Dorsal Eye Selector Pannier (pnr) Suppresses the Eye Fate to Define Dorsal Margin of the Drosophila Eye

Axial patterning is crucial for organogenesis. During Drosophila eye development, dorso-ventral (DV) axis determination is the first lineage restriction event. The eye primordium begins with a default ventral fate, on which the dorsal eye fate is established by expression of the GATA-1 transcription factor pannier (pnr). Earlier, it was suggested that loss of pnr function induces enlargement in the dorsal eye due to ectopic equator formation. Interestingly, we found that in addition to regulating DV patterning, pnr suppresses the eye fate by downregulating the core retinal determination genes eyes absent (eya), sine oculis (so) and dacshund (dac) to define the dorsal eye margin. We found that pnr acts downstream of Ey and affect the retinal determination pathway by suppressing eya. Further analysis of the “eye suppression” function of pnr revealed that this function is likely mediated through suppression of the homeotic gene teashirt (tsh) and is independent of homothorax (hth), a negative regulator of eye. Pnr expression is restricted to the peripodial membrane on the dorsal eye margin, which gives rise to head structures around the eye, and pnr is not expressed in the eye disc proper that forms the retina. Thus, pnr has dual function, during early developmental stages pnr is involved in axial patterning whereas later it promotes the head specific fate. These studies will help in understanding the developmental regulation of boundary formation of the eye field on the dorsal eye margin

Cullin-4 Regulates Wingless and JNK Signaling-Mediated Cell Death in the Drosophila Eye.

In all multicellular organisms, the fundamental processes of cell proliferation and cell death are crucial for growth regulation during organogenesis. Strict regulation of cell death is important to maintain tissue homeostasis by affecting processes like regulation of cell number, and elimination of unwanted/unfit cells. The developing Drosophila eye is a versatile model to study patterning and growth, where complex signaling pathways regulate growth and cell survival. However, the molecular mechanisms underlying regulation of these processes is not fully understood. In a gain-of-function screen, we found that misexpression of cullin-4 (cul-4), an ubiquitin ligase, can rescue reduced eye mutant phenotypes. Previously, cul-4 has been shown to regulate chromatin remodeling, cell cycle and cell division. Genetic characterization of cul-4 in the developing eye revealed that loss-of-function of cul-4 exhibits a reduced eye phenotype. Analysis of twin-spots showed that in comparison with their wild-type counterparts, the cul-4 loss-of-function clones fail to survive. Here we show that cul-4 clones are eliminated by induction of cell death due to activation of caspases. Aberrant activation of signaling pathways is known to trigger cell death in the developing eye. We found that Wingless (Wg) and c-Jun-amino-terminal-(NH2)-Kinase (JNK) signaling are ectopically induced in cul-4 mutant clones, and these signals co-localize with the dying cells. Modulating levels of Wg and JNK signaling by using agonists and antagonists of these pathways demonstrated that activation of Wg and JNK signaling enhances cul-4 mutant phenotype, whereas downregulation of Wg and JNK signaling rescues the cul-4 mutant phenotypes of reduced eye. Here we present evidences to demonstrate that cul-4 is involved in restricting Wg signaling and downregulation of JNK signaling-mediated cell death during early eye development. Overall, our studies provide insights into a novel role of cul-4 in promoting cell survival in the developing Drosophila eye

Novel Neuroprotective Function of Apical-Basal Polarity GeneCrumbs in Amyloid Beta 42 (Aβ42) Mediated Neurodegeneration

Alzheimer\u27s disease (AD, OMIM: 104300), a progressive neurodegenerative disorder with no cure to date, is caused by the generation of amyloid-beta-42 (Aβ42) aggregates that trigger neuronal cell death by unknown mechanism(s). We have developed a transgenic Drosophilaeye model where misexpression of human Aβ42 results in AD-like neuropathology in the neural retina. We have identified an apical-basal polarity gene crumbs (crb) as a genetic modifier of Aβ42-mediated-neuropathology. Misexpression of Aβ42 caused upregulation of Crb expression, whereas downregulation of Crb either by RNAi or null allele approach rescued the Aβ42-mediated-neurodegeneration. Co-expression of full length Crb with Aβ42 increased severity of Aβ42-mediated-neurodegeneration, due to three fold induction of cell death in comparison to the wild type. Higher Crb levels affect axonal targeting from the retina to the brain. The structure function analysis identified intracellular domain of Crb to be required for Aβ42-mediated-neurodegeneration. We demonstrate a novel neuroprotective role of Crb in Aβ42-mediated-neurodegeneration

