Pancreas-specific Transcription Factor 1a Research Articles

Neonatal Diabetes Due to a Mutation in the Distal PTF1A Enhancer: A Case Report and Literature Review

: Biallelic variants in the pancreas-specific transcription factor 1A (PTF1A) gene are a rare cause of permanent neonatal diabetes. We report a case of neonatal diabetes with unique clinical manifestations. The clinical diagnosis of the affected infant was confirmed by insufficient endocrine and exocrine pancreas activity; however, the pancreas was normal in imaging. Molecular analyses identified a novel homozygous single nucleotide variant (Chr10, g.23508441T > G), affecting a highly conserved nucleotide within a distal enhancer of the PTF1A gene. The literature review showed that most of these patients had IUGR and imaging evidence of pancreatic agenesis or hypoplasia. We suggest that pancreatic imaging and evaluation of exocrine pancreas function can help early confirmation of the diagnosis in patients with permanent neonatal diabetes.

Feeder-cell-independent culture of the pig embryonic stem cell-derived exocrine pancreatic cell line, PICM-31.

The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the derivative cell line, PICM-31A1. PICM-31A1 cells were adapted to growth on a polymerized collagen matrix using feeder cell-conditioned medium and were designated PICM-31FF. Like the parental cells, the PICM-31FF cells were small and grew relatively slowly in closely knit colonies that eventually coalesced into a continuous monolayer. The PICM-31FF cells were extensively cultured: 40 passages at 1:2, 1:3, and finally 1:5 split ratios over a 1-yr period. Ultrastructure analysis showed the cells' epithelial morphology and revealed that they retained their secretory granules typical of pancreas acinar cells. The cells maintained their expression of digestive enzymes, including carboxypeptidase A1 (CPA1), amylase 2A (AMY2A), and phospholipase A2 (PLA2G1B). Alpha-fetoprotein (AFP), a fetal cell marker, continued to be expressed by the cells as was the pancreas alpha cell-associated gene, transthyretin. Several pancreas-associated developmental genes were also expressed by the cells, including pancreatic and duodenal homeobox 1 (PDX1) and pancreas-specific transcription factor, 1a (PTF1A). Proteomic analysis of cellular proteins confirmed the cells' production of digestive enzymes and showed that the cells expressed cytokeratin-8 and cytokeratin-18. The PICM-31FF cell line provides an in vitro model of fetal pig pancreatic exocrine cells without the complicating presence of feeder cells.

Derivation and Characterization of a Pig Embryonic-Stem-Cell-Derived Exocrine Pancreatic Cell Line.

The aim of this study was to identify an epithelial cell line isolated from the spontaneous differentiation of totipotent pig epiblast cells. PICM-31 and its colony-cloned derivative cell line, PICM-31A, were established from the culture and differentiation of an epiblast mass isolated from an 8-day-old pig blastocyst. The cell lines were analyzed by transmission electron microscopy, marker gene expression, and mass spectroscopy-based proteomics. The PICM-31 cell lines were continuously cultured and could be successively colony cloned. They spontaneously self-organized into acinarlike structures. Transmission electron microscopy indicated that the cell lines' cells were epithelial and filled with secretory granules. Candidate gene expression analysis of the cells showed an exocrine pancreatic profile that included digestive enzyme expression, for example, carboxypeptidase A1, and expression of the fetal marker, α-fetoprotein. Pancreatic progenitor marker expression included pancreatic and duodenal homeobox 1, NK6 homeobox 1, and pancreas-specific transcription factor 1a, but not neurogenin 3. Proteomic analysis of cellular proteins confirmed the cells' production of digestive enzymes and showed that the cells expressed cytokeratins 8 and 18. The PICM-31 cell lines provide in vitro models of fetal pig pancreatic exocrine cells. They are the first demonstration of continuous cultures, that is, cell lines, of nontransformed pig pancreas cells.

