A deficiency screen of the 3rd chromosome for dominant modifiers of the Drosophila ER integral membrane protein, Jagunal

Abstract The mechanism surrounding chromosome inheritance during cell division has been well documented, however, organelle inheritance during mitosis is less understood. Recently, the endoplasmic reticulum (ER) has been shown to reorganize during mitosis, dividing asymmetrically in proneuronal cells prior to cell fate selection, indicating a programmed mechanism of inheritance. ER asymmetric partitioning in proneural cells relies on the highly conserved ER integral membrane protein, Jagunal (Jagn). Knockdown of Jagn in the compound Drosophila eye displays a pleotropic rough eye phenotype in 48% of the progeny. To identify genes involved in Jagn dependent ER partitioning pathway, we performed a dominant modifier screen of the 3rd chromosome for enhancers and suppressors of this Jagn-RNAi-induced rough eye phenotype. We screened through 181 deficiency lines covering the 3L and 3R chromosomes and identified 12 suppressors and 10 enhancers of the Jagn-RNAi phenotype. Based on the functions of the genes covered by the deficiencies, we identified genes that displayed a suppression or enhancement of the Jagn-RNAi phenotype. These include Division Abnormally Delayed (Dally), a heparan sulfate proteoglycan, the γ-secretase subunit Presenilin, and the ER resident protein Sec63. Based on our understanding of the function of these targets, there is a connection between Jagn and the Notch signaling pathway. Further studies will elucidate the role of Jagn and identified interactors within the mechanisms of ER partitioning during mitosis.


Introduction
During cell division, it is well established that the genetic material in the form of condensed chromosomes is partitioned faithfully to the newly formed daughter cells.Lesser understood is the partitioning and inheritance of cytoplasmic material and organelles during cell division.Early models of organelle inheritance proposed a stochastic bulk inheritance of material, largely based on chance (Warren and Wickner 1996).However, recent studies have indicated a programmed pathway towards the inheritance of organelles similar to other factors necessary for cellular function and cell fate selection (Lowe and Barr 2007).Studies over the past decade have focused largely on the mitotic partitioning of the Golgi apparatus and the endoplasmic reticulum (ER), outlining both their dramatic reorganization of these organelles in frame with the cell cycle and their connection with the cytoskeleton during mitosis (Wei and Seemann 2009;Yagisawa et al. 2013;Bergman et al. 2015;Smyth et al. 2015).Recently, the highly conserved ER transmembrane protein Jagunal (Jagn) was identified to be necessary for the proper asymmetric division of ER during mitosis in proneuronal cells, as inhibition of Jagn led to a symmetrical partitioning of the ER and defects in asymmetric division during early embryonic development (Eritano et al. 2017).Jagn, a highly conserved ER integral membrane protein, has been linked to protein trafficking, cell differentiation, and the hematological disease, severe congenital neutropenia (Lee and Cooley 2007;Boztug et al. 2014;Eritano et al. 2017).Furthermore, null alleles of Jagn display a lethality during early larval stages indicating an essential role during development (Lee and Cooley 2007).However, the molecular mechanism involving Jagn in these functions is poorly understood.To better understand the role of Jagn in ER partitioning and cell fate selection, we sought to identify factors that interact with Jagn using a genetic screening approach.Transcript knockdown of Jagn utilizing RNA interference (RNAi) in the Drosophila compound eye displays a pleotropic rough eye phenotype.To identify genetic interactors, we conducted a dominant modifier screen, using a collection of gene deficiencies along the 3rd chromosome inconjunction with Jagn-RNAi and evaluated https://doi.org/10.1093/g3journal/jkad059Advance Access Publication Date: 18 March 2023

Mutant Screen Report
the rough eye phenotypes.Here, we found several genetic interactions with Jagn, including the glycoprotein Division Abnormally Delayed (Dally), the ER resident protein Sec63, and the γ-secretase subunit Presenilin (Psn).Based on previous studies involving these targets (Tsuda et al. 1999;Struhl and Greenwald 2001), there appears to be a connection between Jagn and established cell signaling pathways, indicating a regulatory connection in mitotic ER partitioning.

