Development and Application of a High-Throughput Screening Method to Evaluate Antifungal Activity against Trichophyton tonsurans.

There exist relatively few drug classes on the market to treat dermatophyte infections. This investigation was designed to develop and validate high-throughput methodology for screening and confirmation of chemicals for activity against Trichophyton tonsurans. Growth characteristics were examined on two platforms (96- and 384-well) in three media at eight spore concentrations over a period of up to 120 h. Microspectrophotometry was used to automate plate reads. The 384-well platform was used to screen more than 7000 compounds from six chemical libraries. Z-scores for optical density relative to positive growth controls were used to flag compounds of interest and activity confirmed in separate assays. The final conditions selected for both screening and confirmation with minimum inhibitory concentration (MIC) determination were growth for 48 h at 32 °C in SabDex with 1 × 10(4) spores per reaction. Sensitivity and specificity averaged 99.2% (range, 95.2%-100%) and 99.8% (range, 99.1%-100%), respectively. MICs for known antifungals were similar to those reported by others using Clinical and Laboratory Standards Institute methods. Several novel compound classes were identified to have activity against T. tonsurans with potency comparable to known antifungals. A robust, reproducible assay is described that permits high-throughput screening in T. tonsurans.

Barrier function of the coelomic epithelium in the developing pancreas.

Tight spatial regulation of extracellular morphogen signaling within the close confines of a developing embryo is critical for proper organogenesis. Given the complexity of extracellular signaling in developing organs, together with the proximity of adjacent organs that use disparate signaling pathways, we postulated that a physical barrier to signaling may exist between organs in the embryo. Here we describe a previously unrecognized role for the embryonic coelomic epithelium in providing a physical barrier to contain morphogenic signaling in the developing mouse pancreas. This layer of cells appears to function both to contain key factors required for pancreatic epithelial differentiation, and to prevent fusion of adjacent organs during critical developmental windows. During early foregut development, this barrier appears to play a role in preventing splenic anlage-derived activin signaling from inducing intestinalization of the pancreas-specified epithelium.

Genetic predictors of susceptibility to cutaneous fungal infections: a pilot genome wide association study to refine a candidate gene search.

BACKGROUND: Trichophyton tonsurans is the foremost fungal pathogen of minority children in the U.S. Despite overwhelming infection rates, it does not appear that this fungus infects children in a non-specific manner. OBJECTIVE: This study was designed to identify genes that may predispose or protect a child from T. tonsurans infection. METHODS: Children participating in an earlier longitudinal study wherein infection rates could be reliably determined were eligible for inclusion. DNA from a subset (n=40) of these children at the population extremes underwent whole genome genotyping (WGG). Allele frequencies between cases and controls were examined and significant SNPs were used to develop a candidate gene list for which the remainder of the cohort (n=115) were genotyped. Cumulative infection rate was examined by genotype and the ability of selected genotypes to predict the likelihood of infection explored by multivariable analysis. RESULTS: 23 genes with a putative mechanistic role in cutaneous infection were selected for evaluation. Of these, 21 demonstrated significant differences in infection rate between genotypes. A risk index assigned to genotypes in the 21 genes accounted for over 60% of the variability observed in infection rate (adjusted r(2)=0.665, p<0.001). Among these, 8 appeared to account for the majority of variability that was observed (r(2)=0.603, p<0.001). These included genes involved in: leukocyte activation and migration, extracellular matrix integrity and remodeling, epidermal maintenance and wound repair, and cutaneous permeability. CONCLUSIONS: Applying WGG to individuals at the extremes of phenotype can help to guide the selection of candidate genes in populations of small cohorts where disease etiology is likely polygenic in nature.

Comparative analysis of secreted enzymes between the anthropophilic-zoophilic sister species Trichophyton tonsurans and Trichophyton equinum.

