PTF1 is a trimeric transcription factor essential to the development of the pancreas and to the maintenance of the differentiated state of the adult exocrine pancreas. It comprises a dimer of P48/PTF1a (a pancreas and neural restricted basic helix-loop-helix [bHLH] protein) and a class A bHLH protein, together with a third protein that we show can be either the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L. In mature acinar cells, PTF1 exclusively contains the RBP-L isoform and is bound to the promoters of acinar specific genes. P48 interacts with the RBP subunit primarily through two short conserved tryptophan-containing motifs, similar to the motif of the Notch intracellular domain (NotchIC) that interacts with RBP-J. The transcriptional activities of the J and L forms of PTF1 are independent of Notch signaling, because P48 occupies the NotchIC docking site on RBP-J and RBP-L does not bind the NotchIC. Mutations that delete one or both of the RBP-interacting motifs of P48 eliminate RBP-binding and are associated with a human genetic disorder characterized by pancreatic and cerebellar agenesis, which indicates that the association of P48 and RBPs is required for proper embryonic development. The presence of related peptide motifs in other transcription factors indicates a broader Notch-independent function for RBPJ/SU(H).

To investigate the role of the HOX-like homeoprotein PDX1 in the formation and maintenance of the pancreas, we have genetically engineered mice so that the only source of PDX1 is a transgene that can be controlled by the application of tetracycline or its analogue doxycycline. In these mice the coding region for the tetracycline-regulated transactivator (tTA _off ) has replaced the coding region of the endogenous Pdx1 gene to ensure correct temporal and spatial expression of the regulatable transactivator. In the absence of doxycycline, tTA _off activates the transcription of a bicistronic transgene encoding PDX1 and an enhanced green fluorescent protein reporter, which acts as a visual marker of transgene expression in living cells. Expression of the transgene-encoded PDX1 rescues the Pdx1 -null phenotype; the pancreata of these mice develop and function normally. The rescue is conditional; doxycycline-mediated repression of the transgenic Pdx1 throughout gestation recapitulates the Pdx1 null phenotype. Moreover, application of doxycycline at mid-pancreogenesis blocks further development. Adult animals of the rescue genotype that were treated with doxycycline for 3 weeks shut off Pdx1 expression, decreased insulin production, and lost the ability to maintain glucose homeostasis. These results demonstrate the feasibility of controlling the formation of an organ during embryogenesis in utero and the maintenance of the mature organ through the experimental manipulation of a key developmental regulator.

PTF1a is an unusual basic helix–loop–helix (bHLH) transcription factor that is required for the development of the pancreas. We show that early in pancreatic development, active PTF1a requires interaction with RBPJ, the vertebrate Suppressor of Hairless, within a stable trimeric DNA-binding complex (PTF1). Later, as acinar cell development begins, RBPJ is swapped for RBPJL, the constitutively active, pancreas-restricted paralog of RBPJ. Moreover, the Rbpjl gene is a direct target of the PTF1 complex: At the onset of acinar cell development when the Rbpjl gene is first induced, a PTF1 complex containing RBPJ is bound to the Rbpjl promoter. As development proceeds, RBPJL gradually replaces RBPJ in the PTF1 complex bound to Rbpjl and appears on the binding sites for the complex in the promoters of other acinar-specific genes, including those for the secretory digestive enzymes. A single amino acid change in PTF1a that eliminates its ability to bind RBPJ (but does not affect its binding to RBPJL) causes pancreatic development to truncate at an immature stage, without the formation of acini or islets. These results indicate that the interaction between PTF1a and RBPJ is required for the early stage of pancreatic growth, morphogenesis, and lineage fate decisions. The defects in pancreatic development phenocopy those of Ptf1a-null embryos; thus, the first critical function of PTF1a is in the context of the PTF1 complex containing RBPJ. Action within an organ-specific transcription factor is a previously unknown function for RBPJ and is independent of its role in Notch signaling.

