Design and Development of a Cell Marking System in Transgenic Mice

The aim of this project was to develop a marker system to analyse cell lineage in mouse skin. This should provide useful information on the usual size and organisation of clones in the epidermis and give some clues as to how cell replacement is normally controlled in the epidermis and to the possible location of the stem and progenitor cells. As there is no marker gene currently available in the murine epidermis a marker gene had to be designed and introduced. This was accomplished by generating transgenic mice with a novel marker gene inserted into their DNA. An Escherichia coli lac Z gene was used as a cell marker and activated in single cells following treating the mice with a chemical mutagen. The marker could be activated by a point mutation in the sequence of an oligonucleotide inserted at the 5' end of the lac Z gene, replacing the ATG initiation codon of the gene. Two different marking strategies were devised. One specific for the chemical mutagen N-nitro-N'-methyl-N-nitosoguanidine (MNNG) and the other for the chemical mutagen acetoxyaminofluorene (AAF). Oligonucleotides were designed, synthesised and cloned in front of the lac Z gene to generate oligollac Z fusion marker genes. Test constructs without initiation codons and control constructs with initiation codons were designed and cloned at the same time for both the MNNG and AAF strategies. The fusion genes were then tested in vitro by transfection in to murine epidermal cell lines. No expression was detected in the inactive marker gene designed to be activated by MNNG, whereas high expression was obtained from two control constructs which both contained and in-frame ATG initiation codon in the sequence of the oligonucleotide cloned into the lac Z gene. The MNNG-responsive marker gene was shown to be activated in stably transfected murine epidermal cell lines in response to treatment with MNNG at a frequency of approximately 4.3 x 10-3 per clonogenic cell. Subsequently the work with the AAF strategy was discontinued as there was insufficient time to pursue both strategies and positive results had been obtained with the MNNG construct. Twenty-two transgenic founders were generated with three marker gene constructs (Act-LacZA, K5-LacZD and K5-LacZE by pronuclear microinjection of fertilised embryos. Lines of transgenic mice were bred from the founders and the expression of the marker genes were analysed. Expression from a control lac Z fusion gene driven by a BKIII (keratin 5) promoter was detected by staining skin tissue with the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal). The expression of the beta-galactosidase protein was observed in the epidermal basal layer and the hair follicles of transgenic mice. The expression was highest in newborn mice and gradually diminished as the mice reached adulthood. It was subsequently shown that targeting the expression of an activated H-ras oncogene to the epidermis with the same promoter results in a high frequency of malignant tumours. The implications of these results to the biology of the skin are discussed. Expression of the MNNG-activated test marker gene under the control of a beta-actin promoter was also detected. Activation of the marker gene was detected in serial sections of murine brain tissue from transgenic mice treated topically with MNNG at birth. No activation was detected in other tissues probably due to the low level of expression of the marker gene. The explanation of these results and the implications for developing a marker system to work efficiently in the epidermis are discussed

(2,0) theory on circle fibrations

We consider (2,0) theory on a manifold M_6 that is a fibration of a spatial S^1 over some five-dimensional base manifold M_5. Initially, we study the free (2,0) tensor multiplet which can be described in terms of classical equations of motion in six dimensions. Given a metric on M_6 the low energy effective theory obtained through dimensional reduction on the circle is a Maxwell theory on M_5. The parameters describing the local geometry of the fibration are interpreted respectively as the metric on M_5, a non-dynamical U(1) gauge field and the coupling strength of the resulting low energy Maxwell theory. We derive the general form of the action of the Maxwell theory by integrating the reduced equations of motion, and consider the symmetries of this theory originating from the superconformal symmetry in six dimensions. Subsequently, we consider a non-abelian generalization of the Maxwell theory on M_5. Completing the theory with Yukawa and phi^4 terms, and suitably modifying the supersymmetry transformations, we obtain a supersymmetric Yang-Mills theory which includes terms related to the geometry of the fibration.Comment: 24 pages, v2 References added, typos correcte

Development of an inducible mouse model of iRFP713 to track recombinase activity and tumour development in vivo

While the use of bioluminescent proteins for molecular imaging is a powerful technology to further our understanding of complex processes, fluorescent labeling with visible light fluorescent proteins such as GFP and RFP suffers from poor tissue penetration and high background autofluorescence. To overcome these limitations, we generated an inducible knock-in mouse model of iRFP713. This model was used to assess Cre activity in a Rosa Cre-ER background and quantify Cre activity upon different tamoxifen treatments in several organs. We also show that iRFP can be readily detected in 3D organoid cultures, FACS analysis and in vivo tumour models. Taken together we demonstrate that iRFP713 is a progressive step in in vivo imaging and analysis that widens the optical imaging window to the near-infrared spectrum, thereby allowing deeper tissue penetration, quicker image acquisition without the need to inject substrates and a better signal to background ratio in genetically engineered mouse models (GEMMs)

The initiator methionine tRNA drives cell migration and invasion leading to increased metastatic potential in melanoma