Drosophila strains and husbandry
Fly stocks and crosses were maintained at 25°C on standard Bloomington Drosophila Stock Center (BDSC) cornmeal medium.Third, left arm (3L) and right arm (3R) Deficiency (Df) chromosome kits were obtained from BDSC.The UAS-Jagn-RNAi [P{KK107655} VIE-260B] was obtained from the Vienna Drosophila Resource Center (VDRC), stock number 108991 (Dietzl et al. 2007).Other fly strains used in this study can be found in Supplementary Table 1.Any strains created in this study are available upon request.

Deficiency screening approach of the 3rd chromosome
The Ey-GAL4 transgenic line was cross to UAS-Jagn-RNAi, and ∼47% of the resulting progeny displayed a rough eye phenotype.A collection of 181 deficiency lines covering the 3rd chromosome were screened for modification of UAS-Jagn-RNAi rough eye phenotype.The UAS-Jagn-RNAi line on the 2nd chromosome was crossed to deficiency lines on the 3rd chromosome (Fig. 3).These transgenic lines were then crossed to Ey-GAL4/Cyo line on the 2nd chromosome, and the eye phenotypes of the progeny were scored.Over 60 flies that were heterozygous for UAS-Jagn-RNAi/Ey-GAL4 and each 3rd chromosome deficiencies were then scored for either a normal eye or rough eye phenotype.Rough eye progeny that fell between 30-60% were considered within control range, while rough eye progeny that were above 60% were considered enhancers, and rough eye progeny below 30% were considered suppressors.To confirm enhancers and suppressors outside the control range (between 30-60%), we used a Pearson's chi-squared test to determine a probability value and if there is a statistically significant difference between the expected (control) percentage and the observed (experimental) percentage.

Target gene screening for potential interactors of Jagunal
A total of 18 genes from suppressors and enhancer 3rd chromosome regions were chosen for further analysis.Several genes covered by the identified deficiencies were eliminated based on overlap with neighboring deficiencies that showed no modification.Genes of interest were selected based on biological roles and Gene Ontology (GO) annotations described in Flybase.org[versions FB2022_05 andFB2022_06] (Gramates et al. 2022) and the Alliance of Genome Resources [versions 5.0.0, 5.1.0,5.2.0, 5.2.1, and 5.3.0](Agapite et al. 2022).The UAS-Jagn-RNAi line on the 2nd chromosome was crossed to mutant lines on the 3rd chromosome (Fig. 4).These transgenic lines were then crossed to Ey-GAL4/Cyo line on the 2nd chromosome.A total of 60 flies that were heterozygous for UAS-Jagn-RNAi/Ey-GAL4 and each 3rd chromosome mutant were then scored for either a normal eye or rough eye phenotype.

Preparation and imaging using field emission scanning electron microscopy
Drosophila lines selected for imaging were placed into 1.5 ml Eppendorf tubes and fixed with 8% paraformaldehyde (Thermo Scientific Chemicals, 416780250) dissolved in PBS (Millipore-Sigma, P4417-100TAB).Samples were kept in the fixative solution at 4°C for 24 hours on a rotating stage.The fixative was removed and the samples were washed 4 times with PBS at 4°C for 10 minutes.To dehydrate the samples, anhydrous ETOH (Fisher Scientific, A405P-4) was added to the PBS solution at 4°C, beginning with a 50% concentration of ETOH.The concentration of ETOH was raised to 70%, 90%, 95%, and 100% with the samples maintained at 4°C on a rotating stage for 24 hours.The final 100% ETOH step was repeated twice.To complete the dehydration process, the ETOH was removed from the samples in a critical point dryer [Tousimis Autosamdri-815(A)].The dehydrated samples were individually mounted on 12.7 mm diameter Al stubs (Ted Pella Inc., 16111-9) using silver epoxy (Ted Pella Inc., 16043), and allowed to harden for 24 hours at room temperature.The samples were then coated with 15 nm of sputter-coated Au/Pd (Cressington 208HR) and stored under vacuum for 24-48 hours.Samples were then examined using a Carl Zeiss Ultra 55 Field Emission Scanning Electron Microscope (FE-SEM).Electron micrographs were recorded using an Everhart-Thornley detector at typical accelerating voltages of 1-2 keV.

