Drosophila optic lobe

Identification of markers for medulla cell fate specification in the Delineating the mechanism s for cellular fate determination in the Drosophila Drosophila optic lobe

Abstract

How progenitor cells develop into mature neurons in the CNS remains to be elusive. Neuroepithelial (NE) cells in flies follow a similar program of neurogenesis as the vertebrate Both retinal progenitor cells (RPC) in vertebrates and neuroepithelial cells (NE) in the medulla of the Drosophila optic lobe have been shown to undergo a similar program of differentiation. They The progenitor cells (RPC and NE) first proliferate extensively to expand cell numbers. This is followed by differentiation where progenitors are specified along a , followed by a series of differentiation into neuronalal or glia lineage. Recently, several regulators that control NE-NB transition have been identified. Previously, we have shown that JAK/STAT and Notch are required for the timely symmetric to asymmetric cell division during larval development. It is still unclear how larval progenitor cells map out to the adult medulla. lUsing the GAL4/UAS system in which [...]; in this study, we identified specific drivers expressed in subsets of progenitor cells during the larva stage and neurons in the adult medulla. Our library of GAL4 drivers will provide a very useful tool to study not only the development of these medulla neurons but the underlying molecular mechanisms that regulate the neural circuitry in the adult medulla. cells (Chenn and McConnell, 1995; Fuerstenberg et al., 1998; Yu et al., 2006). Many different extrinsic factors are shown to play a role in RPC cell fate determination (Yang et al., 2004). To explore what and how different extrinsic factors may also play a role in NE cell fate determination, this study will find good GAL4 drivers showing expression in only specific cell type to be used for cell manipulation. We used immunohistochemistry on different gal4 enhancer trap line to screen for GFP expression only in specific cell type. We found…

Introduction

The visual system is a complex neuronal network which requir es the proper wiring of cell types in a tightly regulated spatial and temporal A fashion. The synchronized progression of cell fate specification in the vertebrate retina is one example of such complex networks. functional vertebrates visual system requires the proper formation of specific cells at specific location within the retinal architecture. RRetinal precursor cells (RPCs) of the retina, the source for all cell types within the retinaproliferate , first needs to proliferate by extensively to give rise to a stable population of cells. This series of symmetrical division is followed by both symmetrical division to generate proper cell number; then by and asymmetrical cell division to sustain a population of stem cells and generate the necessary cellular diversity within the retina architecture to give rise to the many cell types in the tissue (Young 1985). Neuronal cell types that makes up the retina: The many cell types are classified into six different category of neurons -photoreceptors, horizontal, bipolar cells, amacrine interneurons, and ganglion cells, and Muller glia follow a very distinct program of neurogenesis that are regulated by both intrinsic factors (i.e. transcriptional activators/repressors and cell cycle regulators) and extrinsic cues for the cellular environment (reviewed by Ohsawa and Kageyama, 2007).

After the initial proliferation of RPCs, mid-to-late progenitors either divides symmetrically to produce two postmitotic neurons or asymmetrically to produce both a mitotic and post-mitotic daughter cells. The two postmitotic neurons from symmetrical division have its cellular fate determined; whereas the mitotic daughter cell (RPCs) has the same option as late progenitor cells and the postmitotic daughter cells with the possibility of re-entering the cell cycle (Chenn and McConnell, 1995).

For proper formation of the retina in vertebrates, retina precursor cells (RPCs), which give rise to all cell types within the tissue architecture, initially proliferate extensively to increase cell number, followed by the differentiation program to generate six types of neurons and Muller glia cells at three different cellular layers in a precise temporal order (Young 1985) . These six types of neurons include photoreceptor cells (rods and cones), horizontal and bipolar cells, amacrine interneurons, and ganglion cells (reviewed by Ohsawa and Kageyama, 2007) . Clonal analysis and in vitro experiments suggest that initial RPC proliferation occurs through symmetric division whereas mid-to-late progenitors divide both symmetrically to produce two postmitotic neurons and asymmetrically to give rise to one mitotic and post-mitotic daughter cell (Chenn and McConnell, 1995) .

