Biomedical subjects

Ross Cagan

Publications and source records attributed to Ross Cagan.

7 recordsLinked to original sources

PubMed2006

Fast cloning inverted repeats for RNA interference.

Double-stranded RNA (dsRNA) can induce post-transcriptional gene silencing in a wide variety of organisms. Commonly, inverted repeats are used to produce dsRNA to silence genes of interest. However, cloning inverted repeats still remains a rate-limiting step for widely applying this technique. Here we describe a pGEM-T-based vector, pGEM-WIZ, designed to produce inverted repeats for any Drosophila gene. pGEM-WIZ has a high efficiency in assembling inverted repeats and the repeats in this vector are stable in regular Escherichia coli strains. Furthermore, we have developed a method for rapid selection of clones with an inverted repeat based on size and relative copy number of the vector with or without an insert. This method further eases the cloning process. The inverted repeat cassette assembled in pGEM-WIZ can be easily transferred to commonly available expression vectors suitable for stably expressing inverted repeats in vitro and in vivo.

Cloning, Molecular↗

PubMed2005

ZD6474 suppresses oncogenic RET isoforms in a Drosophila model for type 2 multiple endocrine neoplasia syndromes and papillary thyroid carcinoma.

Patients with hereditary medullary thyroid carcinoma (MTC) associated with multiple endocrine neoplasia (MEN) types 2A and 2B and familial MTC (FMTC) have mutations in the RET proto-oncogene. Approximately 40 percent of patients with papillary thyroid carcinoma (PTC) typically have either intrachromosomal or extrachromosomal rearrangements that join the promoter and NH(2)-terminal domains of unrelated genes to the COOH-terminal fragment of RET. The RET point mutations associated with MEN2A, MEN2B, or FMTC, or the chromosomal breakpoints and translocations associated with PTC, typically activate the RET receptor tyrosine kinase (RTK). RET kinase inhibitors are likely to be beneficial for patients with hereditary MTC, where currently there is no effective chemotherapy or radiation therapy. Recently, the low molecular weight tyrosine kinase inhibitor ZD6474 was found to block the enzymatic activity of RET-derived oncoproteins in cultured cell lines. We have developed a Drosophila model for MEN2A and MEN2B diseases by targeting oncogenic forms of RET to the developing Drosophila eye. Here we show that, when fed orally, ZD6474 suppressed RET-mediated phenotypes within the context of this in vivo model. Importantly, ZD6474 showed high efficacy and very low toxicity. This compound failed to significantly suppress an activated form of another RTK, the Drosophila epidermal growth factor receptor, nor did it suppress the activity of downstream components of the RET/Ras pathway. Our results support the view that targeting chemical kinase inhibitors such as ZD6474 to tissues with oncogenic forms of RET is a useful treatment strategy for RET-dependent carcinomas.

Animals↗

PubMed2005

Preferential adhesion mediated by Hibris and Roughest regulates morphogenesis and patterning in the Drosophila eye.

Cell adhesion is essential for morphogenesis; however, the mechanisms by which cell adhesion coordinates precisely regulated morphogenesis are poorly understood. Here we analyze the morphogenetic processes that organize the interommatidial precursor cells (IPCs) of the Drosophila pupal eye. We demonstrate that the Drosophila immunoglobulin superfamily members Hibris and Roughest are essential for IPC morphogenesis in the eye. The two loci are expressed in complementary cell types, and Hibris and Roughest proteins bind directly in vivo. Primary pigment cells employ Hibris to function as organizers in this process; IPCs minimize contacts with neighboring IPCs and utilize Roughest to maximize contacts with primaries. In addition, we provide evidence that interactions between Hibris and Roughest promote junction formation and that levels of Roughest in individual cells determine their capacity for competition. Our results demonstrate that preferential adhesion mediated by heterophilic interacting cell-adhesion molecules can create a precise pattern by minimizing surface free energy.

Animals↗

PubMed2004

klumpfuss regulates cell death in the Drosophila retina.

