Expression-Based Cell Lineage Analysis in Drosophila Through a Course-Based Research Experience for Early Undergraduates.

A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.

The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline.

Removal of cytosine methylation from the genome is critical for reprogramming and transdifferentiation and plays a central role in our understanding of the fundamental principles of embryo lineage development. One of the major models for studying cytosine demethylation is the mammalian germ line during the primordial germ cell (PGC) stage of embryo development. It is now understood that oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is required to remove cytosine methylation in a locus-specific manner in PGCs; however, the mechanisms downstream of 5hmC are controversial and hypothesized to involve either active demethylation or replication-coupled loss. In the current study, we used the aorta-gonad-mesonephros (AGM) organ culture model to show that this model recapitulates germ line reprogramming, including 5hmC reorganization and loss of cytosine methylation from Snrpn and H19 imprinting control centers (ICCs). To directly address the hypothesis that cell proliferation is required for cytosine demethylation, we blocked PI3-kinase-dependent PGC proliferation and show that this leads to a G1 and G2/M cell cycle arrest in PGCs, together with retained levels of cytosine methylation at the Snrpn ICC, but not at the H19 ICC. Taken together, the AGM organ culture model is an important tool to evaluate mechanisms of locus-specific demethylation and the role of PI3-kinase-dependent PGC proliferation in the locus-specific removal of cytosine methylation from the genome.

Cell intrinsic role of COX-2 in pancreatic cancer development.

COX-2 is upregulated in pancreatic ductal adenocarcinomas (PDAC). However, how COX-2 promotes PDAC development is unclear. While previous studies have evaluated the efficacy of COX-2 inhibition via the use of nonsteroidal anti-inflammatory drugs (NSAID) or the COX-2 inhibitor celecoxib in PDAC models, none have addressed the cell intrinsic versus microenvironment roles of COX-2 in modulating PDAC initiation and progression. We tested the cell intrinsic role of COX-2 in PDAC progression using both loss-of-function and gain-of-function approaches. Cox-2 deletion in Pdx1+ pancreatic progenitor cells significantly delays the development of PDAC in mice with K-ras activation and Pten haploinsufficiency. Conversely, COX-2 overexpression promotes early onset and progression of PDAC in the K-ras mouse model. Loss of PTEN function is a critical factor in determining lethal PDAC onset and overall survival. Mechanistically, COX-2 overexpression increases p-AKT levels in the precursor lesions of Pdx1(+); K-ras(G12D)(/+); Pten(lox)(/+) mice in the absence of Pten LOH. In contrast, Cox-2 deletion in the same setting diminishes p-AKT levels and delays cancer progression. These data suggest an important cell intrinsic role for COX-2 in tumor initiation and progression through activation of the PI3K/AKT pathway. PDAC that is independent of intrinsic COX-2 expression eventually develops with decreased FKBP5 and increased GRP78 expression, two alternate pathways leading to AKT activation. Together, these results support a cell intrinsic role for COX-2 in PDAC development and suggest that while anti-COX-2 therapy may delay the development and progression of PDAC, mechanisms known to increase chemoresistance through AKT activation must also be overcome.

PTEN loss accelerates KrasG12D-induced pancreatic cancer development.

KRAS mutations are found in ∼90% of human pancreatic ductal adenocarcinomas (PDAC). However, mice genetically engineered to express Kras(G12D) from its endogenous locus develop PDACs only after a prolonged latency, indicating that other genetic events or pathway alterations are necessary for PDAC progression. The PTEN-controlled phosphatidylinositol 3-kinase (PI3K)/AKT signaling axis is dysregulated in later stages of PDAC. To better elucidate the role of PTEN/PI3K/AKT signaling in Kras(G12D)-induced PDAC development, we crossed Pten conditional knockout mice (Pten(lox/lox)) to mice with conditional activation of Kras(G12D). The resulting compound heterozygous mutant mice showed significantly accelerated development of acinar-to-ductal metaplasia (ADM), malignant pancreatic intraepithelial neoplasia (mPanIN), and PDAC within a year. Moreover, all mice with Kras(G12D) activation and Pten homozygous deletion succumbed to cancer by 3 weeks of age. Our data support a dosage-dependent role for PTEN, and the resulting dysregulation of the PI3K/AKT signaling axis, in both PDAC initiation and progression, and shed additional light on the signaling mechanisms that lead to the development of ADM and subsequent mPanIN and pancreatic cancer.