RNA extraction and quantitative reverse transcriptase PCR
RNA was isolated from Jagn-RNAi/Eye-Gal4 whole-body flies using the PureLink RNA Mini Kit (Invitrogen, 12183025).8-10 flies were lysed using 1 ml of TRIZOL Reagent, and RNA was extracted according to the manufacturer's protocol.RNA was eluted in RNAse-free water, and the concentration of each sample was determined.qRT-PCR was performed using a CFX96 Touch Real-Time PCR Detection System.Extracted RNA was used for the qRT-PCR with an application by SYBR Green PCR master mix (Luna Universal One-Step RT-qPCR Kit, E3005).Transcript levels were normalized to Actin.The primer pairs for Actin42A are described in Ponton et al. (2011).Jagunal primer forward is CTACTTCTCGGACGTGTGGG and Jagunal primer reverse is AAGGCATACCAGAAGACGCC.

Inhibition of Jagunal leads to a rough eye phenotype
Studies over the past 30 years have shown that the Drosophila compound eye is an excellent model for investigating cell signaling and cell fate determination (Freeman 1997;St Johnston 2002;Kumar 2018).In order to investigate if Jagn disruption affects eye development, we expressed the transgenic line, UAS-Jagn-RNAi with the Eyeless-GAL4 (Ey-GAL4) driver (Brand and Dormand 1995).Eyeless is a transcription factor that is highly conserved and described as a master regulator of eye development (Halder et al. 1995).The Ey-GAL4 transgenic line has eyeless enhancer sequences upstream of the GAL4 transgene, to induce tissue specific GAL4 expression in the compound eye.Inhibition of Jagn within the eye displayed a pleotropic phenotype with ∼52% expressing a normal eye indistinguishable from controls, while 48% displayed rough eye phenotype, with 5% producing a severe eye phenotype including several that were eyeless (Fig. 1).In addition, we measured the extent of the Ey-GAL4/ UAS-Jagn-RNAi effect on Jagn transcript levels using quantitative RT-PCR for mRNA levels (Supplementary Fig. 1).There was a significant reduction in Jagn transcript levels with activation of the Jagn-RNAi construct using the Ey-GAL4 driver.Closer inspection of Ey-GAL4/UAS-Jagn-RNAi (referred to as Jagn-RNAi moving forward) rough eye phenotype displayed several defects involved in cell fate selection and asymmetric division during eye development (Ready et al. 1976;Wolff and Ready 1991;Leshko-Lindsay and Corces 1997).Specifically, we observed defects in the Drosophila compound eye including size, shape, and patterning of the ommatidia when Jagn-RNAi was expressed (Fig. 2a-d).There were several incidences of multiple bristles (Fig. 2a and d arrowheads) and multiple sockets (Fig. 2b arrows), as well as missing bristles and sockets (Fig. 2b-d asterisk).Additionally, there were examples where cell borders were not clearly defined (Fig. 2c) indicating possible defects in polarity or cell division (Kumar 2012).These defects led us to hypothesize that Jagn may play a role in mitotic orientation (i.e.spindle positioning) and/or cell fate selection.