The cell's repertoire of transfer RNAs (tRNAs) has been linked to cancer. Recently, levels of the initiator methionine tRNA (tRNAiMet) in stromal fibroblasts have been shown to influence extracellular matrix (ECM) secretion to drive tumour growth and angiogenesis. Here we show that increased tRNAiMet within cancer cells does not influence tumour growth, but drives cell migration and invasion via a mechanism that is independent from ECM synthesis and dependent on α5β1 integrin and levels of the translation initiation ternary complex. In vivo and ex vivo migration (but not proliferation) of melanoblasts is significantly enhanced in transgenic mice which express additional copies of the tRNAiMet gene. We show that increased tRNAiMet in melanoma drives migratory, invasive behaviour and metastatic potential without affecting cell proliferation and primary tumour growth, and that expression of RNA polymerase III-associated genes (which drive tRNA expression) are elevated in metastases by comparison with primary tumours. Thus specific alterations to the cancer cell tRNA repertoire drive a migration/invasion programme that may lead to metastasis

The initiator methionine tRNA drives secretion of type II collagen from stromal fibroblasts to promote tumor growth and angiogenesis

Summary: Expression of the initiator methionine tRNA (tRNAi Met) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi Met expression levels influence tumor progression. We have found that tRNAi Met expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi Met in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi Met contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi Met gene (2+tRNAi Met mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi Met mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi Met mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi Met significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi Metoverexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl- 3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi Met-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi Met mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi Met levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis

Differential requirements for MDM2 E3 activity during embryogenesis and in adult mice

The p53 tumor suppressor protein is a potent activator of proliferative arrest and cell death. In normal cells, this pathway is restrained by p53 protein degradation mediated by the E3-ubiquitin ligase activity of MDM2. Oncogenic stress releases p53 from MDM2 control, so activating the p53 response. However, many tumors that retain wild-type p53 inappropriately maintain the MDM2-p53 regulatory loop in order to continuously suppress p53 activity. We have shown previously that single point mutations in the human MDM2 RING finger domain prevent the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2's E3 activity without preventing p53 binding. Here, we show that an analogous mouse MDM2 mutant (MDM2 I438K) restrains p53 sufficiently for normal growth but exhibits an enhanced stress response in vitro. In vivo, constitutive expression of MDM2 I438K leads to embryonic lethality that is rescued by p53 deletion, suggesting MDM2 I438K is not able to adequately control p53 function through development. However, the switch to I438K expression is tolerated in adult mice, sparing normal cells but allowing for an enhanced p53 response to DNA damage. Viewed as a proof of principle model for therapeutic development, our findings support an approach that would inhibit MDM2 E3 activity without preventing MDM2/p53 binding as a promising avenue for development of compounds to activate p53 in tumors with reduced on-target toxicities

Intravital FRAP imaging using an E-cadherin-GFP mouse reveals disease- and drug-dependent dynamic regulation of cell-cell junctions in live tissue

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments

Towards a tensionless string field theory for the N=(2,0) CFT in d=6

We describe progress in using the field theory of tensionless strings to arrive at a Lagrangian for the six-dimensional $\mathcal N=(2,0)$ conformal theory. We construct the free part of the theory and propose an ansatz for the cubic vertex in light-cone superspace. By requiring closure of the $(2,0)$ supersymmetry algebra, we fix the cubic vertex up to two parameters.Comment: 46 pages, 2 figures. V2: references added; minor changes and improvement

Lectures on Non-BPS Dirichlet branes

A comprehensive introduction to the boundary state approach to Dirichlet branes is given. Various examples of BPS and non-BPS Dirichlet branes are discussed. In particular, the non-BPS states in the duality of Type IIA on K3 and the heterotic string on T4 are analysed in detail.Comment: 46 pages, 5 figures, LaTeX; lectures given at the TMR network school on `Quantum aspects of gauge theories, supersymmetry and quantum gravity', Torino, 26 January - 2 February 2000, and at the `Spring workshop on Superstrings and related matters', Trieste, 27 March - 4 April 2000; references adde

Amplitudes for Multiple M5 Branes

We study N=(m,0) super-Poincare invariant six-dimensional massless and five-dimensional massive on-shell amplitudes. We demonstrate that in six dimensions, all possible three-point amplitudes involving tensor multiplets are necessarily embedded in gravitational theories. For non-gravitational amplitudes we consider instead five-dimensional massive amplitudes with N=(2,0) supersymmetry, aiming at describing the world volume theory of multiple M5 branes compactified on M^{4,1}x S^1. We find non-gravitational amplitudes whose on-shell degrees of freedom are shown to match those of the massive particle states that arise from self-dual strings wrapping a circle. Along the way we find interesting hints of a fermionic symmetry in the (2,0) theory, which accompanies the self-dual tensor gauge symmetry. We also discuss novel theories with (3,0) and (4,0) supersymmetry.Comment: 49 pages, 4 figures, v2: Better organization and more details in the section on KK interaction