Trichophyton tonsurans (TT) and Trichophyton equinum (TE) are two closely related dermatophytes with very different host preferences. This study was designed to examine the genetic and transcript level variations of secreted enzymes between TT and TE. Thirty-one genes representing 10 gene families were selected for comparison and complete genomic and cDNA sequences were elucidated. Sequence analyses of the selected genes identified 104 polymorphisms between the two dermatophytes, 37 of which are expected to encode changes in their polypeptide sequence. Quantitative RT-PCR was used to examine the differences in levels of transcript between TT and TE grown over 14d in aqueous keratin medium. Differences in transcript expression between TT and TE were gene specific and ranged from 1.1-fold to 33-fold. Intra-specific variability across all genes ranged from 41% to 250%. Despite their overall genetic similarity, TT and TE exhibit a moderate degree of variability in the genomic make-up of their secreted enzymes and the extent to which they are transcribed when grown in an aqueous keratin medium. Such differences may contribute to how these genetically similar organisms have adapted to infect divergent host organisms.

The prevalence of infections with Trichophyton tonsurans in schoolchildren: the CAPITIS study.

BACKGROUND: Although Trichophyton tonsurans has become the leading cause of tinea capitis in the United States, reported infection rates vary widely, and prevalence estimates for the pediatric population at large remain poorly characterized. METHODS: A prospective, cross-sectional, surveillance study of children attending kindergarten through fifth grade in 44 schools across the bi-state (Kansas/Missouri), Kansas City metropolitan area was conducted. Fungal cultures were collected from all participants, and molecular analyses were used to characterize the patterns of infection within the population. RESULTS: Of 10,514 children (age: 8.3 +/- 1.9 years) examined for the presence of T tonsurans on their scalps, 6.6% exhibited positive cultures. Infection rates at participating schools ranged from 0% to 19.4%, exceeding 30% at a given grade level in some schools. Black children demonstrated the highest rates of infection (12.9%), with prevalence estimates for the youngest members of this racial group approaching 18%. Infection rates for Hispanic (1.6%) and white (1.1%) children were markedly lower. A single genetic strain of T tonsurans was identified in only 16.6% of classrooms, whereas each child harbored a unique genetic strain in 51.4%. CONCLUSIONS: We report a large-scale, citywide, surveillance study of T tonsurans infection rates among children in primary school in a metropolitan area. The striking prevalence rates and genetic heterogeneity among the fungal isolates confirm the relatively large degree to which this pathogen has become integrated into metropolitan communities.

Divergence among an international population of Trichophyton tonsurans isolates.

Trichophyton tonsurans is a widely distributed pathogen that demonstrates a significant degree of genetic and phenetic heterogeneity. To date, the degree of genetic relatedness among geographically segregated isolates has not been explored. This investigation evaluates the extent of genetic variation among an international population of T. tonsurans isolates and examines the relatedness of isolates within and between countries. Molecular strain typing was performed on 198 isolates obtained from 14 countries. A mixed-marker strategy utilizing 27 sequence variations in 13 gene loci was applied to all isolates and cluster analysis was performed to examine the relationship between strains. Phylogenetic analysis was used to corroborate the findings of the cluster analysis with T. equinum strains serving as an out-group. In total, 47 distinct strain types were identified represented by seven clusters and one singleton. There appeared to be a moderate degree of clustering among isolates obtained from North America, Asia and Australia, although European isolates were uniformly distributed among the majority of clusters. The degree of genetic variation observed in this study coupled with the geographic localization would support the argument for allopatric divergence within this species.

A large outbreak of Trichophyton tonsurans among health care workers in a pediatric hospital.

BACKGROUND: Although Trichophyton tonsurans remains a major cause of dermataophytoses in US children, nosocomial spread may go unrecognized in health care settings. We describe a staff outbreak of T tonsurans infection among health care workers in a freestanding pediatric hospital. METHODS: Epidemiologic evaluation (retrospective and prospective) was performed in the health care providers and ancillary staff assigned to a 27-bed inpatient medical unit in which the suspected outbreak occurred. RESULTS: Twenty-one individuals, including staff, a hospital volunteer, and a patient, developed tinea corporis during a 5-month period. All infections coincided with multiple admissions of a 2-year-old suspected index patient who demonstrated persistent infections of the scalp and arm. Fungal isolates obtained from the index patient and affected staff (when available) were subjected to multilocus strain typing, which revealed an identical genetic match between the index case and infected hospital personnel. CONCLUSION: T tonsurans can spread widely among staff members caring for children with recalcitrant dermatophyte infections. Recognition that workplace transmission may be the etiology of a succession of infections occurring in a single inpatient unit is necessary to limit the number of infected individuals.