Screening the 3rd chromosome deficiency collection
To identify possible genes that interact with Jagn, we employed a genetic approach using a dominant modifier screen for targets that either enhance or suppress the Jagn-RNAi rough eye phenotype.Our prior study (Eritano et al. 2017) suggested that the asymmetric partitioning of the ER transmembrane protein Jagn may play a role in cell fate selection.An earlier investigation into the connection between Jagn and the genetic condition, severe congenital neutropenia (SCN) employed a tandem affinity purification approach to identify interacting partners with Jagn (Boztug et al. 2014).While this approach was successful in demonstrating a link between Jagn and the secretory pathway, there was little evidence towards downstream activity and a role in the generation of cell diversity.Dominant modifier screening approaches using the Drosophila compound eye have been very successful in identifying missing components of developmental pathways including connections to cell signaling networks than other traditional genetic screening approaches (Simon 1994).Typically, traditional loss-of-function screening methods rely on use of a homozygous mutant which, if necessary for development, can lead to an early lethality, as is the case for Jagn (Lee and Cooley 2007).Dominant modifier screens using the compound eye allow for the investigation of Jagn interactors because the eye is not required for viability and fertility.In addition, this approach has been very successful in identifying components of conserved cell signaling pathways and patterning during development that traditional biochemical approaches were unable to accomplish (Banerjee et al. 1987;Simon et al. 1991;Halder et al. 1995;Lee et al. 2001).However, there are some caveats with performing a screen of this nature, including the presence of additional or second site modifiers being present in the genetic background which could confound the results (Brand and   crossing strategy that expressed the Jagn-RNAi transgenic line in combination with deficiency lines covering the 3rd chromosome (Fig. 3).These deficiencies are included in a defined kit provided by the BDSC and cover ∼96% of the genes found on the 3rd chromosome (see Methods and materials).These fly populations were scored for any changes in the population expressing the Jagn-RNAi rough eye phenotype indicating either an enhancement or suppression based on the genes disrupted by the defined deficiency.Here, we screened 104 deficiency lines covering the 3rd right (3R) arm of the chromosome and 77 lines covering the 3rd left (3L) arm of the chromosome.To identify modifiers of the Jagn-RNAi-induced rough eye phenotype, we set a defined range of percentages of progeny that show the rough eye phenotype between 30-60%.Any number of progeny below 30% showing a rough eye phenotype was considered suppressors, while any progeny showing a rough eye phenotype above 60% were considered to be enhancers.From our screening efforts, 159 deficiencies fell within the control range (between 30-60%), and 22 were outside of the control range.To determine if these observations were significant, we performed a Pearson's chi-squared analysis and determined the probability based on the expected (Jagn-RNAi control 47%) and the observed percentage of rough eye phenotype in combination with a deficiency line.Based on these criteria, we identified 12 deficiency lines that displayed a suppression of the rough eye phenotype, and 10 deficiency lines that displayed an enhancement of the rough eye phenotype (Table 1 and Fig. 4).