Background & Aims The mature pancreatic acinar cell is dedicated to the production of very large amounts of digestive enzymes. The early stages of pancreatic development require the Rbpj form of the trimeric Pancreas Transcription Factor 1 complex (PTF1-J). As acinar development commences, Rbpjl gradually replaces Rbpj; in the mature pancreas, PTF1 contains Rbpjl (PTF1-L). We investigated whether PTF1-L controls the expression of genes that complete the final stage of acinar differentiation. Methods We analyzed acinar development and transcription in mice with disrupted Rbpjl (Rbpjl_ko/ko mice). We performed comprehensive analyses of the messenger RNA population and PTF1 target genes in pancreatic acinar cells from these and wild-type mice. Results In Rbpjl_ko/ko mice, acinar differentiation was incomplete and characterized by decreased expression (as much as 99%) of genes that encode digestive enzymes or proteins of regulated exocytosis and mitochondrial metabolism. Whereas PTF1-L bound regulatory sites of genes in normal adult pancreatic cells, the embryonic form (PTF1-J) persisted in the absence of Rbpjl and replaced PTF1-L; the extent of replacement determined gene expression levels. Loss of PTF1-L reduced expression (>2-fold) of only about 50 genes, 90% of which were direct targets of PTF1-L. The magnitude of the effects on individual digestive enzyme genes correlated with the developmental timing of gene activation. Absence of Rbpjl increased pancreatic expression of liver-restricted messenger RNA. Conclusions Replacement of Rbpj by Rbpjl in the PTF1 complex drives acinar differentiation by maximizing secretory protein synthesis, stimulating mitochondrial metabolism and cytoplasmic creatine-phosphate energy stores, completing the packaging and secretory apparatus, and maintaining acinar-cell homeostasis.

The basic helix-loop-helix (bHLH) transcription factor PTF1a is critical to the development of the embryonic pancreas. It is required early for the formation of the undifferentiated tubular epithelium of the nascent pancreatic rudiment and then becomes restricted to the differentiating acinar cells, where it directs the transcriptional activation of the secretory digestive enzyme genes. Here we report that the complex temporal and spatial expression of Ptf1a is controlled by at least three separable gene-flanking regions. A 14.8-kb control domain immediately downstream of the last Ptf1a exon is highly conserved among mammals and directs expression in the dorsal part of the spinal cord but has very little activity in the embryonic or neonatal pancreas. A 13.4-kb proximal promoter domain initiates limited expression in cells that begin the acinar differentiation program. The activity of the proximal promoter domain is complemented by an adjacent 2.3-kb autoregulatory enhancer that is able to activate a heterologous minimal promoter with high-level penetrance in the pancreases of transgenic mice. During embryonic development, the enhancer initiates expression in the early precursor epithelium and then superinduces expression in acinar cells at the onset of their development. The enhancer contains two evolutionarily conserved binding sites for the active form of PTF1a, a trimeric complex composed of PTF1a, one of the common bHLH E proteins, and either RBPJ or RBPJL. The two sites are essential for acinar cell-specific transcription in transfected cell lines and mice. In mature acinar cells, the enhancer and PTF1a establish an autoregulatory loop that reinforces and maintains Ptf1a expression. Indeed, the trimeric PTF1 complex forms dual autoregulatory loops with the Ptf1a and Rbpjl genes that may maintain the stable phenotype of pancreatic acinar cells.

The orphan nuclear receptor NR5A2 is necessary for the stem-like properties of the epiblast of the pre-gastrulation embryo and for cellular and physiological homeostasis of endoderm-derived organs postnatally. Using conditional gene inactivation, we show that Nr5a2 also plays crucial regulatory roles during organogenesis. During the formation of the pancreas, Nr5a2 is necessary for the expansion of the nascent pancreatic epithelium, for the subsequent formation of the multipotent progenitor cell (MPC) population that gives rise to pre-acinar cells and bipotent cells with ductal and islet endocrine potential, and for the formation and differentiation of acinar cells. At birth, the NR5A2-deficient pancreas has defects in all three epithelial tissues: a partial loss of endocrine cells, a disrupted ductal tree and a >90% deficit of acini. The acinar defects are due to a combination of fewer MPCs, deficient allocation of those MPCs to pre-acinar fate, disruption of acinar morphogenesis and incomplete acinar cell differentiation. NR5A2 controls these developmental processes directly as well as through regulatory interactions with other pancreatic transcriptional regulators, including PTF1A, MYC, GATA4, FOXA2, RBPJL and MIST1 (BHLHA15). In particular, Nr5a2 and Ptf1a establish mutually reinforcing regulatory interactions and collaborate to control developmentally regulated pancreatic genes by binding to shared transcriptional regulatory regions. At the final stage of acinar cell development, the absence of NR5A2 affects the expression of Ptf1a and its acinar specific partner Rbpjl, so that the few acinar cells that form do not complete differentiation. Nr5a2 controls several temporally distinct stages of pancreatic development that involve regulatory mechanisms relevant to pancreatic oncogenesis and the maintenance of the exocrine phenotype.