Multilocus genotyping identifies infections by multiple strains of Trichophyton tonsurans.

Acquisition of multiple genetic strains of a single dermatophyte species should not be unexpected in areas of high endemicity, and yet multistrain infections are infrequently reported. This communication details mixed Trichophyton tonsurans infections and highlights the need to confirm the presence of multiple strains in a clinical single isolate by use of a multilocus approach.

Faulty bone morphogenetic protein signaling in esophageal atresia with tracheoesophageal fistula.

BACKGROUND: The organogenesis of esophageal atresia with tracheoesophageal fistula remains unclear. We have previously demonstrated that the fistula tract develops from a trifurcation of the embryonic lung bud and displays pulmonary lineage traits. Unlike the lung, the fistula grows without branching. Bone morphogenetic proteins (BMPs) are known to be important in lung branching. We studied possible BMP signaling defects as a potential cause for the absence of branching in the fistula tract. METHODS: Adriamycin was administered to pregnant rats on days 6-9 of gestation to induce tracheoesophageal fistula. Microdissection was performed at E13 and E17 isolating the foregut. Tissues were analyzed using immunohistochemistry for BMP ligand (BMP2, BMP4, BMP7) and receptor (BMPRIA, BMPRIB, BMPRII) expression. RESULTS: Immunohistochemistry revealed the presence of all 3 BMP ligands at E13, localized specifically to the esophageal mucosa but absent in the fistula and lung. At E17, the ligands were again present in the esophageal mucosa, and additionally in the fistula tract mucosa, but remained absent in the lung. At E17, all of the BMP receptors were also localized to the luminal surface of esophagus and fistula. However, in the lung epithelium, only BMPRII was found, whereas BMPRIA and BMPRIB remained absent. CONCLUSIONS: The normal expression pattern of BMP4 was increased at the branch tips and low between branches. Among other results, we show here a constant expression level of BMP ligands throughout the entire epithelium of the fistula tract. This diffuse expression suggests defective BMP signaling in the fistula tract and explains its nonbranching phenotype.

Mesenchymal epimorphin is important for pancreatic duct morphogenesis.

Epithelial-mesenchymal interactions are crucial for the proper development of many organs, including the pancreas. Within the pancreas, the ducts are thought to harbor stem/progenitor cells, and possibly to give rise to pancreatic ductal carcinoma. Little is known about the mechanism of formation of pancreatic ducts in the embryo. Pancreatic mesenchyme contains numerous soluble factors which help to sustain the growth and differentiation of exocrine and endocrine structures. Here, we report that one such morphoregulatory mesenchymal protein, epimorphin, plays an important role during pancreatic ductal proliferation and differentiation. We found that epimorphin is expressed in pancreatic mesenchyme during early stages of development, and at mesenchymal-epithelial interfaces surrounding the ducts at later stages. Strong upregulation of epimorphin expression was seen during in vitro pancreatic duct differentiation. Similarly, in vitro pancreatic duct formation was inhibited by a neutralizing antibody against epimorphin, whereas addition of recombinant epimorphin partially rescued duct formation. Together, our study demonstrates the role of epimorphin in pancreatic ductal morphogenesis.

Bone morphogenetic protein expression patterns in human esophageal atresia with tracheoesophageal fistula.

The organogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains unknown. The fistula tract appears to develop from a non-branching trifurcation of the embryonic lung bud. The non-branching growth of the fistula differs from the other lung buds and suggests a deficiency in bone morphogenetic protein (BMP) signaling, since BMPs are critical to proper lung development and branching. With IRB approval, portions of newborn human proximal esophageal pouch and distal fistula samples were recovered at the time of surgical repair of EA/TEF. The tissues were processed for immunohistochemistry. Commercially available fetal tissues were used as controls. In control tissues, BMP ligands (BMP 2, 4, and 7) were all present in the esophagus but absent in the trachea. BMPRIA was absent in both tissues. BMPRIB was detected in trachea but not in esophagus and BMPRII was detected in esophagus but not in trachea. In the EA/TEF specimens, all BMP ligands were present in the proximal esophageal pouch but absent in the fistula tract. BMPRIA and BMPRIB were not detected in either tissue. However, BMPRII was found in both fistula tract and proximal pouch. The submucosa of the fistula appears to maintain a mixed (identical neither to lung, esophagus, or trachea) BMP signaling pattern, providing one mechanism which could potentially explain the esophageal dismotility and lack of lung branching seen in the fistula/distal esophagus.

Synergistic endocrine induction by GLP-1 and TGF-beta in the developing pancreas.

OBJECTIVES: Glucagon-like peptide-1 (GLP-1) is known to stimulate glucose-dependent insulin production and secretion by pancreatic beta-cells. Preliminary evidence suggests that GLP-1 may also influence endocrine differentiation from pancreatic progenitor cells. Additionally, TGF-beta signaling can also control endocrine differentiation by both inhibiting proliferation and enhancing differentiation of endocrine progenitor cells to become mature beta-cells. Here we document synergy of these two signaling pathways in the differentiation of endocrine cells in the developing pancreas. METHODS: Embryonic pancreas was harvested from mice at day 11.5 and cultured for six days with GLP-1 agonist, exendin-4, and/or TGF-beta1 ligand. Also, a pan-neutralizing TGF-beta isoform antibody was used alone or with exendin-4 to study TGF-beta inhibition in this system. Pancreatic cultures were processed for immunohistochemistry. RESULTS: Exogenous TGF-beta1 and exendin-4 each individually enhanced both insulin and glucagon differentiation dose-dependently. However, when combined there was an additive effect to a 4.5-fold increase in insulin-positive differentiation. We also saw suppression of amylase-positive differentiation. Surprisingly, TGF-beta pan-neutralizing antibody also gave an augmentation of endocrine differentiation by 1.5 to 2-fold, but no synergistic effect was seen with exendin-4. CONCLUSION: We conclude that TGF-beta isoforms have a specific synergistic role with GLP-1 pathway signaling in early pancreatic development, toward endocrine differentiation and away from acinar differentiation.

Cross-talk between bone morphogenetic protein and transforming growth factor-beta signaling is essential for exendin-4-induced insulin-positive differentiation of AR42J cells.

A key goal of cellular engineering is to manipulate progenitor cells to become beta-cells, allowing cell replacement therapy to cure diabetes mellitus. As a paradigm for cell engineering, we have studied the molecular mechanisms by which AR42J cells become beta-cells. Bone morphogenetic proteins (BMPs), implicated in a myriad of developmental pathways, have not been well studied in insulin-positive differentiation. We found that the canonical intracellular mediators of BMP signaling, Smad-1 and Smad-8, were significantly elevated in AR42J cells undergoing insulin-positive differentiation in response to exendin-4 treatment, suggesting a role for BMP signaling in beta-cell formation. Similarly, endogenous BMP-2 ligand and ALK-1 receptor (activin receptor-like kinase-1; known to activate Smads 1 and 8) mRNAs were specifically up-regulated in exendin-4-treated AR42J cells. Surprisingly, Smad-1 and Smad-8 levels were suppressed by the addition of BMP-soluble receptor inhibition of BMP ligand binding to its receptor. Here, insulin-positive differentiation was also ablated. BMP-2 ligand antisense also strongly inhibited Smad-1 and Smad-8 expression, again with the abolition of insulin-positive differentiation. These results demonstrate a previously unrecognized key role for BMP signaling in mediating insulin-positive differentiation through the intracellular Smad signaling pathway. In short, BMP signaling may represent a novel downstream target of exendin-4 (glucagon-like peptide 1) signaling and potentially serve as an upstream regulator of transforming growth factor-beta isoform signaling to differentiate the acinar-like AR42J cells into insulin-secreting cells.