Drosophila Eye Model to Study Neuroprotective Role of CREB Binding Protein (CBP) in Alzheimer’s Disease

Background: The progressive neurodegenerative disorder Alzheimer’s disease (AD) manifests as loss of cognitive functions, and finally leads to death of the affected individual. AD may result from accumulation of amyloid plaques. These amyloid plaques comprising of amyloid-beta 42 (Aβ42) polypeptides results from the improper cleavage of amyloid precursor protein (APP) in the brain. The Aβ42 plaques have been shown to disrupt the normal cellular processes and thereby trigger abnormal signaling which results in the death of neurons. However, the molecular-genetic mechanism(s) responsible for Aβ42 mediated neurodegeneration is yet to be fully understood. Methodology/Principal Findings: We have utilized Gal4/UAS system to develop a transgenic fruit fly model for Aβ42 mediated neurodegeneration. Targeted misexpression of human Aβ42 in the differentiating photoreceptor neurons of the developing eye of transgenic fly triggers neurodegeneration. This progressive neurodegenerative phenotype resembles Alzheimer’s like neuropathology. We identified a histone acetylase, CREB Binding Protein (CBP), as a genetic modifier of Aβ42 mediated neurodegeneration. Targeted misexpression of CBP along with Aβ42 in the differentiating retina can significantly rescue neurodegeneration. We found that gain-of-function of CBP rescues Aβ42 mediated neurodegeneration by blocking cell death. Misexpression of Aβ42 affects the targeting of axons from retina to the brain but misexpression of full length CBP along with Aβ42 can restore this defect. The CBP protein has multiple domains and is known to interact with many different proteins. Our structure function analysis using truncated constructs lacking one or more domains of CBP protein, in transgenic flies revealed that Bromo, HAT and polyglutamine (BHQ) domains together are required for the neuroprotective function of CBP. This BHQ domain of CBP has not been attributed to promote survival in any other neurodegenerative disorders. Conclusions/Significance: We have identified CBP as a genetic modifier of Aβ42 mediated neurodegeneration. Furthermore, we have identified BHQ domain of CBP is responsible for its neuroprotective function. These studies may have significant bearing on our understanding of genetic basis of AD

Activation of JNK Signaling Mediates Amyloid-ß-Dependent Cell Death

Alzheimer's disease (AD) is an age related progressive neurodegenerative disorder. One of the reasons for Alzheimer's neuropathology is the generation of large aggregates of Aß42 that are toxic in nature and induce oxidative stress, aberrant signaling and many other cellular alterations that trigger neuronal cell death. However, the exact mechanisms leading to cell death are not clearly understood.We employed a Drosophila eye model of AD to study how Aß42 causes cell death. Misexpression of higher levels of Aß42 in the differentiating photoreceptors of fly retina rapidly induced aberrant cellular phenotypes and cell death. We found that blocking caspase-dependent cell death initially blocked cell death but did not lead to a significant rescue in the adult eye. However, blocking the levels of c-Jun NH(2)-terminal kinase (JNK) signaling pathway significantly rescued the neurodegeneration phenotype of Aß42 misexpression both in eye imaginal disc as well as the adult eye. Misexpression of Aß42 induced transcriptional upregulation of puckered (puc), a downstream target and functional read out of JNK signaling. Moreover, a three-fold increase in phospho-Jun (activated Jun) protein levels was seen in Aß42 retina as compared to the wild-type retina. When we blocked both caspases and JNK signaling simultaneously in the fly retina, the rescue of the neurodegenerative phenotype is comparable to that caused by blocking JNK signaling pathway alone.Our data suggests that (i) accumulation of Aß42 plaques induces JNK signaling in neurons and (ii) induction of JNK contributes to Aß42 mediated cell death. Therefore, inappropriate JNK activation may indeed be relevant to the AD neuropathology, thus making JNK a key target for AD therapies