Mechanistic insights into the activity of Ptf1-p48 (pancreas transcription factor 1a): probing the interactions levels of Ptf1-p48 with E2A-E47 (transcription factor E2-alpha) and ID3 (inhibitor of DNA binding 3)

Ptf1-p48 (Pancreas specific transcription factor 1a) is transcription regulatory protein known for the activation of exocrine specific genes. Downregulation of its expression formulates early stages of pancreatic adenocarcinoma as deduced by its association with oncogenic bHLH (Basic Helix–Loop–Helix) protein ID3 (Inhibitor of DNA binding 3) protein whose overexpression induces cytoplasmic mislocalization of Ptf1-p48. The precise mechanism and/or functional role of Ptf1-p48in promoting pancreatic cancer is vague. The structural features of the Ptf1-p48 and its dimerization with E47 (Transcription factor E2-alpha) and ID3 mediated by their HLH (Helix–Loop–Helix) domain were perceived through MD (Molecular Dynamics) simulations of 50 ns. The interactions formed by the HLH domain in both Ptf1-E47 and Ptf1-ID3 complexes are favored by the synergistic movement of their domain helices. Accordingly, in the Ptf1-E47 complex α7 of Ptf1-p48 and α1 helix of E47 along with the loop residues of their HLH domain exhibit transitions marked by inward movement toward each other and forms polar and charged interactions. In the Ptf1-ID3 complex, α8 of Ptf1-p48 moves toward the α3 helix of ID3 and forms hydrogen bonds. The interface analysis also reveals better interface in the Ptf1-p48 complex than the Ptf1-ID3 evident by energetics and number of hydrogen bonds. The interactions in each of these complexes, supported by angular displacement and mode vector analyzes, comprehensibly describe the considerable structural changes induced upon dimer formation. It thereby gives an insight into the interfaces that could help in designing of potential inhibitors for ID3 to curb the cancer cell growth.

Orphan nuclear hormone receptor Small Heterodimer Partner plays a protective role in diet‐induced islet dysfunction

Our early study found that deficiency of small heterodimer partner (SHP, NR0B2) protected the mice from diet‐induced obesity due to increased fatty acid oxidation but exacerbated glucose tolerance due to impaired glucose‐stimulated insulin secretion upon a 6‐month western diet regimen. Current study explored potential roles of SHP in the development of glucose intolerance furthermore. We assessed islet function using illumina beadchip array and morphometric measurement of pancreatic sections. Beadchip array revealed that many exocrine specific genes (for example, CUB and Zona Pellucida‐Like Domains (cuzd1), pancreas specific transcription factor 1a (ptf1a), trypsin 4 (try4) and pancreatic carboxyl ester lipase (cel) etc.) were markedly down‐regulated in the islets of SHP null mice in either chow or western diet‐fed condition. However, expression of major genes involved in insulin secretion and islet function were similar between WT and SHP null mice. Results from quantitative real‐time PCR analysis agreed with majority of beadchip array results except Glut2, which was decreased in SHP null islets. Corroborating with impaired insulin secretion and lower Glut2 mRNA level, glucose‐stimulated Ca2+ influx was greatly diminished in SHP null islets. Islet size was also decreased in SHP null mice fed either diet. The observed diabetic phenotype and pancreatic exocrine insufficiency in whole body SHP−/−mice was recapitulated in the pancreas specific SHPKO (PSHPKO) mice, where SHP deletion is confirmed in both endocrine and exocrine tissues. Interestingly the lean phenotype was reproduced only in liver‐specific SHPKO mice but not in PSHPKO mice. Even though detailed underlying molecular mechanisms for the observed phenotypes by SHP deletion remain to be elucidated, current data suggest that SHP plays distinctive and tissue‐specific role in the development of obesity and diabetes.Support or Funding InformationThis work had been supported by the National Institutes of Health 344 Grant R01DK093774 (to YKL)

Uncovering stem cell differentiation factors for salivary gland regeneration by quantitative analysis of differential proteomes.

