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.

Molecular mechanisms underlying phenotypic degeneration in Cordyceps militaris: insights from transcriptome reanalysis and osmotic stress studies.

Phenotypic degeneration in Cordyceps militaris poses a significant concern for producers, yet the mechanisms underlying this phenomenon remain elusive. To address this concern, we isolated two strains that differ in their abilities to form fruiting bodies. Our observations revealed that the degenerated strain lost the capacity to develop fruiting bodies, exhibited limited radial expansion, increased spore density, and elevated intracellular glycerol levels. Transcriptome reanalysis uncovered dysregulation of genes involved in the MAPK signaling pathway in the degenerate strain. Our RT-qPCR results demonstrated reduced expression of sexual development genes, along with upregulation of genes involved in asexual sporulation, glycerol synthesis, and MAPK regulation, when compared to the wild-type strain. Additionally, we discovered that osmotic stress reduced radial growth but increased conidia sporulation and glycerol accumulation in all strains. Furthermore, hyperosmotic stress inhibited fruiting body formation in all neutralized strains. These findings indicate dysregulation of the MAPK signaling pathway, the possibility of the activation of the high-osmolarity glycerol and spore formation modules, as well as the downregulation of the pheromone response and filamentous growth cascades in the degenerate strain. Overall, our study sheds light on the mechanisms underlying Cordyceps militaris degeneration and identifies potential targets for improving cultivation practices.

Key transcriptional effectors of the pancreatic acinar phenotype and oncogenic transformation.

Proper maintenance of mature cellular phenotypes is essential for stable physiology, suppression of disease states, and resistance to oncogenic transformation. We describe the transcriptional regulatory roles of four key DNA-binding transcription factors (Ptf1a, Nr5a2, Foxa2 and Gata4) that sit at the top of a regulatory hierarchy controlling all aspects of a highly differentiated cell-type-the mature pancreatic acinar cell (PAC). Selective inactivation of Ptf1a, Nr5a2, Foxa2 and Gata4 individually in mouse adult PACs rapidly altered the transcriptome and differentiation status of PACs. The changes most emphatically included transcription of the genes for the secretory digestive enzymes (which conscript more than 90% of acinar cell protein synthesis), a potent anabolic metabolism that provides the energy and materials for protein synthesis, suppressed and properly balanced cellular replication, and susceptibility to transformation by oncogenic KrasG12D. The simultaneous inactivation of Foxa2 and Gata4 caused a greater-than-additive disruption of gene expression and uncovered their collaboration to maintain Ptf1a expression and control PAC replication. A measure of PAC dedifferentiation ranked the effects of the conditional knockouts as Foxa2+Gata4 > Ptf1a > Nr5a2 > Foxa2 > Gata4. Whereas the loss of Ptf1a or Nr5a2 greatly accelerated Kras-mediated transformation of mature acinar cells in vivo, the absence of Foxa2, Gata4, or Foxa2+Gata4 together blocked transformation completely, despite extensive dedifferentiation. A lack of correlation between PAC dedifferentiation and sensitivity to oncogenic KrasG12D negates the simple proposition that the level of differentiation determines acinar cell resistance to transformation.

Drosophila CtBP regulates proliferation and differentiation of eye precursors and complexes with Eyeless, Dachshund, Dan, and Danr during eye and antennal development.

Specification factors regulate cell fate in part by interacting with transcriptional co-regulators like CtBP to regulate gene expression. Here, we demonstrate that CtBP forms a complex or complexes with the Drosophila melanogaster Pax6 homolog Eyeless (Ey), and with Distal antenna (Dan), Distal antenna related (Danr), and Dachshund to promote eye and antennal specification. Phenotypic analysis together with molecular data indicate that CtBP interacts with Ey to prevent overproliferation of eye precursors. In contrast, CtBP,dan,danr triple mutant adult eyes have significantly fewer ommatidia than CtBP single or dan,danr double mutants, suggesting that the CtBP/Dan/Danr complex functions to recruit ommatidia from the eye precursor pool. Furthermore, CtBP single and to a greater extent CtBP,dan,danr triple mutants affect the establishment and maintenance of the R8 precursor, which is the founding ommatidial cell. Thus, CtBP interacts with different eye specification factors to regulate gene expression appropriate for proliferative vs. differentiative stages of eye development.

MIST1 and PTF1 Collaborate in Feed-Forward Regulatory Loops That Maintain the Pancreatic Acinar Phenotype in Adult Mice.