The early phase of vertebrate neurogenesis, in which RPCs proliferate to increase cell number closely models that of Drosophila optic lobe neuroepithelial-to-neuroblast (NE-NB) transition . Drosophila larval central nervous system (CNS) comprises of several types of neuroblast (NB) which include ventral nerve cord (VNC) NB, central brain NB, mushroom bodies (MB) NB, and optic lobe NB (Betschinger et al., 2004 ; Egger et al., 2007; Ito and Hotta, 1992; Truman and Bate, 198 8) . Unlike central brain NBs, tThe optic lobe NB is an interesting model to study since it follows a distinct pattern of neurogenesis in which [...]; unlike VNC, central brain, and MB NBs which have a predetermined cell fate upon embryogenesis. The optic lobe NBs are derived from two neuroepithelia layers, called the inner and outer optic anlage (IOA and OOA), that originatinge from a region in the head epidermis called the optic lobe placode (White and Kankel, 1978).

The OOA, composed of a homogenous population of neuroepithelial (NE) cells that eventually differentiate give rise to the outer medulla and lamina neurons, while the IOA give rise to the lobular and inner medullary neurons. During early development (first-to-second instar),

m edial Similar to vertebrates' retinal formation, the development in the medulla of the Drosophila optic lobe comprises of both symmetr ical and asymmetrical cell division. As with RPCs in vertebrates, NE n euroepithelial cells (NE) divide by symmetric division to located in the medulla of the Drosophila optic lobe first proliferates to increase in cell numbers. By 50 hrs post-hatching, at the early third instar stage; . At the early-to-late third instar stage, NE cells begins to transition to differentiating into NBs in a synchronized manner, where they morphologically change from a columnar to spherical appearance. Each NB asymmetrically divides, although the exact mechanism of medial NB differentiation is still unclear. It is suspected that NB divide asymmetrically to produce a mitotic NB and a developmentally-restricted ganglion mother cell (GMC), which divides again to generate two glial or neuronal cells (Chia et al., 2008; Knoblich, 2008). Whereas the mechanism of the transition from symmetrical to asymmetrical cell division in vertebrates is difficult to identify due to the lack of a well-ordered transition in space and time, the

NE-NB transition in the medulla progresses in a synchronized and orderly manner, sweeping from the medial to lateral ed ge known as the proneural wave (Yasugi et al., 2008) . Unlike larval NE-NB transition,

In embryonic neurogenesis of Drosophila, NBs are specified through the lateral inhibition mechanism. They are selected are induced from proneural cluster (PNC) of the neuroepithelial layer a population of NE cells in a proneural cluster (PNC) that expresses a series of proneural genes such as l(1)sc, atonal, achaete, and scute. These proneural genes, part of the Achaete-Scute complex (ASC), encode for basic helix-loop-helix (bHLH) transcription factors that dimerize with another bHLH protein, Daughterless (Da) (Jarman et al., 1993; Murre et al., 1989; Villares and Cabrera, 1987). The selection of NB from proneural cluster works by a mechanism known as lateral inhibition. The Notch signaling pathway has been shown to mediate this lateral inhibition mechanism in which the selected NBs, through the expression of Notch, that represses the proneural genes and, inhibit the neural potential in neighboring cells causing them to adopt an epidermal fate (Artavanis-Tsakonas and Simpson, 1991; Hassan and Vaessin, 1996). In this manner, NB delaminates from the epithelial layer. The Notch signaling pathway consists of the Delta ligand, the Notch receptor, and the CBF1/Su(H)/LAG1 (CSL) CSL transcription factor Suppressor of Hairless [Su(H)] (Kopan et al., 2009). The Delta (Dl) ligand interaction with the extracellular domain of the transmembrane Notch receptor results in the cleavage of the Notch receptor by a member of the metalloprotease family, releasing its the intracellular domain of Notch (Nintra). The Nintra then enters the nucleus to associate with CSL transcription factor, Su(H), which in . This association causes the CSL protein to change from transcriptional repressor to transcriptional activator that turn activates downstream target genes. The inhibition of neuronalal potential in neighboring cells is mediated by the Enhancer of split Complex [E(spl)-C]. E(spl)-C, dependent upon Notch activation , is a group of proneural genes that makes all cells in the PNC equipotent to either become a neural or epidermal cell (Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995).