Severe xerostomia (dry mouth) compromises the quality of life in patients with Sjögren’s syndrome or radiation therapy for head and neck cancer. A clinical management of xerostomia is often unsatisfactory as most interventions are palliative with limited efficacy. Following up our previous study demonstrating that mouse BM-MSCs are capable of differentiating into salivary epithelial cells in a co-culture system, we further explored the molecular basis that governs the MSC reprogramming by utilizing high-throughput iTRAQ-2D-LC-MS/MS-based proteomics. Our data revealed the novel induction of pancreas-specific transcription factor 1a (PTF1α), muscle, intestine and stomach expression-1 (MIST-1), and achaete-scute complex homolog 3 (ASCL3) in 7 day co-cultured MSCs but not in control MSCs. More importantly, a common notion of pancreatic-specific expression of PTF1 α was challenged for the first time by our verification of PTF1 α expression in the mouse salivary glands. Furthermore, a molecular network simulation of our selected putative MSC reprogramming factors demonstrated evidence for their perspective roles in salivary gland development. In conclusion, quantitative proteomics with extensive data analyses narrowed down a set of MSC reprograming factors potentially contributing to salivary gland regeneration. Identification of their differential/synergistic impact on MSC conversion warrants further investigation.

Mutations in the noncoding genome.

Clinical diagnostic sequencing currently focuses on identifying causal mutations in the exome, wherein most disease-causing mutations are known to occur. The rest of the genome is mostly comprised of regulatory elements that control gene expression, but these have remained largely unexplored in clinical diagnostics due to the high cost of whole genome sequencing and interpretive challenges. The purpose of this review is to illustrate examples of diseases caused by mutations in regulatory elements and introduce the diagnostic potential for whole genome sequencing. Different classes of functional elements and chromatin structure are described to provide the clinician with a foundation for understanding the basis of these mutations. The utilization of whole-genome sequence data, epigenomic maps and induced pluripotent stem (IPS) cell technologies facilitated the discovery that mutations in the pancreas-specific transcription factor 1a enhancer can cause isolated pancreatic agenesis. High resolution array comparative genomic hybridisation (CGH), whole-genome sequencing, maps of 3-D chromatin architecture, and mouse models generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas were used to show that disruption of topological-associated domain boundary elements cause limb defects. Structural variants that reposition enhancers in somatic cells have also been described in cancer. Although not ready for diagnostics, new technologies, epigenomic maps, and improved knowledge of chromatin architecture will soon enable a better understanding and diagnostic solutions for currently unexplained genetic disorders.

Ectopic Ptf1a Expression in Murine ESCs Potentiates Endocrine Differentiation and Models Pancreas Development In Vitro

Besides its role in exocrine differentiation, pancreas-specific transcription factor 1a (PTF1a) is required for pancreas specification from the foregut endoderm and ultimately for endocrine cell formation. Examining the early role of PTF1a in pancreas development has been challenging due to limiting amounts of embryonic tissue material for study. Embryonic stem cells (ESCs) which can be differentiated in vitro, and without limit to the amount of experimental material, can serve as a model system to study these early developmental events. To this end, we derived and characterized a mouse ESC line with tetracycline-inducible expression of PTF1a (tet-Ptf1a mESCs). We found that transient ectopic expression of PTF1a initiated the pancreatic program in differentiating ESCs causing cells to activate PDX1 expression in bud-like structures resembling pancreatic primordia in vivo. These bud-like structures also expressed progenitor markers characteristic of a developing pancreatic epithelium. The epithelium differentiated to generate a wave of NGN3+ endocrine progenitors, and further formed cells of all three pancreatic lineages. Notably, the insulin+ cells in the cultures were monohormonal, and expressed PDX1 and NKX6.1. PTF1a-induced cultures differentiated into significantly more endocrine and exocrine cells and the ratio of endocrine-to-exocrine cell differentiation could be regulated by retinoic acid (RA) and nicotinamide (Nic) signaling. Moreover, induced cultures treated with RA and Nic exhibited a modest glucose response. Thus, this tet-Ptf1a ESC-based in vitro system is a valuable new tool for interrogating the role of PTF1a in pancreas development and in directing differentiation of ESCs to endocrine cells.