The deficiency line D(3L)ED208 displayed a strong enhancement of the Jagn rough eye phenotype (P value = 6.3 × 10 −9 ) and includes several genes involved in GPI anchor biogenesis in the ER and oogenesis development.This is in line with previous studies on Jagn involvement in oogenesis growth (Lee and Cooley 2007).The deficiency Df(3L)BSC800 displayed an enhancement of the rough eye phenotype at 88.3% (P value = 5.9 × 10 −8 ) of which, two genes of interest were identified, alphaCOP (αCOP) and neuronal synaptobrevin (nSyb).αCOP is part of the COPI coat complex involved in retrograde transport and is located at the ER-Golgi intermediate complex (ERGIC) (Girod et al. 1999), while nSyb is a neuronal SNAP protein involved in vesicle fusion (Kidokoro 2003).Both targets are in line with a role of Jagn in ER retention and the connection to the secretory pathway demonstrated in the Boztug et al. (2014) study.In addition, these targets are further supported by Jagn possessing a di-lysine ER retention motif (K(X) KXX) on its C-terminus indicating a role in retrograde transport (Lee and Cooley 2007).Df(3L)ED4421 showed a suppression of the rough eye phenotype (Table 1 and Fig. 4, P value = 0.002) and has several genes of interest including Dally, a heparan sulfate proteoglycan involved in germline stem cell maintenance and Klp67A, a kinesin motor involved in chromosome segregation and mitotic spindle assembly (Nakato et al. 1995;Savoian and Glover 2010).These targets are of interest based on the role that Jagn is involved in partitioning of the ER during mitosis (Eritano et al. 2017) and may associate with mitotic molecular motors like Klp67A.Dally was originally identified based on its role in cell division in the larval brain and was shown to be a member of the glypican family of integral membrane proteoglycans (Nakato et al. 1995).Also, in the Boztug et al. (2014) study involving the human ortholog of Jagn, JAGN1, there were defects in cell surface proteoglycans with JAGN1 mutated, which is in line with a connection with Dally cell signaling activity (Jackson et al. 1997;Takeo et al. 2005).The deficiency Df(3L)BSC839 displayed a strong enhancement of the Jagn rough eye phenotype (P value = 1.3 × 10 −7 ) with 87.30% (Table 1) containing the deleted gene, Psn, as well as 50 other deleted genes.In examination of two flanking Fig. 4. Modification of Jagn-RNAi rough eye phenotypes.Images of modification of the Jagn-RNAi rough eye phenotype with genes that were selected as possible genetic interactors.Examples of modification involving deficiency ED4421 crossed to a line containing Ey-Gal4/UAS-Jagn-RNAi and examined for modification of the Jagn-RNAi rough eye phenotype (see Fig. 3 for crossing strategy).ED4421 displayed a suppression of the Jagn-RNAi rough eye phenotype.Genes including Dally, Sec63, and Psn [C4] crossed to Ey-Gal4/UAS-Jagn-RNAi displaying a modification of the Jagn-RNAi rough eye phenotype (see Fig. 5 for crossing strategy).Dally showed a suppression of the rough eye phenotype, while Sec63 and Psn [C4] showed an enhancement of the rough eye phenotype.Fig. 3. Crossing strategy for Dominant modifier screen of the 3rd chromosome.In order to identify dominant modifiers of the Jagn RNAi induced eye phenotype, we used the following crossing strategy to screen the collection of deficiency lines on the 3rd chromosome.After two generations, a transgenic line was developed which included the Jagn RNAi line, ey-Gal4 and a 3rd chromosome deficiency.This line was screened for any enhancement or suppression of the Jagn RNAi eye defect.
deficiencies to Df(3L)BSC839, Df(3L)4858, and Df(3L)BSC797, they showed no significant changes to the rough eye phenotype, 33% and 44% respectively.This is within the control percentages, indicating no modification of the Jagn-induced rough eye phenotype by these flanking deficiencies.This led us to examine a smaller section of the Df(3L)BSC839 containing only 15 genes, with 7 being unannotated and 3 being non coding genes.The other 36 genes were removed from consideration based on their location on the flanking deficiencies which showed no significant changes in eye phenotype.This helped in narrowing down possible candidates, which included the γ-secretase subunit, Psn as an interactor with Jagn.