Genetic mechanisms involved in axial patterning and neurodegeneration in Drosophila eye

Complex network of genetic and molecular mechanisms governing the process of organogenesis have an important bearing on development of organisms. We are using an established model of Drosophila melanogaster commonly referred to as fruit fly in order to understand these mechanisms. We have used Drosophila eye to discern genetic hierarchy controlling the (i) event of axial patterning, and (ii) to study neurodegeneration in the developing eye. Axial patterning involves generation of dorsal-ventral (DV), anterior-posterior (AP) and proximal-distal (PD) axes in the organ primordium and is considered crucial for transformation of monolayer epithelium into a three dimensional organ. Any abnormalities in expression patterns of axial patterning genes may result in complete loss of organ. Drosophila eye develops from a default ventral state conferred by expression of genes Lobe (L) and Serrate (Ser). It has been found that antagonistic interaction of dorsal and ventral genes helps generation of midline or the equator which is essential for growth and differentiation of the eye field. Loss-of-function of L/Ser results in complete or loss-of-ventral eye depending on time axis involved. In a genetic modifier screen performed for search for modifiers of L mutant phenotypes, an E3 ubiquitin ligase, Cullin-4 (Cul-4) and GATA-1 transcription factor Pannier (Pnr) were identified. In the current study, we have characterized Cul-4, in promoting cell survival in the ventral domain of developing eye via downregulation of Wingless (Wg) signaling. Cul-4 also regulates JNK signaling to prevent cell death in the developing eye. We thus place the Cul-4 in the hierarchy of ventral genes involved in eye development.We also present the role of GATA-1 transcription factor Pnr in defining the dorsal eye margin boundary by suppressing the eye fate. Pnr downregulates retinal determination gene machinery via zinc finger transcription factor teashirt (tsh). We thus provide a novel mechanism involved in defining dorsal margins of the eye during early stages of organogenesis and an eye suppression function, as a late role of pnr in the developing eye. Identification and characterization of these genes in the dorsal and ventral domains of the eye may help enrich our understanding of the genetic hierarchy and the complex interactions of genes involved in axial patterning in the eye during organogenesis. Since the genetic machinery is highly conserved from flies to humans, these studies will have direct implications on higher vertebrates as well. Other than patterning and growth studies, Drosophila eye has been widely used to study genetic and molecular basis of neurodegeneration. A part of current study is to test the mechanisms involved in the neuronal cell death caused during the course of Alzheimer\u27s disease (AD). AD is caused due to accumulation of Aß-42 peptide which is a product formed because of incorrect cleavage of Amyloid Precursor Protein (APP). Accumulation of Aß-42 results in formation of amyloid plaques which eventually results into stress and the neuronal cell death. We have found that JNK signaling pathway is induced upon Aß-42 accumulation and causes cell death of the neurons in the brain. Our study provides a new mechanistic insight from the perspective of identifying the new targets of AD neuropathy

Molecular Genetic Mechanisms of Axial Patterning: Mechanistic Insights into Generation of Axes in the Developing Eye

All multicellular organisms require axial patterning to transform a single-layer organ primordium to a three-dimensional organ. It involves delineation of anteroposterior (AP), dorsoventral (DV), and proximodistal (PD) axes. Any deviation in this fundamental process results in patterning and growth defects during organogenesis. The Drosophila eye is an excellent model to study axial patterning. In the Drosophila eye, DV lineage is the first axis to be determined, which is followed by generation of the AP axis. The default state of the Drosophila early eye primordium is ventral, and the dorsal fate is established by onset of expression of dorsal eye fate selector pannier (pnr)in a group of cells on the dorsal eye margin. The boundary between dorsal and ventral compartments is the site for activation of Notch (N) signaling and is referred to as the equator. Activation of N signaling is crucial for initiating the cell proliferation and differentiation in the developing Drosophila eye imaginal disc. This chapter will focus on (a) how axial patterning occurs in the developing Drosophila eye, (b) how the developing eye field gets divided into dorsal and ventral cell populations, and (c) how DV patterning genes contribute toward the growth and patterning of the fly retina