Repression of neuronal differentiation is mediated through the Enhancer of split Complex [ E(spl) -C], encoding a group of genes that are activated by Notch via CSL-binding sites (Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995 Whereas embryonic In contrast to the NB selection are stochastic, NE-NB in the Drosophila embryo, the transition from NE to NB in the larval optic lobe stage Drosophila is a well-ordered process, going from medial to lateral starting at the medial edgend resultsing in a proneural wave of differentiation . l(1)sc, part of the Achaete-Scute Complex ASC (and the homologue of Mash1 in vertebrates), is found to be present at the forefront of the proneural wave critical to the NE-NB transition. At the most lateral edge, the unpaired (upd) ligand inhibits the proneural wave through activation of the with Janus Kinase/signal transducer and activator of transcription (JAK/STAT) pathway negatively regulating the proneural wave (Yasugi et al., 2008). In vertebrates, Hes1 [Drosophila E(spl)-C]of the Achaete Scute Complex is required to maintain the population of of early progenitor cells. Drosophila enhancer of split, homologue of vertebrates' hes1, Llikewise, E(spl)-C are also present in the early stages of NE-NB transition in the medulla. The JAK/STAT signaling pathway is composed of the ligands: Unpaired (Upd), the receptor, Domeless (Dome); the JAK kinase, Hopscotch (Hop); and the transcription factor, Stat92E (Agaisse et al., 2003; Binari and Perrimon, 1994; Brown et al., 2001; Chen et al., 2002; Gilbert et al., 2005; Harrison et al., 1998; Hombria et al., 2005; Hou et al., 1996; Yan et al., 1996). Upd ligand binding with the Dome receptor causes a conformational change within the protein that resultsting in the formation of phosphorylate tyrosine residue on the cytokine receptor. Stat92E with the ability to bind phosphotyrosine residues is recruited to the receptor and gets phosphorylated by Hop. The phosphotyrosine residue on Stat92E acts as docking site for other Stat92E phosphotyrosine residue leading to the dimerization of Stat92E. These Stat92E dimers translocate to the nucleus and activate downstream target genes. In tThe optic lobe, JAK/STAT is required requires the negative regulation of JAK/STAT to prevent premature NE-NB differentiation. The lateral movement of JAK/STAT signaling frees medial NE cells from its the negative regulation; thus, enabling medial NE cells to express l(1)sc initiating NE-NB transition (Yasugi et al., 2008).

In addition to JAK/STAT, Notch has been identified as a critical regulator of NE-NB transition. During early development, Recent study in the early stage of NE-NB , early first to second instar stages, has shown that both Notch signaling and Jak/StatJAK/STAT is expressed in the OOA signaling occurring concomitantly in the early larval medulla to prevent premature NE-NB transition. (Ngo et al., manuscript in preparation), where JAK/STAT functionsThrough functional analysis, JAK/STAT was shown to act upstream of Notch. As the OOA expansion is in progresses, JAK/STAT is transiently expressed throughout the entire OOA, remaining only in the lateral edge. Using the novel lineage-tracing system, G-TRACE to visualize both the real-time expression and lineage-traced expression. G-TRACE uses an enhancer trap construct GAL4/UAS system, in which GAL4 activates an upstream activating sequence (UAS) to promote the expression of downstream reporter gene RFP and the FLP recombinase. Through cis-homologous recombination, FLP removes an FRT-flanked termination cassette that is inserted between an ubiquitin promoter (Ubiquitin-p63E). Once removed, the EGFP downstream of the promoter leads to the expression of GFP in all subsequent daughter cells even without the presence of GAL4 (Evans et al., 2009). With this construct, real-time expression can be visualized with RFP while lineage-traced expression is visualized with GFP. Using G-TRACE, upd This transient ubiquitous expression helps to activate, or maintain, a more stable expression of Notch in the medial edge, thus preventing premature NE from differentiating until the OOA has fully expanded, leaving a stable population of NE cells from which NB can be derived.

In that study, using immunohistochemistry (IHC), the OOA was shown to be composed of a homogenous population of NE cells proliferating through symmetrical division. Once the OOA finish expanding at approximately 48-60 hrs, the NE cells inititates differentiation into NB cells. This transition can be easily visualized through IHC as NE cells where columnar shape cells begin to change to a more spherical shape characteristic of NB cells. As one would expected, columnar cells are found in the lateral edge while spherical cells are found medially, thus corresponding to the concept that that the NE-NB transition occurs as a proneural wave progressing medially to laterally.