Creating a targeted list of genes as potential interactors of Jagn
Based on our screening efforts described above, we identified several regions of the 3rd chromosome that contain genes as possible interactors with Jagn (Table 1, Supplementary Table 2).Upon further examination of the genes found in these regions, there were several potential targets that we selected for further analysis.Identified genes that were found in deficiencies that showed an enhancement or suppression of the Jagn-RNAi-induced rough eye phenotype were tested based on information involving the Gene Orthology (GO) annotations provided by the Alliance of Genome Resources (Agapite et al. 2022) (Supplementary Table 2).Identification of these genes was also aided by a previous study involving an investigation of binding partners for the human ortholog, JAGN1 (Boztug et al. 2014).Studies involving human patients have linked JAGN1 to SCN, and an affinity purification approach was performed identifying several factors including machinery involved in COPI vesicle formation, microtubule binding, and membrane trafficking pathways.In addition, SCN is categorized by defects in N-linked glycosylation in primary neutrophils, which shares roles with targets, the proteoglycan Dally and the Notch regulator Psn (Nakato et al. 2002;Schäffer and Klein 2007;Sorensen and Conner 2010;Zhao et al. 2020).We identified several genes that are located in the deficiencies that showed a modification of the Jagn-RNAi-induced rough eye phenotype and sought to Listed are the deficiency lines covering the 3rd chromosome that indicated either an enhancement or suppression of the Jagn-RNAi-induced rough eye phenotype.Percentages of rough eye phenotype above 60% were considered enhancers, while percentages of rough eye phenotype below 30% were considered suppressors.Control numbers of Jagn-RNAi-induced rough eye phenotype without a deficiency were ∼47%.N = 60 eye counts for each cross.P values were determined using Pearson's chi-squared analysis.
Fig. 5. Crossing strategy of selected genes involved in modification of the Jagn-RNAi-induced eye phenotype.To examine specific genes involved in the modification of the Jagn-RNAi (red)-induced eye phenotype, we used the following crossing strategy to screen the mutant alleles of (purple) on the 3rd chromosome.After two generations, a transgenic line was developed which included the Jagn-RNAi line, ey-Gal4 (yellow) and the selected mutant allele.This line was screened for any enhancement or suppression of the Jagn-RNAi eye defect.
G. Ascencio et al. | 5 test these individual genes as possible interactors with Jagn.Supplementary Table 2 lists the genes selected to test based on the GO annotations describing their biological function, and mutations were genetically crossed with Jagn-RNAi line, and the eye phenotype was scored (Fig. 5).Several of the selected targets did not show any modification of the Jagn-RNAi rough eye phenotype (Table 2) including αCOP (P value = 0.1), Rab11 (P value = 0.2), αTub67c (P value = 0.02), mir-Ban (P value = 0.2), Arl61P1 (P value = 0.2), and mir-282 (P value = 0.9).These genes were selected based on their role in the secretory pathway, mitosis, and neural development (Urbé et al. 1993;Schroder-Kohne et al. 1998;Matthies et al. 1999;Saito et al. 2005;Wakil et al. 2019).In addition, there were a couple of predicted genes, CG32264 and CG739 that were of interest.CG32264 is predicted to be involved in actin binding activity and reorganization, while CG739 was a protein involved in mitochondrial translocation (Agapite et al. 2022).Unfortunately, both did not show any modification of the rough eye phenotype.We did see a suppression of the Jagn-RNAi rough eye phenotype (17%, P value = 4.8 × 10 −6 ) with the mutation Dally (Fig. 4, Table 2).We also saw a suppression (28%, P value = 0.002) with the mutation Eip63C, a cyclin-dependent kinase that interacts with CycY and is essential for development (Liu and Finley 2010).Mutations in Ccn (19%, P value = 1.9 × 10 −5 ), a gene predicted to enable heparin/integrin binding activity (Holbourn et al. 2008), also displayed a strong suppression of the rough eye phenotype.Interestingly, there was an enhancement (88%, P value = 5.1 × 10 −8 ) of the Jagn-RNAi rough eye phenotype with the Sec63 mutation (Fig. 4, Table 2).Sec63 is part of a complex of proteins that are involved in the translocation of mRNAs into the ER lumen (Linxweiler et al. 2017;Jung and Kim 2021) and interacts with ER chaperone protein, BiP.However, the role of Sec63 in ER translocation is still poorly understood, and substrates of Sec63 still remain to be identified.We also examined UAS-Jagn-RNAi expression in another tissue, the scutellum region using the P{GawB}455.2GAL4 line.