Evidence for annular pancreas as an associated anomaly in the VATER/VACTERL association and investigation of the gene encoding pancreas specific transcription factor 1A as a candidate gene

Evidence for annular pancreas as an associated anomaly in the VATER/VACTERL association and investigation of the gene encoding pancreas specific transcription factor 1A as a candidate gene

Recessive mutations in a distal PTF1A enhancer cause isolated pancreatic agenesis.

The contribution of cis-regulatory mutations to human disease remains poorly understood. Whole genome sequencing can identify all non-coding variants, yet discrimination of causal regulatory mutations represents a formidable challenge. We used epigenomic annotation in hESC-derived embryonic pancreatic progenitor cells to guide the interpretation of whole genome sequences from patients with isolated pancreatic agenesis. This uncovered six different recessive mutations in a previously uncharacterized ~400bp sequence located 25kb downstream of PTF1A (pancreas-specific transcription factor 1a) in ten families with pancreatic agenesis. We show that this region acts as a developmental enhancer of PTF1A and that the mutations abolish enhancer activity. These mutations are the most common cause of isolated pancreatic agenesis. Integrating genome sequencing and epigenomic annotation in a disease-relevant cell type can uncover novel non-coding elements underlying human development and disease.

Distinct enhancers of ptf1a mediate specification and expansion of ventral pancreas in zebrafish

Development of the pancreas and cerebellum require Pancreas-specific transcription factor-1a (Ptf1a), which encodes a subunit of the transcription factor complex PTF1. Ptf1a is required in succession for specification of the pancreas, proper allocation of pancreatic progenitors to endocrine and exocrine fates, and the production of digestive enzymes from the exocrine acini. In several neuronal structures, including the cerebellum, hindbrain, retina and spinal cord, Ptf1a is transiently expressed and promotes inhibitory neuron fates at the expense of excitatory fates. Transcription of Ptf1a in mouse is maintained in part by PTF1 acting on an upstream autoregulatory enhancer. However, the transcription factors and enhancers that initially activate Ptf1a expression in the pancreas and in certain structures of the nervous system have not yet been identified. Here we describe a zebrafish autoregulatory element, conserved among teleosts, with activity similar to that described in mouse. In addition, we performed a comprehensive survey of all non-coding sequences in a 67kb interval encompassing zebrafish ptf1a, and identified several neuronal enhancers, and an enhancer active in the ventral pancreas prior to activation of the autoregulatory enhancer. To test the requirement for autoregulatory control during pancreatic development, we restored ptf1a function through BAC transgenesis in ptf1a morphants, either with an intact BAC or one lacking the autoregulatory enhancer. We find that ptf1a autoregulation is required for development of the exocrine pancreas and full rescue of the ptf1a morphant phenotype. Similarly, we demonstrate that a ptf1a locus lacking the early enhancer region is also capable of rescue, but only supports formation of a hypoplastic exocrine pancreas. Through our dissection of the complex regulatory control of ptf1a, we identified separate cis-regulatory elements that underlie different aspects of its expression and function, and further demonstrated the requirement of maintained ptf1a expression for normal pancreatic morphogenesis. We also identified a novel enhancer that mediates initiation of ptf1a expression in the pancreas, through which the signals that specify the ventral pancreas are expected to exert their action.