Interplay of glucagon-like peptide-1 and transforming growth factor-beta signaling in insulin-positive differentiation of AR42J cells.

The differentiation of pancreatic exocrine AR42J cells into insulin-expressing endocrine cells has served as an important model for both endogenous in vivo beta-cell differentiation as well as potential application to beta-cell engineering of progenitor cells. Exogenous activin, possibly working through intracellular smad 2 and/or smad 3, as well as exogenous exendin-4 (a long-acting glucagon-like peptide-1 agonist) have both been shown to induce insulin-positive/endocrine differentiation in AR42J cells. In this study, we present evidence of significant interplay and interdependence of these two pathways as well as potential synergy between the pathways. In particular, insulin-positive differentiation seems to entail an exendin-4-induced drop in smad 2 and elevation in smad 3 in RNA levels. The latter appears to be dependent on endogenous transforming growth factor (TGF)-beta isoform release by the AR42J cells and may serve as a mechanism to promote beta-cell maturation. The drop in smad 2 may mediate early endocrine commitment. The coapplication of exogenous exendin-4 and, specifically, low-dose exogenous TGF-beta1 led to a dramatic 20-fold increase in insulin mRNA levels, supporting a novel synergistic and codependent relationship between exendin-4 signaling and TGF-beta isoform signaling.

Multifaceted pancreatic mesenchymal control of epithelial lineage selection.

Mouse pancreatic development is critically dependent on epithelial-mesenchymal interactions. The pancreas differs from other epithelial-mesenchymal organs in that the epithelium gives rise to both epithelial exocrine cells and non-epithelial endocrine cells. We studied the nature of the interactions between the epithelium and mesenchyme with respect to the decision between exocrine and endocrine lineages. We show here a tripartite influence of mesenchyme on the developing epithelium. First, close proximity or contact of mesenchyme with the epithelium induces exocrine differentiation. Second, this mesenchymal proximity to the epithelium suppresses endocrine differentiation. Third, mesenchyme has an overall enhancing effect on the degree of insulin differentiation, suggesting a pro-endocrine effect in those epithelial cells at a distance from the mesenchyme. Proximity or contact between the mesenchyme and epithelium appeared to be necessary for the pro-exocrine effects of mesenchyme. We found that, in a co-culture system, NIH3T3 cells were able to substitute for mesenchyme in exocrine induction as well as in both the endocrine induction and endocrine inhibition, implying that the responsible molecules are not unique to pancreatic mesenchyme. Laminin appears to be a key molecule mediating the epithelial-mesenchymal interactions that lead to exocrine differentiation, since inhibition of laminin expression resulted in blockage of the pro-exocrine induction of mesenchyme.

Aberrant fibroblast growth factor receptor 2 signalling in esophageal atresia with tracheoesophageal fistula.

BACKGROUND: Although the pathogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains unknown, it has been shown that despite its esophageal appearance, the fistula tract originates from respiratory epithelium. The authors now hypothesize that defects in fibroblast growth factor (FGF) signaling contribute to the esophaguslike phenotype of the fistula tract. FGF2R is critical to normal lung morphogenesis and occurs in 2 isoforms (FGF2RIIIb and FGF2RIIIc), each with different ligand-binding specificity. To characterize FGF signaling in the developing EA/TEF, the authors analyzed levels of FGF2R splice variants in experimental EA/TEF. METHODS: The standard Adriamycin-induced EA/TEF model in rats was used. Individual foregut components from Adriamycin-treated and control embryos were processed for real-time, fluorescence-activated semiquantitative reverse transcriptase polymerase chain reaction on gestational days 12.5 and 13.5. RESULTS: Both fistula tract and Adriamycin-treated or normal esophagus showed significantly lower levels of FGF2RIIIb than either Adriamycin-treated lung buds (E12.5, P =.02; E13.5, P <.005) or normal lung buds (E12.5, P <.005; E13.5, P <.01). At E13.5, the fistula tract had lower levels of FGF2RIIIc than either treated (P <.01) or normal lung (P <.05). CONCLUSIONS: Levels of FGF2R in the developing fistula tract resemble that of distal esophagus rather than developing lung. This defect in FGF2RIIIb signaling may account for the nonbranching, esophaguslike phenotype of the fistula, despite its respiratory origin.