This morphological transition with NE cells differentiating into NB cells correlates with an orderly expression of a group of proneural genes being transiently expressed in differentiating cells. The characterized proneural gene, l(1)sc, was used as an indicator of the proneural activation. The proneural wave needs a precise mechanism of regulation to which the NE cells need to abide to for functional neurogenesis. As such the recent study has shown that both the JAK/STAT and Notch signaling pathway being responsible. JAK/STAT activity within the medulla was characterized at different time point in the larval stage using a recently developed lineage tracing system, G-Trace, to visualize both the real-time expression and lineage-traced expression. G-Trace uses an enhancer trap construct GAL4/UAS system, in which GAL4 activates an upstream activating sequence that promotes the expression of downstream reporter gene RFP. While at the same time, the GAL4 initiates FLP recombinase that removes an FRT-flanked termination cassette. The removal of the FRT-flanked termination cassette inserted between an ubiquitin promoter (Ubiquitin-p63E) and the EGFP downstream of the promoter leads to the expression of GFP. EGFP expression will be maintained in later daughter cells in which the termination cassette has been removed even without the presence of GAL4 (Evan et al., 2009) . With this construct, RFP will show the real-time expression of JAK/STAT and GFP will show the lineage-traced expression. With IHC, upd (ligand for JAK/STAT) expression was found to be constrained to a small population of cells in the early OOA. By the late third instar, Over time upd expression is lineage-traced in the majority of expressed in all cells throughout the in the entire optic lobe. , shown using lineage traced expression. However, Iinterestingly, in that studies, JAK/STAT was found to be restricted to the lateral edge prior to even before the activation of the proneural wave. An earlier study proposed that JAK/STAT plays an indirect role in proneural wave progression, but with the data from this recent study, another mechanism has to be involved in addition to the JAK/STAT signaling pathway. Notch signaling pathway was the primary suspect for the second mechanism.

Although NNotch has been known to be a major part of the lateral inhibition mechanism governing NE-NB transition in embryonic central nervous system (CNS) of DrosophilaDrosophila; . However, not much is known about its function in the OOA during the in later larval stages. We have examined To explore the possibility of Notch involvement in NE-NB transition in early larval stages, Notch expression was examined alongside JAK/STAT using transcriptional reporters: E(spl)m8-LacZ and Stat92EGFP, respectively. At the onset of the proneural wave activation, in third instar larval stage, E(spl) expression was restricted to the medial region whereas Stat92E expression was restricted to the lateral region. Both Notch and JAK/STAT were confined to a localized region on either side of the proneural wave. As the proneural wave progresses the gap between the two domains decreases shrinked. Nevertheless, Even though the two regions are moving closer to each other, they the Notch and JAK/STAT expression domains still maintain distinct boundaries throughout proneural wave activation, with E(spl) expression present medially to JAK/STAT. Both expression and functional analyses established the requirement of From the study, Delta (Dl), the ligand for Notch signaling, appears to be the signal bridging the gap between the two distinct regions. Dl expression was localized to the NE cells in the region between Notch and JAK/STAT expression.

With both Notch and JAK/STAT during shown to play a role in the early transition from NE cells to NBs cells, where they the recent study further showed that both Notch and JAK/STAT are needed have to be present for the proper timing and orderly progression of the NE-NB transition. Loss-of-function of both Notch and JAK/STAT leads to the differentiation of premature NBs. Stat92E loss-of-function with Notch also leads to the improper development of NB. Likewise, Notch loss-of-function leads to the same result as Stat92E loss-of-function.

Our previous The results indicate that during the timeframe of the proneural wave progression, both signals Notch and JAK/STAT are confined to either both sides of the proneural wave, with Notch being expressed at the medial end and JAK/STAT expression at the lateral edge. This two opposing inhibitory forces, Notch and JAK/STAT, confines the NE-NB transition to a very limited and precise zone. The cells expressing DeltaDl, transiently expressing l(1)sc, initiating NE-NBes the transition from NE-NB turn on and are confined to the proneural wave region between Notch and JAK/STAT. Notch expression at the medial end make sure NE cells in the progress of the transition, and those already that have made the transition to NB, are prevented from further differentiationunnecessarily reactivating the transition. The JAK/STAT at the lateral end prevents premature NE from differentiating to NB. With this regulatory mechanism, NE-NB transition is carefully regulated.

The Drosophila optic lobe contains approximately 60,000 cells divided into four distinct neuropiles called the lamina, medulla, lobula, and lobula plate. The medulla is the largest structure of the four neuropiles consisting of approximately 40,000 neurons. The medulla neuropile is stratified into ten different layers (M1-M10). The M1-M10 receives input from the R1-R6 photoreceptor via lamina monopolar neurons and the M3/M6 layers receive inputs from the R7/R8 inner photoreceptor for color vision (Morante and Desplan, 2004; Fishbach and Dittrich, 1989). Recently, Morante and Desplan, using Marcum and enhancer traps-Gal4 lines, have reconstructed elements of the neuronal network in the optic lobe, in which they have identified different markers for different cellular subtypes in the medulla of the optic lobe (2008). Even though different subtype of cells in the medulla are known, not much is known about what determine the different cell differentiation path in the medulla.