An isoform of retinoid-related orphan receptor β directs differentiation of retinal amacrine and horizontal interneurons

Amacrine and horizontal interneurons integrate visual information as it is relayed through the retina from the photoreceptors to the ganglion cells. The early steps that generate these interneuron networks remain unclear. Here we show that a distinct RORβ1 isoform encoded by the retinoid-related orphan nuclear receptor β gene (Rorb) is critical for both amacrine and horizontal cell differentiation in mice. A fluorescent protein cassette targeted into Rorb revealed RORβ1 as a novel marker of immature amacrine and horizontal cells and of undifferentiated, dividing progenitor cells. RORβ1-deficient mice lose expression of pancreas-specific transcription factor 1a (Ptf1a) but retain forkhead box n4 factor (Foxn4), two early-acting factors necessary for amacrine and horizontal cell generation. RORβ1 and Foxn4 synergistically induce Ptf1a expression, suggesting a central role for RORβ1 in a transcriptional hierarchy that directs this interneuron differentiation pathway. Moreover, ectopic RORβ1 expression in neonatal retina promotes amacrine cell differentiation.

Pancreas-specific deletion of mouse Gata4 and Gata6 causes pancreatic agenesis

Pancreatic agenesis is a human disorder caused by defects in pancreas development. To date, only a few genes have been linked to pancreatic agenesis in humans, with mutations in pancreatic and duodenal homeobox 1 (PDX1) and pancreas-specific transcription factor 1a (PTF1A) reported in only 5 families with described cases. Recently, mutations in GATA6 have been identified in a large percentage of human cases, and a GATA4 mutant allele has been implicated in a single case. In the mouse, Gata4 and Gata6 are expressed in several endoderm-derived tissues, including the pancreas. To analyze the functions of GATA4 and/or GATA6 during mouse pancreatic development, we generated pancreas-specific deletions of Gata4 and Gata6. Surprisingly, loss of either Gata4 or Gata6 in the pancreas resulted in only mild pancreatic defects, which resolved postnatally. However, simultaneous deletion of both Gata4 and Gata6 in the pancreas caused severe pancreatic agenesis due to disruption of pancreatic progenitor cell proliferation, defects in branching morphogenesis, and a subsequent failure to induce the differentiation of progenitor cells expressing carboxypeptidase A1 (CPA1) and neurogenin 3 (NEUROG3). These studies address the conserved and nonconserved mechanisms underlying GATA4 and GATA6 function during pancreas development and provide a new mouse model to characterize the underlying developmental defects associated with pancreatic agenesis.

Microarray analysis of Xenopus endoderm expressing Ptf1a

Pancreas-specific transcription factor 1a (Ptf1a), a bHLH transcription factor, has two temporally distinct functions during pancreas development; initially it is required for early specification of the entire pancreas, while later it is required for proper differentiation and maintenance of only acinar cells. The importance of Ptf1a function was revealed by the fact that loss of Ptf1a leads to pancreas agenesis in humans. While Ptf1a is one of the most important pancreatic transcription factors, little is known about the differences between the regulatory networks it controls during initial specification of the pancreas as opposed to acinar cell development, and to date no comprehensive analysis of its downstream targets has been published. In this article, we use Xenopus embryos to identify putative downstream targets of Ptf1a. We isolated anterior endoderm tissue overexpressing Ptf1a at two early stages, NF32 and NF36, and compared their gene expression profiles using microarrays. Our results revealed that Ptf1a regulates genes with a wide variety of functions, providing insight into the complexity of the regulatory network required for pancreas specification.

Mutations in PTF1A are not a common cause for human VATER/VACTERL association or neural tube defects mirroring Danforth's short tail mouse.

Danforth's short tail (Sd) mutant mice exhibit defects of the neural tube and other abnormalities, which are similar to the human vertebral anomalies, anal atresia, cardiac defects, tracheosophageal fistula and/or esophageal atresia, renal and radial abnormalities, and limb defects (VATER/VACTERL) association, including defects of the hindgut. Sd has been shown to underlie ectopic gene expression of murine Ptf1a, which encodes pancreas-specific transcription factor 1A, due to the insertion of a retrotansposon in its 5' regulatory domain. In order to investigate the possible involvement of this gene in human VATER/VACTERL association and human neural tube defects (NTDs), a sequence analysis was performed. DNA samples from 103 patients with VATER/VACTERL and VATER/VACTERL‑like association, all presenting with anorectal malformations, and 72 fetuses with NTDs, where termination of pregnancy had been performed, were included in the current study. The complete PTF1A coding region, splice sites and 1.5 kb of the 5' flanking promotor region was sequenced. However, no pathogenic alterations were detected. The results of the present study do not support the hypothesis that high penetrant mutations in these regions of PTF1A are involved in the development of human VATER/VACTERL association or NTDs, although rare mutations may be detectable in larger patient samples.

