IntroductionStudies of genes associated with retrovirus-induced neoplasia form the basis of much of our present knowledge of oncogenes, and have contributed to our understanding of both gene function and the neoplastic process. 1 Friend murine leukemia virus (F-MuLV) does not contain any oncogenic sequences. Instead it induces tumors by activating or inactivating oncogenes or tumor suppressor genes, respectively, through a process known as retroviral insertional mutagenesis. 2 Previous studies in our laboratory and others have demonstrated that the pivotal genetic event associated with erythroleukemia initiation is the insertional activation of the Ets-related gene Fli-1. [3][4][5] Progression toward more malignant stages has been shown to be associated with insertional inactivation of either p53 or p45 NFE2. [6][7][8][9][10] To further investigate the role of p53 in F-MuLV-induced erythroleukemia, p53-deficient mice were infected with F-MuLV. While the loss of p53 in primary erythroleukemias accelerated tumor initiation and immortalization, 11 10% of these tumors did not acquire an insertional activation of the Fli-1 locus, and the cell lines established from these erythroleukemias did not express endogenous Fli-1. The transformation of these cells is attributable to insertional activation or inactivation of another gene, since F-MuLV-induced erythroleukemia usually requires Fli-1 activation for erythroid transformation. 3 Therefore, the investigation of a novel site of proviral integration is necessary to uncover the mechanism of transformation in the Fli-1-negative erythroleukemias. This study describes the identification of a novel integration site, designated Friend murine leukemia integration site 3 (Fli-3), containing sequences identical to the miRNA gene family.miRNAs are small, non-protein-encoding RNAs that play important roles in a variety of biologic processes. They act by binding to complementary sequences in the 3Ј untranslated region (UTR) of the mRNA of their target gene. This binding process reduces the stability of these mRNAs, thereby altering the protein expression. 12,13 Some miRNA genes exist in clusters that can be expressed as single transcriptional polycistron. 14 Emerging evidence suggests the potential involvement of altered regulation of miRNA in the pathogenesis of human cancers. [15][16][17][18][19][20] Fli-3 is a murine homologue of the human C13orf25 gene, a newly identified target gene for 13q31-q32 chromosomal amplification in B-cell-type lymphomas. 21 C13orf25 contains sequences corresponding to the mir-17-92 polycistron. He et al 22 have recently reported that mir-17-92 is highly expressed in B-cell lymphomas and its expression in c-myc-overexpressing transgenic mice accelerated tumorigenicity by an as-yet-undefined process. Hayashita et al 18 also showed that mir-17-92 is overexpressed in human lung cancer and enhances cell proliferation. Interestingly, the Fli-3 transcript contains the identical mir-17-92 sequence, suggesting that this murine homologue may play a similar rol...

Loss of Heterozygosity (LOH) typically refers to a phenomenon in which diploid cells that are heterozygous for a mutant allele lose their wild type allele through mutations. LOH is implicated in oncogenesis when it affects the remaining wild type copy of a tumor suppressor. Drosophila has been a useful model to identify genes that regulate the incidence of LOH, but most of these studies use adult phenotypic markers such as multiple wing hair ( mwh ). Here, we describe a cell-autonomous fluorescence-based system that relies on the QF/QS transcriptional module to detect LOH, which may be used in larval, pupal and adult stages and in conjunction with the GAL4/UAS system. Using the QF/QS system, we were able to detect the induction of cells with LOH by X-rays in a dose-dependent manner in the larval wing discs, and to monitor their fate through subsequent development in pupa and adult stages. We tested the genetic requirement for changes in LOH, using both classical mutants and GAL4/UAS-mediated RNAi. Our results identify two distinct culling phases that eliminate cells with LOH, one in late larval stages and another in the pupa. The two culling phases are genetically separable, showing differential requirement for pro-apoptotic genes of the H99 locus and transcription factor Srp. A direct comparison of mwh LOH and QF/QS LOH suggests that cells with different LOH events are distinguished from each other in a p53-dependent manner and are retained to different degrees in the final adult structure. These studies reveal previously unknown mechanisms for the elimination of cells with chromosome aberrations.

Ionizing radiation (IR) is used to treat half of all cancer patients because of its ability to kill cells. IR, however, can induce stem cell-like properties in non-stem cancer cells, potentiating tumor regrowth and reduced therapeutic success. We identified previously a subpopulation of cells in Drosophila larval wing discs that exhibit IR-induced stem cell-like properties. These cells reside in the future wing hinge, are resistant to IR-induced apoptosis, and are capable of translocating, changing fate, and participating in regenerating the pouch that suffers more IR-induced apoptosis. We used here a combination of lineage tracing, FACS-sorting of cells that change fate, genome-wide RNAseq, and functional testing of 42 genes, to identify two key changes that are required cell-autonomously for IR-induced hinge-to-pouch fate change: (1) repression of hinge determinants Wg (Drosophila Wnt1) and conserved zinc-finger transcription factor Zfh2 and (2) upregulation of three ribosome biogenesis factors. Additional data indicate a role for Myc, a transcriptional activator of ribosome biogenesis genes, in the process. These results provide a molecular understanding of IR-induced cell fate plasticity that may be leveraged to improve radiation therapy.

No abstract

The neuromuscular junction (NMJ) of larval Drosophila is widely used as a genetic model for basic neuroscience research. The presynaptic side of the NMJ is formed by axon terminals of motor neurons, the soma of which reside in the ventral ganglion of the central nervous system (CNS). Here we describe a streamlined protocol for dissection and immunostaining of the Drosophila CNS and NMJ that allows processing of multiple genotypes within a single staining tube. We also present a computer script called Automated Image Analysis with Background Subtraction which facilitates identification of motor nuclei, quantification of pixel intensity, and background subtraction. Together, these techniques provide a pipeline for neuroscientists to compare levels of different biomolecules in motor nuclei. We conclude that these methods should be adaptable to a variety of different cell and tissue types for the improvement of efficiency, reproducibility, and throughput during data quantification.