Diana Zitserman

The evolutionarily conserved TSC/Rheb pathway activates Notch in tuberous sclerosis complex and Drosophila external sensory organ development

Mutations in either of the genes encoding the tuberous sclerosis complex (TSC), TSC1 and TSC2, result in a multisystem tumor disorder characterized by lesions with unusual lineage expression patterns. How these unusual cell-fate determination patterns are generated is unclear. We therefore investigated the role of the TSC in the Drosophila external sensory organ (ESO), a classic model of asymmetric cell division. In normal development, the sensory organ precursor cell divides asymmetrically through differential regulation of Notch signaling to produce a pIIa and a pIIb cell. We report here that inactivation of Tsc1 and overexpression of the Ras homolog Rheb each resulted in duplication of the bristle and socket cells, progeny of the pIIa cell, and loss of the neuronal cell, a product of pIIb cell division. Live imaging of ESO development revealed this cell-fate switch occurred at the pIIa-pIIb 2-cell stage. In human angiomyolipomas, benign renal neoplasms often found in tuberous sclerosis patients, we found evidence of Notch receptor cleavage and Notch target gene activation. Further, an angiomyolipoma-derived cell line carrying biallelic TSC2 mutations exhibited TSC2- and Rheb-dependent Notch activation. Finally, inhibition of Notch signaling using a gamma-secretase inhibitor suppressed proliferation of Tsc2-null rat cells in a xenograft model. Together, these data indicate that the TSC and Rheb regulate Notch-dependent cell-fate decision in Drosophila and Notch activity in mammalian cells and that Notch dysregulation may underlie some of the distinctive clinical and pathologic features of TSC.

Frequent Unanticipated Alleles of lethal giant larvae in Drosophila Second Chromosome Stocks

Forty years ago, a high frequency of lethal giant larvae (lgl) alleles in wild populations of Drosophila melanogaster was reported. This locus has been intensively studied for its roles in epithelial polarity, asymmetric neural divisions, and restriction of tissue proliferation. Here, we identify a high frequency of lgl alleles in the Bloomington second chromosome deficiency kit and the University of California at Los Angeles Bruinfly FRT40A-lethal P collection. These unrecognized aberrations confound the use of these workhorse collections for phenotypic screening or genetic mapping. In addition, we determined that independent alleles of insensitive, reported to affect asymmetric cell divisions during sensory organ development, carry lgl deletions that are responsible for the observed phenotypes. Taken together, these results encourage the routine testing of second chromosome stocks for second-site alleles of lgl.

dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions

Drosophila EHBP1 is a novel regulator of Notch signaling that may function as an adaptor protein during the exocytosis and recycling of the Notch ligand Delta.

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway. Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Sanpodo controls sensory organ precursor fate by directing notch trafficking and binding γ-secretase

In sensory organ precursor cells, Sanpodo can enhance or suppress Notch signaling by promoting interaction with Presenilin or driving receptor internalization, respectively.

The TSC1/2 complex controls Drosophila pigmentation through TORC1-dependent regulation of catecholamine biosynthesis.

In Drosophila, the pattern of adult pigmentation is initiated during late pupal stages by the production of catecholamines DOPA and dopamine, which are converted to melanin. The pattern and degree of melanin deposition is controlled by the expression of genes such as ebony and yellow as well as by the enzymes involved in catecholamine biosynthesis. In this study, we show that the conserved TSC/TORC1 cell growth pathway controls catecholamine biosynthesis in Drosophila during pigmentation. We find that high levels of Rheb, an activator of the TORC1 complex, promote premature pigmentation in the mechanosensory bristles during pupal stages, and alter pigmentation in the cuticle of the adult fly. Disrupting either melanin synthesis by RNAi knockdown of melanogenic enzymes such as tyrosine hydroxylase (TH), or downregulating TORC1 activity by Raptor knockdown, suppresses the Rheb-dependent pigmentation phenotype in vivo. Increased Rheb activity drives pigmentation by increasing levels of TH in epidermal cells. Our findings indicate that control of pigmentation is linked to the cellular nutrient-sensing pathway by regulating levels of a critical enzyme in melanogenesis, providing further evidence that inappropriate activation of TORC1, a hallmark of the human tuberous sclerosis complex tumor syndrome disorder, can alter metabolic and differentiation pathways in unexpected ways.

Numb regulates the balance between Notch recycling and late-endosome targeting in Drosophila neural progenitor cells

Steady-state and pulse-labeling techniques are used to follow Notch receptors in sensory organ precursor cells in Drosophila. Numb and L(2)gl antagonize a pool of Notch receptors, and Numb promotes Notch targeting to late endosomes in Drosophila neural progenitors to regulate Notch signaling and cell fate.