Adaptive gene regulation of pyruvate dehydrogenase kinase isoenzyme 4 in hepatotoxic chemical-induced liver injury and its stimulatory potential for DNA repair and cell proliferation

The processes involved in the adaptation of animals to environmental factors remain unclear. We examined the mechanisms underlying the adaptive potential of the mouse against hepatotoxic chemical-induced injury. Microarray analysis revealed that ethylbenzene, a hepatotoxic chemical, upregulated PDK4 (encoding pyruvate dehydrogenase kinase isoenzyme 4) in mouse livers and that the upregulation was enhanced by previous exposure to the chemical. Although PDK4 is an energy resource regulator induced by starvation, expression of other fasting-inducible genes was unaffected. PDK4 induced by chemical stress developed hepatic accumulation of sirtuin 1 by regulating pyruvate concentration and activated the Nbn and ATM, which are critical for DNA repair and checkpoint activation. PDK4 overexpression on carbon tetrachloride (CCl4)-induced liver injury resulted in delayed necrotic tissue recovery with cell cycle arrest and decreased γH2AX foci and micronucleus formation. PDK4 silencing on CCl4-induced liver injury accelerated necrotic tissue recovery and increased γH2AX foci and micronucleus formation, indicating the essential role of PDK4 in DNA repair and checkpoint activation. PDK4 overexpression induced pancreas-specific transcription factor 1a (Ptf1a) upregulation and transcriptional activation of several pancreatic genes in the liver. Ptf1a overexpression by adenoviral gene delivery resulted in accelerated tissue recovery on CCl4-induced liver injury. Our data identified PDK4 as a novel pivotal factor in adaptation to chemical stress.

P/CAF modulates the activity of the transcription factor p48/Ptf1a involved in pancreatic acinar differentiation

p48, also called Ptf1a (pancreas-specific transcription factor 1a), is a tissue-restricted bHLH (basic helix loop helix) transcription factor which is critical for pancreatic commitment during development and for the activation and maintenance of the acinar differentiation programme in the exocrine pancreas. High-level expression of exocrine digestive enzymes, a hallmark of mature acinar cells, depends largely on the trimeric complex PTF1, formed by p48, RBP-L (recombination signal-binding protein 1-like) and a class A bHLH protein. In addition, p48 induces cell-cycle exit by controlling G(1)/S-phase progression. However, the mechanisms that mediate PTF1-dependent gene activation are poorly understood. In the present study, we report that p48 increases transcription through two activation domains located in its N-terminal region by recruiting transcriptional co-activators. The histone acetyltransferase cofactor p/CAF {p300/CBP [CREB (cAMP-response-element-binding protein)-binding protein]-associated factor} interacts with p48 in acinar cells in vivo and is associated with the promoter region of acinar genes targeted by the PTF1 complex. p/CAF potentiates PTF1 transcriptional activity by enhancing selectively the p48 transactivation activity. p/CAF promotes the nuclear accumulation of p48 and its in vivo acetylation in Lys(200). The K200R mutation abolishes the transcriptional activity of p48, as well as its capacity to functionally co-operate with RBP-L to ensure effective PTF1-driven transcription, indicating that p/CAF-mediated acetylation of p48 is required for the full transcriptional activity of PTF1. In contrast, p/CAF did not co-operate with p48 in its growth regulatory effects. These results support a critical and selective role of p/CAF in PTF1-dependent gene activation during acinar differentiation.