Defective sonic hedgehog signaling in esophageal atresia with tracheoesophageal fistula.

BACKGROUND: The pathogenesis of esophageal atresia and tracheoesophageal fistula (EA/TEF) remains unknown. We have found previously that an initial esophageal atresia, followed by an abnormal (absent) branching pattern of the middle branch of a trifurcation of the lung/tracheal bud, leads to the neonatal finding of TEF. Mice null mutant for hedgehog signaling can experience the development of EA/TEF, but the mechanism for this development is also unknown. Given that EA/TEF in humans appears not to be due to genetic defects, a hedgehog mutation cause seems very unlikely. However, defective hedgehog signaling that is caused by environmental effects in the human embryo likely could be implicated. We studied a teratogen-induced model of EA/TEF to determine the mechanism by which defective hedgehog signaling may lead to EA/TEF. METHODS: We injected Adriamycin into pregnant rats to induce EA/TEF in rat embryos. We first quantified sonic hedgehog (Shh) signaling pathway molecule expression using real-time, semiquantitative reverse-transcriptase polymerase chain reaction for Shh, Shh receptors (patched and smoothened), and downstream intracellular targets of those receptors (Gli family members). On the basis of these findings, we then developed an in vitro culture system for the day-12 embryonic TEF and manipulated Shh signaling using either exogenous Shh or Shh inhibitors. RESULTS: By reverse transcriptase-polymerase chain reaction, a unique difference between the fistula tract and control tissues was that Gli-2 (downstream signaling molecule of Shh) messenger RNA levels were much lower in the fistula tract than in the adjacent esophagus (P =.002). Surprisingly, in the culture experiments, the fistula tract was induced to branch by exogenous Shh. Such branching of the fistula was unexpected and further supports the presumed respiratory origin of the fistula tract because the normal lung, but not normal esophagus, branched in response to Shh. The Shh inhibitor had no effect, which indicated that defective signaling, rather than hyperfunctioning Shh, is critical to the nonbranching phenotype of the fistula tract in TEF. CONCLUSIONS: The recapitulation of respiratory developmental morphogenesis by the fistula tract of TEF in the presence of exogenous Shh, together with the quantitative reduction in normal, endogenous levels of Gli-2, strongly suggests that 1 mechanism for the formation of the fistula tract is the lack of proper Shh signaling because of Gli-2 deficiency, with subsequent straight, nonbranching caudal growth of the fistula tract. This deficiency can be rescued by excess exogenous Shh, thus reestablishing respiratory morphogenesis.

Fibroblast growth factor signaling in the developing tracheoesophageal fistula.