Programmed cell death (PCD) plays a central role in the sculpting and maturation of developing epithelia. In adult tissue, PCD plays a further role in the prevention of malignancy though removal of damaged cells. Here, we report that mutations in klumpfuss result in an excess of support cells during maturation of the developing Drosophila pupal retina. These ectopic cells are the result of a partial and specific failure of apoptotic death during normal cell fate selection. klumpfuss is required and differentially expressed in the cells that choose the life or death cell fate. We also provide genetic and biochemical evidence that klumpfuss regulates this process through down-regulation of the Epidermal Growth Factor Receptor/dRas1 signaling pathway. Based on its sequence Klumpfuss is an EGR-class nuclear factor, and our results suggest a mechanism by which mutations in EGR-class factors such as Wilms' Tumor Suppressor-1 may result in oncogenic events such as pediatric kidney tumors.

Animals↗

PubMed2004

A role for wingless in an early pupal cell death event that contributes to patterning the Drosophila eye.

Programmed cell death (PCD) is utilized in a wide variety of tissues to refine structure in developing tissues and organs. However, little is understood about the mechanisms that, within a developing epithelium, combine signals to selectively remove some cells while sparing essential neighbors. One popular system for studying this question is the developing Drosophila pupal retina, where excess interommatidial support cells are removed to refine the patterned ommatidial array. In this paper, we present data indicating that PCD occurs earlier within the pupal retina than previously demonstrated. As with later PCD, this death is dependent on Notch activity. Surprisingly, altering Drosophila Epidermal Growth Factor Receptor or Ras pathway activity had no effect on this death. Instead, our evidence indicates a role for Wingless signaling to provoke this cell death. Together, these signals regulate an intermediate step in the selective removal of unneeded interommatidial cells that is necessary for a precise retinal pattern.

Animals↗

PubMed2003

The signals that drive kidney development: a view from the fly eye.

PURPOSE OF REVIEW: Development of the mammalian kidney is a complex process involving numerous signals and signaling pathways. Other complex tissues have benefited enormously from studies in lower, simpler organisms. The present review provides an update on what we have learned from the fruitfly Drosophila melanogaster, and argues that Drosophila is an important but under-utilized organism for study of renal development. RECENT FINDINGS: The Malpighian tubules provide renal function to the fly. These require a number of signaling pathways for their development that are also seen in vertebrate kidney development, including the Notch, Ras, and Wnt signaling pathways, as well as nuclear factors such as Krüppel and Cut/Cux-1. Many of these factors are shared between early Malpighian tubule development and ureteric bud formation. The Ret signaling receptor, which is central to mammalian renal development, is poorly understood in flies, although its expression pattern is intriguing. Surprisingly, other signaling factors such as Neph-1, Pax2, and Wilms' tumor suppressor-1 appear to work within later fly retinal development, providing a surprising link between these two disparate tissues. SUMMARY: Drosophila offers a powerful palate of tools for dissecting developmental processes. Importantly, these tools can often be examined at the level of single cells, permitting us to address issues of differentiation with high resolution. If we are to take full advantage of Drosophila, however, then we must target specific issues and gain a better understanding of the details of Malpighian tubule development.

Animals↗

PubMed2002

Morgue mediates apoptosis in the Drosophila melanogaster retina by promoting degradation of DIAP1.

Inhibitor of apoptosis proteins (IAPs) provide a critical barrier to inappropriate apoptotic cell death through direct binding and inhibition of caspases. We demonstrate that degradation of IAPs is an important mechanism for the initiation of apoptosis in vivo. Drosophila Morgue, a ubiquitin conjugase-related protein, promotes DIAP1 down-regulation in the developing retina to permit selective programmed cell death. Morgue complexes with DIAP1 in vitro and mediates DIAP1 degradation in a manner dependent on the Morgue UBC domain. Reaper (Rpr) and Grim, but not Hid, also promote the degradation of DIAP1 in vivo, suggesting that these proteins promote cell death through different mechanisms.

Amino Acid Sequence↗