Exdpf Is a Key Regulator of Exocrine Pancreas Development Controlled by Retinoic Acid and ptf1a in Zebrafish

Both endocrine and exocrine pancreatic cells arise from pancreatic-duodenal homeobox 1 (pdx1)-positive progenitors. The molecular mechanisms controlling cell fate determination and subsequent proliferation, however, are poorly understood. Unlike endocrine cells, less is known about exocrine cell specification. We report here the identification and characterization of a novel exocrine cell determinant gene, exocrine differentiation and proliferation factor (exdpf), which is highly expressed in the exocrine cell progenitors and differentiated cells of the developing pancreas in zebrafish. Knockdown of exdpf by antisense morpholino caused loss or significant reduction of exocrine cells due to lineage-specific cell cycle arrest but not apoptosis, whereas the endocrine cell mass appeared normal. Real-time PCR results demonstrated that the cell cycle arrest is mediated by up-regulation of cell cycle inhibitor genes p21Cip, p27Kip, and cyclin G1 in the exdpf morphants. Conversely, overexpression of exdpf resulted in an overgrowth of the exocrine pancreas and a severe reduction of the endocrine cell mass, suggesting an inhibitory role for exdpf in endocrine cell progenitors. We show that exdpf is a direct target gene of pancreas-specific transcription factor 1a (Ptf1a), a transcription factor critical for exocrine formation. Three consensus Ptf1a binding sites have been identified in the exdpf promoter region. Luciferase assay demonstrated that Ptf1a promotes transcription of the exdpf promoter. Furthermore, exdpf expression in the exocrine pancreas was lost in ptf1a morphants, and overexpression of exdpf successfully rescued exocrine formation in ptf1a-deficient embryos. Genetic evidence places expdf downstream of retinoic acid (RA), an instructive signal for pancreas development. Knocking down exdpf by morpholino abolished ectopic carboxypeptidase A (cpa) expression induced by RA. On the other hand, exdpf mRNA injection rescued endogenous cpa expression in embryos treated with diethylaminobenzaldehyde, an inhibitor of RA signaling. Moreover, exogenous RA treatment induced anterior ectopic expression of exdpf and trypsin in a similar pattern. Our study provides a new understanding of the molecular mechanisms controlling exocrine cell specification and proliferation by a novel gene, exdpf. Highly conserved in mammals, the expression level of exdpf appears elevated in several human tumors, suggesting a possible role in tumor pathogenesis.

Interactions between Hairy/Enhancer of Split-related proteins and the pancreatic transcription factor Ptf1-p48 modulate function of the PTF1 transcriptional complex

In the developing pancreas, the onset of exocrine differentiation is driven by the activity of the PTF1 (pancreas transcription factor 1) transcriptional complex, which is comprised of the class II bHLH (basic helix-loop-helix) protein, Ptf1-p48 [also known as Ptf1a (pancreas specific transcription factor 1a)], and a class I E-box binding partner. Activity of the PTF1 complex is normally inhibited by the Notch signalling pathway, a process mediated by Notch effector proteins in the HES (Hairy/Enhancer of Split) family of bHLH transcriptional repressors. In the present study, we show that this inhibitory effect occurs through direct interaction between HES family members and Ptf1-p48. The HES family members Hey1 (hairy/enhancer-of-split related with YRPW motif 1) and Hey2 co-immunoprecipitate with Ptf1-p48, and Ptf1-p48 binding by Hes1 is also evident in yeast two-hybrid and GST (glutathione S-transferase) pull-down assays. The ability of Hes1 to interact with Ptf1-p48 resides within a fragment comprised of the bHLH, Orange and C-terminal domains, and does not require the N-terminal or WRPW elements. The ability of truncated versions of Hes1 to bind Ptf1-p48 correlates with their ability to down-regulate the activity of the PTF1 transcriptional complex, defining Ptf1-p48 binding as the most likely mechanism by which Notch effector proteins delay exocrine pancreatic differentiation.