of these genes only partially overlap, thus providing distinct readouts of Hh pathway stimulation. We also show that chick Ptc2 is expressed in the posterior apical ectodermal ridge (AER) of the limb bud in a pattern similar to Fgf4 and that the induction of Ptc2 within the AER, like that of Fgf4, is mediated via antagonism of BMP signaling. The differential responsiveness of cells to Hh pathway stimulation (as marked by the differential induction of Ptc genes) suggests heterogeneity in the mechanisms by which Hh signals are transduced within different populations of receiving cells.     

Regulation of pancreas development by hedgehog signaling

Pancreas organogenesis is regulated by the interaction of distinct signaling pathways that promote or restrict morphogenesis and cell differentiation. Previous work has shown that activin, a TGF(beta+) signaling molecule, permits pancreas development by repressing expression of Sonic hedgehog (Shh), a member of the hedgehog family of signaling molecules that antagonize pancreas development. Here we show that Indian hedgehog (Ihh), another hedgehog family member, and Patched 1 (Ptc1), a receptor and negative regulator of hedgehog activity, are expressed in pancreatic tissue. Targeted inactivation of Ihh in mice allows ectopic branching of ventral pancreatic tissue resulting in an annulus that encircles the duodenum, a phenotype frequently observed in humans suffering from a rare disorder known as annular pancreas. Shh(-)(/)(-) and Shh(-)(/)(-) Ihh(+/)(-) mutants have a threefold increase in pancreas mass, and a fourfold increase in pancreatic endocrine cell numbers. In contrast, mutations in Ptc1 reduce pancreas gene expression and impair glucose homeostasis. Thus, islet cell, pancreatic mass and pancreatic morphogenesis are regulated by hedgehog signaling molecules expressed within and adjacent to the embryonic pancreas. Defects in hedgehog signaling may lead to congenital pancreatic malformations and glucose intolerance.     

Altered differentiation and paracrine stimulation of mammary epithelial cell proliferation by conditionally activated Smoothened

The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation.     

α-Amylase expressed in human small intestinal epithelial cells is essential for cell proliferation and differentiation

α-Amylase, which plays an essential role in starch degradation, is expressed mainly in the pancreas and salivary glands. Human α-amylase is also detected in other tissues, but it is unclear whether the α-amylase is endogenously expressed in each tissue or mixed exogenously with one expressed by the pancreas or salivary glands. Furthermore, the biological significance of these α-amylases detected in tissues other than the pancreas and salivary glands has not been elucidated. We discovered that human α-amylase is expressed in intestinal epithelial cells and analyzed the effects of suppressing α-amylase expression. α-Amylase was found to be expressed at the second-highest messenger RNA level in the duodenum in human normal tissues after the pancreas. α-Amylase was detected in the cell extract of Caco-2 intestinal epithelial cells but not secreted into the culture medium. The amount of α-amylase expressed increased depending on the length of the culture of Caco-2 cells, suggesting that α-amylase is expressed in small intestine epithelial cells rather than the colon because the cells differentiate spontaneously upon reaching confluence in culture to exhibit the characteristics of small intestinal epithelial cells rather than colon cells. The α-amylase expressed in Caco-2 cells had enzymatic activity and was identified as AMY2B, one of the two isoforms of pancreatic α-amylase. The suppression of α-amylase expression by small interfering RNA inhibited cell differentiation and proliferation. These results demonstrate for the first time that α-amylase is expressed in human intestinal epithelial cells and affects cell proliferation and differentiation. This α-amylase may induce the proliferation and differentiation of small intestine epithelial cells, supporting a rapid turnover of cells to maintain a healthy intestinal lumen.     

Epidermal hyperplasia and expansion of the interfollicular stem cell compartment in mutant mice with a C-terminal truncation of Patched1

Hedgehog (Hh) signaling is conserved from flies to humans and is indispensable in embryogenesis and adulthood. Patched (Ptc) encodes a receptor for Hh ligands and functions as a tumor suppressor. PTCH1 mutations in humans are found in basal cell carcinoma (BCC) and irradiated Ptc1(+/-) mice recapitulate this phenotype. However, due to embryonic lethality associated with the Ptc1 null mutation, its normal function in embryonic and adult skin remains unknown. Here we describe the epidermal phenotypes of a spontaneous and viable allele of Ptc1, Ptc1(mes), in which the C-terminal domain (CTD) is truncated. Ptc1(mes/mes) embryos display normal epidermal and hair follicle development. Postnatal Ptc1(mes/mes) skin displays severe basal cell layer hyperplasia and increased proliferation, while stratification of the suprabasal layers is mostly normal. Interestingly, truncation of the Ptc1 CTD did not result in skin tumors. However, long term labeling studies revealed a greater than three-fold increase in label-retaining cells in the interfollicular epidermis of Ptc1(mes/mes) adults, indicating possible expansion of the epidermal stem cell compartment. Increased expression of regulators of epidermal homeostasis, c-Myc and p63, was also observed in Ptc1(mes/mes) adult skin. These results suggest that the CTD of Ptc1 is involved in regulating epidermal homeostasis in mature skin.     

Understanding human cancer using Drosophila: Tid47, a cytosolic product of the DnaJ-like tumor suppressor gene l2Tid,is a novel molecular partner of patched related to skin cancer

Recessive mutations of the Drosophila gene lethal(2)-tumorous imaginal discs (l(2)tid) cause neoplastic growth of the anlagen of the adult organs, the imaginal discs. Here we report that the three proteins encoded by this evolutionarily conserved gene, Tid50, Tid47, and Tid40, identified as members of the DnaJ cochaperone family, are destined for different cellular compartments, build complexes with many proteins in a developmental stage-specific manner, and are likely to be involved in different cellular processes. We show that the cytosolic Tid47 molecule is a novel component of the Hedgehog (Hh)-Patched (Ptc) signaling regulating cell/tissue polarity and spatial patterning during development and is associated with human tumors such as basal cell carcinoma (BCC) and medulloblastoma. We provide functional evidence for its direct in vivo interaction with the Hh-bound Ptc receptor during signal transmission. Because loss of l(2)tid causes neoplastic transformation of Hh-responsive cells, we suggest that Tid47 may at least act as a guardian of the Hh signaling gradient by regulating Ptc homeostasis in the tissue. Finally, we show that the expression of htid-1, the human counterpart of l(2)tid, is altered in human BCCs. We demonstrate that in BCCs loss of htid expression correlates with loss of differentiation capacity of the neoplastic cells similar to that found in the Drosophila tumor model.     

Reduced white fat mass in adult mice bearing a truncated Patched 1

Hedgehog (Hh) signaling emerges as a potential pathway contributing to fat formation during postnatal development. In this report, we found that Patched 1 (Ptc1), a negative regulator of Hh signaling, was expressed in the epididymal fat pad of adult mice. Reduced total white fat mass and epididymal adipocyte cell size were observed in naturally occurring spontaneous mesenchymal dysplasia (mes) adult mice (Ptc1(mes/mes)), which carry a deletion of Ptc1 at the carboxyl-terminal cytoplasmic region. Increased expression of truncated Ptc1, Ptc2 and Gli1, the indicators of ectopic activation of Hh signaling, was observed in epididymal fat pads of adult Ptc1(mes/mes) mice. In contrast, expression of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, adipocyte P2 and adipsin were reduced in epididymal fat pads of adult Ptc1(mes/mes) mice. Taken together, our results indicate that deletion of carboxyl-terminal tail of Ptc1 can lead to the reduction of white fat mass during postnatal development.