While we have characterized the dynamics during early development of the OOA, not much has been established regarding how the final medullar cell fate is determined in the adult. Furthermore, not many markers that have been identified in the medulla or medulla primordium such as NE cells, NBs, or the mature neurons. Thus, one of the aims of this One of the first aim of our project is to find specific markers for specific cell types. Using the GAL4/UAS system, we identified some drivers that preferentially marks To be able to track cell development through time, certain GAL4/UAS lines need to be screen for expression of GFP in specific cell types in the medulla. The GALal4/UAS system is based on the GAL4 transcriptional activator in, developed by Brand and Perrimon uses an enhancer trap construct that uses the Saccharomyces cerevisiae transcriptional activator, Gal4, as a reporter gene (Brand and Perrimon, 1993). The activity of the Gal4 protein is monitored by a second reporter protein, GFP, under the control of a Gal4-responsive upstream activating sequence. Many GAL4 enhancer-trap libraries have been created, such as the Kyoto Stock Center. As GAL4 is controlled by the specific regulatory elements within the genome, each GAL4 insertion will show variable pattern of expression, some being ubiquitous while other may be more specific to the tissue and different cell types within the tissue architecture. We have examined 50+ GAL4 enhancer trap lines to look for expression in specific cell types within the larval and adult optic lobe. Here, we present the results of our findings, where [...] shows expression in specific cell types in the medulla.Many different Gal4 enhancer trap lines are available with different insertion site of Gal4 using P-element. Different insertion sites result in expression of different enhancer. The different expression of Gal4 leads to the second reporter protein being expressed differently across Gal4 enhancer trap line. Thus along with a good Gal4 enhancer trap line showing GFP expression with only one type of specific cell in the medulla, that one type of specific cells can be easily manipulated by with different extrinsic factors being insert downstream of UAS; GAL4 activates UAS which activates downstream genes including GFP. For example, Notch could be inserted just downstream of UAS and IHC can be performed. GFP expression in only one type of cells indicate that Notch is only being expressed in those types of cells. IHC allows for the visualization of whether the manipulation had any effect on the cells. With this tool, we can explore the possibility of what roles extrinsic factors play in determining cell fates in the medulla of the optic lobe.

Along with the marker tools being used to explore the role of extrinsic factors in cell fate determination, a mosaic analysis with a repressible marker (MARCM) will be performed on Drosophila at different stages . MARCM, a genetic tool used for Drosophila to label single cell or a cluster of cell (clones) sharing a single progenitor, can be used to track cells over a period of time ( Wu and Luo, 2006 ) . This is important for the purpose of determining whether a cell spatial arrangement or the time when a cell is born are factor s in cell fate determination.

The MARCM construct relies on the use of the GAL80 repressor protein, GAL4/UAS system, and FLP recombination target (FRT) system-mediated mitotic recombination. GAL80 when present represses the expression of GAL4/UAS. Thus, the purpose of this construct is to generate homozygous mutant cells from heterozygous precursors with both GAL80 and GAL4/UAS present. Two FRT sites located at the same site on homologous chromosomes are used along with heat shock-dependent FLP recombinase. A sample with both GAL80 and GAL4/UAS will be heat shocked to activate FLP recombinase, which in turn will activate mitotic recombination at two FRT site located at the same position on homologous chromosomes. This results in the random marking by GFP of a cell or a cluster of cell due to GAL80 being flipped out. The cluster of cells originates from a single progenitor cell that had GAL80 flipped out.

Materials and Methods

Genetics

Flies were grown at 25 ° C unless otherwise noted. The following Gal4 enhancer trap lines with different insertion site were used in this study: 103509, 103701, 103879, 103869, and 103884 and the reporter lines: UASm cd 8GFP(II).

Immunohistochemistry

Samples were fixed in 4% EM-grade formaldehyde in phosphate buffer saline (Fisher-Scientific, pH=7.4) with 0.3% Triton X-100. Immunohistochemistry was performed using standard protocol. The following antibodies were provided by the Developmental Studies Hybridoma Bank: BP104 anti-mouse (1:30) and NCAD2 anti-rat (1:50). Secondary antibodies (Jackson) were used at the following dilutions: anti-mouse Alexa 546 (1:250) and anti-rat Cy5 (1:500). Specimens were mounted with Vectashield mounting medium (Vector) and v iewed on a confocal microscope.

