Michael A. Hale

Experimental control of pancreatic development and maintenance

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 (tTAoff) 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, tTAoff 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.

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.

Transcriptional Autoregulation Controls Pancreatic Ptf1a Expression during Development and Adulthood

ABSTRACT 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 nuclear hormone receptor family member NR5A2 controls aspects of multipotent progenitor cell formation and acinar differentiation during pancreatic organogenesis

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.

Transcriptional Maintenance of Pancreatic Acinar Identity, Differentiation, and Homeostasis by PTF1A.

ABSTRACT Maintenance of cell type identity is crucial for health, yet little is known of the regulation that sustains the long-term stability of differentiated phenotypes. To investigate the roles that key transcriptional regulators play in adult differentiated cells, we examined the effects of depletion of the developmental master regulator PTF1A on the specialized phenotype of the adult pancreatic acinar cell in vivo. Transcriptome sequencing and chromatin immunoprecipitation sequencing results showed that PTF1A maintains the expression of genes for all cellular processes dedicated to the production of the secretory digestive enzymes, a highly attuned surveillance of unfolded proteins, and a heightened unfolded protein response (UPR). Control by PTF1A is direct on target genes and indirect through a ten-member transcription factor network. Depletion of PTF1A causes an imbalance that overwhelms the UPR, induces cellular injury, and provokes acinar metaplasia. Compromised cellular identity occurs by derepression of characteristic stomach genes, some of which are also associated with pancreatic ductal cells. The loss of acinar cell homeostasis, differentiation, and identity is directly relevant to the pathologies of pancreatitis and pancreatic adenocarcinoma.

Dominant and context-specific control of endodermal organ allocation by Ptf1a

The timing and gene regulatory logic of organ-fate commitment from within the posterior foregut of the mammalian endoderm is largely unexplored. Transient misexpression of a presumed pancreatic-commitment transcription factor, Ptf1a, in embryonic mouse endoderm (Ptf1aEDD) dramatically expanded the pancreatic gene regulatory network within the foregut. Ptf1aEDD temporarily suppressed Sox2 broadly over the anterior endoderm. Pancreas-proximal organ territories underwent full tissue conversion. Early-stage Ptf1aEDD rapidly expanded the endogenous endodermal Pdx1-positive domain and recruited other pancreas-fate-instructive genes, thereby spatially enlarging the potential for pancreatic multipotency. Early Ptf1aEDD converted essentially the entire glandular stomach, rostral duodenum and extrahepatic biliary system to pancreas, with formation of many endocrine cell clusters of the type found in normal islets of Langerhans. Sliding the Ptf1aEDD expression window through embryogenesis revealed differential temporal competencies for stomach-pancreas respecification. The response to later-stage Ptf1aEDD changed radically towards unipotent, acinar-restricted conversion. We provide strong evidence, beyond previous Ptf1a inactivation or misexpression experiments in frog embryos, for spatiotemporally context-dependent activity of Ptf1a as a potent gain-of-function trigger of pro-pancreatic commitment.

A rapid in vivo screen for pancreatic ductal adenocarcinoma therapeutics

ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related deaths in the United States, and is projected to be second by 2025. It has the worst survival rate among all major cancers. Two pressing needs for extending life expectancy of affected individuals are the development of new approaches to identify improved therapeutics, addressed herein, and the identification of early markers. PDA advances through a complex series of intercellular and physiological interactions that drive cancer progression in response to organ stress, organ failure, malnutrition, and infiltrating immune and stromal cells. Candidate drugs identified in organ culture or cell-based screens must be validated in preclinical models such as KIC (p48Cre;LSL-KrasG12D;Cdkn2af/f) mice, a genetically engineered model of PDA in which large aggressive tumors develop by 4 weeks of age. We report a rapid, systematic and robust in vivo screen for effective drug combinations to treat Kras-dependent PDA. Kras mutations occur early in tumor progression in over 90% of human PDA cases. Protein kinase and G-protein coupled receptor (GPCR) signaling activates Kras. Regulators of G-protein signaling (RGS) proteins are coincidence detectors that can be induced by multiple inputs to feedback-regulate GPCR signaling. We crossed Rgs16::GFP bacterial artificial chromosome (BAC) transgenic mice with KIC mice and show that the Rgs16::GFP transgene is a KrasG12D-dependent marker of all stages of PDA, and increases proportionally to tumor burden in KIC mice. RNA sequencing (RNA-Seq) analysis of cultured primary PDA cells reveals characteristics of embryonic progenitors of pancreatic ducts and endocrine cells, and extraordinarily high expression of the receptor tyrosine kinase Axl, an emerging cancer drug target. In proof-of-principle drug screens, we find that weanling KIC mice with PDA treated for 2 weeks with gemcitabine (with or without Abraxane) plus inhibitors of Axl signaling (warfarin and BGB324) have fewer tumor initiation sites and reduced tumor size compared with the standard-of-care treatment. Rgs16::GFP is therefore an in vivo reporter of PDA progression and sensitivity to new chemotherapeutic drug regimens such as Axl-targeted agents. This screening strategy can potentially be applied to identify improved therapeutics for other cancers. Editors choice - Drug Discovery Collection: The study describes a rapid in vivo screen for novel strategies to treat pancreatic ductal adenocarcinoma (PDA).