Ajay Srivastava

Antagonizing Scalloped With a Novel Vestigial Construct Reveals an Important Role for Scalloped in Drosophila melanogaster Leg, Eye and Optic Lobe Development

Scalloped (SD), a TEA/ATTS-domain-containing protein, is required for the proper development of Drosophila melanogaster. Despite being expressed in a variety of tissues, most of the work on SD has been restricted to understanding its role and function in patterning the adult wing. To gain a better understanding of its role in development, we generated sd47M flip-in mitotic clones. The mitotic clones had developmental defects in the leg and eye. Further, by removing the VG domains involved in activation, we created a reagent (VGΔACT) that disrupts the ability of SD to form a functional transcription factor complex and produced similar phenotypes to the flip-in mitotic clones. The VGΔACT construct also disrupted adult CNS development. Expression of the VGΔACT construct in the wing alters the cellular localization of VG and produces a mutant phenotype, indicating that the construct is able to antagonize the normal function of the SD/VG complex. Expression of the protein:protein interaction portion of SD is also able to elicit similar phenotypes, suggesting that SD interacts with other cofactors in the leg, eye, and adult CNS. Furthermore, antagonizing SD in larval tissues results in cell death, indicating that SD may also have a role in cell survival.

Understanding Lunasin’s biology and potential as a cancer therapeutic by utilizing Drosophila genetics

Soy contains many bioactive molecules known to elicit anticancer effects. One such peptide, Lunasin, has been shown to selectively act on newly transformed cells while having no cytotoxic effect on non-tumorigenic or established cancer cell lines. While this effect on in vitro systems is promising, Lunasin’s efficacy in an in vivo system is yet to be assessed. In this review, we discuss the state of knowledge with respect to Lunasin and then review some of the powerful genetic tools available in Drosophila. The availability of a sophisticated genetic tool box makes Drosophila an excellent genetic model well suited to studying the biology of Lunasin and its effect on tumor progression in an in vivo model organism.

Further developmental roles of the Vestigial/Scalloped transcription complex during wing development in Drosophila melanogaster.

The Drosophila homologue of the human TEF-1 gene, scalloped (sd), is required for wing development. The SD protein forms part of a transcriptional activation complex with the protein encoded by vestigial (vg) that, in turn, activates target genes important for wing formation. One sd function involves a regulatory feedback loop with vg and wingless (wg) that is essential in this process. The dorsal-ventral (D/V) margin-specific expression of wg is lost in sd mutant wing discs while the hinge-specific expression appears normal. In the context of wing development, a VG::sdTEA domain fusion produces a protein that mimics the wild-type SD/VG complex and restores the D/V boundary-specific expression of wg in a sd mutant background. Further, targeted expression of wg at the D/V boundary in the wing disc was able to partially rescue the sd mutant phenotype. This infers that sd could function in either the maintenance or induction of wg at the D/V border. Another functional role for sd is the establishment of sensory organ precursors (SOP) of the peripheral nervous system at the wing margin. Thus, the relationship between sd and senseless (sens) in the development of these cells is also examined, and it appears that sd must be functional for proper sens expression, and ultimately, for sensory organ precursor development.

A Cathepsin-L is required for invasive behavior during Air Sac Primordium development in Drosophila melanogaster.

TheDrosophila Air Sac Primordium (ASP) has emerged as an important structure where cellular, genetic and molecular events responsible for invasive behavior and branching morphogenesis can be studied. In this report we present data which demonstrate that a Cathepsin‐L encoded by the gene CP1 inDrosophila is necessary for invasive behavior during ASP development. We find that CP1 is expressed in ASP and knockdown of CP1 results in suppression of migratory and invasive behavior observed during ASP development. We further show that CP1 possibly regulates invasive behavior by promoting degradation of Basement Membrane. Our data provide clues to the possible role of Cathepsin L in human lung development and tumor invasion, especially, given the similarities between human lung andDrosophila ASP development.

A protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of Drosophila melanogaster.

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases (1-7). Progression of tumors from a benign to a metastatic state is a complex event (8) and has been modeled in Drosophila to help us better understand the genetic basis of this disease (9). Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system (10) and exploits the cooperation between an activated oncogene, Ras(V12) and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors (9). I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

A soy protein Lunasin can ameliorate amyloid-beta 42 mediated neurodegeneration in Drosophila eye

Alzheimer’s disease (AD), a fatal progressive neurodegenerative disorder, also results from accumulation of amyloid-beta 42 (Aβ42) plaques. These Aβ42 plaques trigger oxidative stress, abnormal signaling, which results in neuronal death by unknown mechanism(s). We misexpress high levels of human Aβ42 in the differentiating retinal neurons of the Drosophila eye, which results in the Alzheimer’s like neuropathology. Using our transgenic model, we tested a soy-derived protein Lunasin (Lun) for a possible role in rescuing neurodegeneration in retinal neurons. Lunasin is known to have anti-cancer effect and reduces stress and inflammation. We show that misexpression of Lunasin by transgenic approach can rescue Aβ42 mediated neurodegeneration by blocking cell death in retinal neurons, and results in restoration of axonal targeting from retina to brain. Misexpression of Lunasin downregulates the highly conserved cJun-N-terminal Kinase (JNK) signaling pathway. Activation of JNK signaling can prevent neuroprotective role of Lunasin in Aβ42 mediated neurodegeneration. This neuroprotective function of Lunasin is not dependent on retinal determination gene cascade in the Drosophila eye, and is independent of Wingless (Wg) and Decapentaplegic (Dpp) signaling pathways. Furthermore, Lunasin can significantly reduce mortality rate caused by misexpression of human Aβ42 in flies. Our studies identified the novel neuroprotective role of Lunasin peptide, a potential therapeutic agent that can ameliorate Aβ42 mediated neurodegeneration by downregulating JNK signaling.

A novel link between FMR gene and the JNK pathway provides clues to possible role in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive form of thoracic cancer with poor prognosis. While some studies have identified the molecular alterations associated with MPM, little is known about their role in MPM. For example, fragile X mental retardation (FMR) gene is up‐regulated in MPM but its role in MPM is unknown. Here, utilizingDrosophila genetics, I investigate the possible roleFMR may be playing in MPM. I provide evidence which suggests that FMR may contribute to tumorigenesis by up‐regulating a matrix metalloprotease (MMP) and by degrading the basement membrane (BM), both important for tumor metastasis. I also demonstrate a novel link between FMR and the JNK pathway and suggest that the effects of FMR in MPM could in part be mediated by up‐regulation of the JNK pathway.