Result and Discussion

In vertebrates, intrinsic and extrinsic factors have been shown to precisely control the differentiation ofThrough the screening of different GAL4 enhancer trap lines, if good Gal4 drivers are found, they will be used along with MARCM for cell fate mapping. With good GAL4 drivers, different cells can be manipulated to determine how extrinsic factors, such as Notch and JAK/STAT, plays a role in cell fate determination. Additionally, MARCM allows for the marking and identification of single cell or cluster of cells originating from a single progenitor cell. Using MARCM along with different specific markers that mark different cell types in the medulla, specific cells can be track over time.

Whereas it is known in the vertebrate system, the birth of neural progenitors at each layer. Within each layer, both time and space information is needed to encode a different cell type of the vertebrate retina. The Drosophila central brain, on the other hand, deviates from such programmed neurogenesis. In the central brain, each primary lineage, derived from the primary NB of the embryo, are born and specified irrespective of time. We suspect the mechanism of neurogenesis in the medulla, however, to not follow that of the central brain, but that of the vertebrate retinaat different time and at specific location along the z-axis will differentiate into specific cell type due to different environmental signal at different location and time, not much is known about the Drosophila model in that aspect. However, we suspect that the same concept applies to the Drosophila medulla.

To elucidate the mechanism of neuronal cell specific in the Drosophila medulla, we utilize mosaic analysis with a repressible marker (MARCM) technique . MARCM have been to label single cell or a cluster of cell (clones) sharing a single progenitor and can be used to track cells over a period of time (Wu and Luo, 2006). This is important to determine whether spatial arrangement or the time when a cell is born play a pivotal role in regulating cell fate specification.

MARCM relies on construct relies on the use of the GAL80 repressor protein, GAL4/UAS system, and FLP recombination target (FRT) system to induce mitotic recombination. GAL80 when present represses the expression of GAL4/UAS. By placing the expression of FLP recombinase under the control of a heat-shock promoter, we can [...] Activation of FLP will induce mitotic recombination at the two FRT s equences located at the same position on homologous chromosomes. This results in excision of GAL80 in the twin-spot daughter cells, activation of GAL4 and its reporter gene(s).

Thus, t he cluster of cells originates from a single progenitor cell that had GAL80 flipped out. Using MARCM to identif y of single cell or cluster of cells originating from a single progenitor cell , we hope to determine whether Using MARCM to induce single-cell clones, we can determine whether NBs formed at certain position contribute to only one cell type similar to the vertebrate system or there areis another mechanisms such as temporal factors behind cell fate determination. Therefore, if cell fate determination works in the same way for Drosophila as vertebrates, inducing clones at any time point at a specific position along the z-axis should result in the same cell type. However, if different cell types are found at different time point, then the different time window when a cell is born may play a role in cell fate determination.

The result of this cell fate mapping is significan ce i n its application toward understanding how cell fate determination works in Drosophila . The development of the medulla of the DrosophilaDrosophila optic lobe appears to be very similar has been shown to be similar to that of RPCs in vertebrates, where progenitor cells first proliferate to expand cell number followed by both symmetrical and asymmetrical differentiation. SuchThis similarity makes the DrosophilaDrosophila optic as a potentially useful model a possible model organism to be used in the to study of molecular mechanism RPC development in vertebrates. Drosophila is a model organism that can be easily manipulated and studied due to the variety and extensive arsenal of genetic tools as well the easiness of its care and maintenance compared to other vertebrate model organism. With many human genes for diseases conserved in Drosophila, this makes Drosophila a viable model organism for research.

Materials and Methods

Genetics

Flies were grown at 25 ° C unless otherwise noted. The following Gal4 enhancer trap lines with different insertion site were used in this study: 103509, 103701, 103879, 103869, and 103884 and the repor ter lines: UAS-mCD8GFP (second chromosome) .

Immunohistochemistry

Samples were fixed in 4% EM-grade formaldehyde in phosphate buffer saline (Fisher-Scientific, pH=7.4) with 0.3% Triton X-100. Immunohistochemistry was performed using standard procedures . The following antibodies were provided by the Developmental Studies Hybridoma Bank: BP104 anti-mouse (1:30) and NCAD2 anti-rat (1:50). Secondary antibodies (Jackson Immuno Research ) were used at the following dilut ions: anti-mouse Alexa 546 (1 :50 0) and anti-rat Cy5 (1: 1 00). Specimens were mounted with Vectashield mounting medium (Vector) and viewed on a confocal microscope.

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