Much remains unknown regarding the regulatory networks formed by transcription factors in mature, differentiated mammalian cells in vivo, despite many studies of individual DNA-binding transcription factors. We report a constellation of feed-forward loops formed by the pancreatic transcription factors MIST1 and PTF1 that govern the differentiated phenotype of the adult pancreatic acinar cell. PTF1 is an atypical basic helix-loop-helix transcription factor complex of pancreatic acinar cells and is critical to acinar cell fate specification and differentiation. MIST1, also a basic helix-loop-helix transcription factor, enhances the formation and maintenance of the specialized phenotype of professional secretory cells. The MIST1 and PTF1 collaboration controls a wide range of specialized cellular processes, including secretory protein synthesis and processing, exocytosis, and homeostasis of the endoplasmic reticulum. PTF1 drives Mist1 transcription, and MIST1 and PTF1 bind and drive the transcription of over 100 downstream acinar genes. PTF1 binds two canonical bipartite sites within a 0.7-kb transcriptional enhancer upstream of Mist1 that are essential for the activity of the enhancer in vivo MIST1 and PTF1 coregulate target genes synergistically or additively, depending on the target transcriptional enhancer. The frequent close binding proximity of PTF1 and MIST1 in pancreatic acinar cell chromatin implies extensive collaboration although the collaboration is not dependent on a stable physical interaction.

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

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.

MIST1 Links Secretion and Stress as both Target and Regulator of the Unfolded Protein Response.

Transcriptional networks that govern secretory cell specialization, including instructing cells to develop a unique cytoarchitecture, amass extensive protein synthesis machinery, and be embodied to respond to endoplasmic reticulum (ER) stress, remain largely uncharacterized. In this study, we discovered that the secretory cell transcription factor MIST1 (Bhlha15), previously shown to be essential for cytoskeletal organization and secretory activity, also functions as a potent ER stress-inducible transcriptional regulator. Genome-wide DNA binding studies, coupled with genetic mouse models, revealed MIST1 gene targets that function along the entire breadth of the protein synthesis, processing, transport, and exocytosis networks. Additionally, key MIST1 targets are essential for alleviating ER stress in these highly specialized cells. Indeed, MIST1 functions as a coregulator of the unfolded protein response (UPR) master transcription factor XBP1 for a portion of target genes that contain adjacent MIST1 and XBP1 binding sites. Interestingly, Mist1 gene expression is induced during ER stress by XBP1, but as ER stress subsides, MIST1 serves as a feedback inhibitor, directly binding the Xbp1 promoter and repressing Xbp1 transcript production. Together, our findings provide a new paradigm for XBP1-dependent UPR regulation and position MIST1 as a potential biotherapeutic for numerous human diseases.

The acinar differentiation determinant PTF1A inhibits initiation of pancreatic ductal adenocarcinoma.

Understanding the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) may provide therapeutic strategies for this deadly disease. Recently, we and others made the surprising finding that PDAC and its preinvasive precursors, pancreatic intraepithelial neoplasia (PanIN), arise via reprogramming of mature acinar cells. We therefore hypothesized that the master regulator of acinar differentiation, PTF1A, could play a central role in suppressing PDAC initiation. In this study, we demonstrate that PTF1A expression is lost in both mouse and human PanINs, and that this downregulation is functionally imperative in mice for acinar reprogramming by oncogenic KRAS. Loss of Ptf1a alone is sufficient to induce acinar-to-ductal metaplasia, potentiate inflammation, and induce a KRAS-permissive, PDAC-like gene expression profile. As a result, Ptf1a-deficient acinar cells are dramatically sensitized to KRAS transformation, and reduced Ptf1a greatly accelerates development of invasive PDAC. Together, these data indicate that cell differentiation regulators constitute a new tumor suppressive mechanism in the pancreas.

Ectopic Noggin in a Population of Nfatc1 Lineage Endocardial Progenitors Induces Embryonic Lethality.

The initial heart is composed of a myocardial tube lined by endocardial cells. The TGFß superfamily is known to play an important role, as BMPs from the myocardium signal to the overlying endocardium to create an environment for EMT. Subsequently, BMP and TGFß signaling pathways synergize to form primitive valves and regulate myocardial growth. In this study, we investigated the requirement of BMP activity by transgenic over-expression of extracellular BMP antagonist Noggin. Using Nfatc1Cre to drive lineage-restricted Noggin within the endocardium, we show that ectopic Noggin arrests cardiac development in E10.5-11 embryos, resulting in small hearts which beat poorly and die by E12.5. This is coupled with hypoplastic endocardial cushions, reduced trabeculation and fewer mature contractile fibrils in mutant hearts. Moreover, Nfatc1Cre -mediated diphtheria toxin fragment-A expression in the endocardium resulted in genetic ablation and a more severe phenotype with lethality at E11 and abnormal linear hearts. Molecular analysis demonstrated that endocardial Noggin resulted in a specific alteration of TGFß/BMP-mediated signal transduction, in that, both Endoglin and ALK1 were downregulated in mutant endocardium. Combined, these results demonstrate the cell-autonomous requirement of the endocardial lineage and function of unaltered BMP levels in facilitating endothelium-cardiomyocyte cross-talk and promoting endocardial cushion formation.