BACKGROUND/PURPOSE: The Adriamycin-induced rat model of esophageal atresia and tracheoesophageal fistula (EA/TEF) provides a reliable system for the study of EA/TEF pathogenesis. The authors previously hypothesized that faulty branching lung morphogenesis pathways were a critical component of its pathogenesis. The authors have found evidence for faulty fibroblast growth factor (FGF) signaling related to epithelial-mesenchymal interactions in the fistula tract. To better define FGF signaling, the differential expression of FGF ligands and their receptors between lung, fistula tract, and esophagus are described. METHODS: Time-dated pregnant, Sprague-Dawley rats were injected with Adriamycin (2 mg/kg intraperitoneally) on days 6 through 9 of gestation. Tissues were processed for histology and reverse transcriptase polymerase chain reaction. FGF-1, -7 and -10 were measured from whole lung, fistula tract, and esophagus of TEF or normal embryos. Expression of FGF2RIIIb and FGF2RIIIc receptors was measured in isolated epithelium and mesenchyme of lung and fistula tract of TEF embryos as well as lung and esophagus from normal controls. RESULTS: FGF-1 mRNA was present in the fistula tract and normal and Adriamycin-exposed lung but absent from whole esophagus. Interestingly, FGF-7 mRNA was present only in normal lung. FGF-10 was present in all tissues examined. FGF2RIIIb mRNA was absent in fistula mesenchyme but present in all other tissues examined. However, the splice variant FGF2RIIIc mRNA was present in all tissues examined. CONCLUSIONS: These findings support defective FGF signaling in the rat model of EA/TEF. Absence of FGF-7 mRNA in Adriamycin-exposed tissues suggests the primary effect of Adriamycin may be to inhibit FGF-7 expression. Moreover, absence of FGF2RIIIb in fistula mesenchyme may be caused by loss of positive feedback from FGF-7, its normal obligate ligand. Understanding these specific defects in FGF signaling may provide insight into faulty mechanisms of EA/TEF.

Glucagon is required for early insulin-positive differentiation in the developing mouse pancreas.

The embryonic pancreas is thought to develop from pluripotent endodermal cells that give rise to endocrine and exocrine cells. A key guidance mechanism for pancreatic development has previously been found to be epithelial-mesenchymal interaction. Interactions within the epithelium, however, have not been well studied. Glucagon is the earliest peptide hormone present at appreciable levels in the developing pancreatic epithelium (embryonic day [E]-9.5 in mouse). Insulin accumulation begins slightly later (E11 in mouse), followed by a rapid accumulation during the "second wave" of insulin differentiation ( approximately E15). Here we found that blocking early expression and function of glucagon, but not GLP-1, an alternate gene product of preproglucagon mRNA, prevented insulin-positive differentiation in early embryonic (E11) pancreas. These results suggest a novel concept and a key role for glucagon in the paracrine induction of differentiation of other pancreatic components in the early embryonic pancreas.

Retinoid signaling controls mouse pancreatic exocrine lineage selection through epithelial-mesenchymal interactions.

BACKGROUND & AIMS: The early embryonic pancreas gives rise to exocrine (ducts and acini) and endocrine lineages. Control of exocrine differentiation is poorly understood, but may be a critical avenue through which to manipulate pancreatic ductal carcinoma. Retinoids have been shown to change the character of pancreatic ductal cancer cells to a less malignant phenotype. We have shown that 9-cis retinoic acid (9cRA) inhibits acinar differentiation in the developing pancreas, in favor of ducts, and we wanted to determine the role of retinoids in duct versus acinar differentiation. METHODS: We used multiple culture systems for the 11-day embryonic mouse pancreas. RESULTS: Retinoic acid receptor (RAR)-selective agonists mimicked the acinar suppressive effect of 9cRA, suggesting that RAR-RXR heterodimers were critical to ductal differentiation. RARalpha was only expressed in mesenchyme, whereas RXRalpha was expressed in epithelium and mesenchyme. Retinaldehyde dehydrogenase 2, a critical enzyme in retinoid synthesis, was expressed only in pancreatic epithelium. 9cRA did not induce ductal differentiation in the absence of mesenchyme, implicating a requirement for mesenchyme in 9cRA effects. Mesenchymal laminin is necessary for duct differentiation, and retinoids are known to enhance laminin expression. In 9cRA-treated pancreas, immunohistochemistry for laminin showed a strong band of staining around ducts, and blockage of laminin signaling blocked all 9cRA effects. Western blot and RT-PCR of pancreatic mesenchyme showed laminin-beta1 protein and mRNA induction by 9cRA. CONCLUSIONS: Retinoids regulate exocrine lineage selection through epithelial-mesenchymal interactions, mediated through up-regulation of mesenchymal laminin-1.