Here, we saw that expression of Jagn-RNAi displayed a modest defect in scutellum bristle formation (Supplementary Fig. 2b).We then examined Jagn-RNAi expression in unison with the identified suppressors BSC419, Dally, and Psn [C4] .Similar to our observations in the Drosophila compound eye, we saw a suppression of the bristle phenotype with Dally, Psn [C4] , and BSC419 (Supplementary Fig. 2c, d, and e).
Based on our examination of Df(3L)BSC839 and identification of the region including Psn, we sought to investigate if Psn displays a modification of the Jagn-RNAi-induced rough eye phenotype.Support for this interaction stems from the abovementioned study involving human JAGN1 and its interaction with the protein, adipocyte plasma membrane-associated protein (APMAP), an inhibitor of amyloid-beta (Aβ) aggregates (Boztug et al. 2014).Additionally, a recent study also showed that APMAP interacts with γ-secretase and involves in modulating its activity (Mosser et al. 2015).Here, we crossed mutations in Psn, the Drosophila ortholog of γ-secretase (Struhl and Greenwald 1999), with the Jagn-RNAi-induced rough eye phenotype and examine the progeny for a modification of the eye phenotype (Fig. 4, Table 2).We saw a strong enhancement of the rough eye phenotype (75.4%, P value = 0.0003) in the presence of the Psn mutation, indicating a genetic interaction between Psn and Jagn.
Overall, our screening efforts have identified several interactors with Jagn including Dally, Sec63, Eip63C, Ccn, and Psn.While this is not an exhaustive list of interactors along the 3rd chromosome, these modifiers do shed some light onto the role of Jagn and indicate a possible mechanism involved in the signaling and distribution of ER membrane in cells as they adopt their cell fate.Of particular interest is the HSP protein, Dally.Dally was initially identified as an integral membrane proteoglycan required for cell division during patterning formation in development (Nakato et al. 1995), and studies connected Dally function to Frizzled function in transducing Wg signaling (Lin and Perrimon 1999).However, over the past several years, studies have also liked Dally function to other signaling pathways including TGF-beta and JAK/STAT signaling.Recently, Dally also has been linked to Notch signaling involving maintenance of the germline stem cell niche (Zhao et al. 2020).This indicates that Dally plays a role in several signaling pathways and is upstream of Notch signaling involving stem cell development.
Further support involving a connection between Jagn and the Notch signaling pathway is the identification of the γ-secretase Drosophila ortholog Psn as a genetic interactor with Jagn.There have been several studies demonstrating that γ-secretase is responsible for cleavage and processing of the Notch receptor for transcription of genes involving the generation and differentiation of neuronal cells (Roncarati et al. 2002;Sorensen and Conner 2010).In addition, γ-secretase has also been linked to disorders including Alzheimer's disease and several types of cancer (Miele et al. 2006;De Strooper et al. 2012).
Future studies of Jagn function in proneuronal cells and its role in cell fate selection will focus on the connection to the Notch signaling pathway and the generation of cell diversity in the central nervous system.

Fig. 1 .
Fig. 1.Inhibition of Jagn displays pleotropic defects in eye development.Jagn-RNAi transgenic lines were crossed with the eyeless (Ey) Gal4 driver to inhibit Jagn function during eye development.Scanning Electron Micrographs (SEM) were taken of the Drosophila compound eyes and when Jagn is inhibited, 52% of progeny displayed eyes similar to controls (a), while 48% displayed pleotropic eye defects (b-e).In comparison with control eyes (a), inhibition of Jagn displayed a range of defects including moderate disruption of eye shape and size (b and c), to more severe defects in eye development shown in (d) and (e).Scale bar ∼100 μm.

Fig. 2 .
Fig. 2. Jagn deficient compound eyes display defects in cell division and development.Examination of individual ommatidium in eyes deficient for Jagn displayed defects in cell fate selection and cell division.These include ommatidia with multiple bristles (a and d arrowheads), or severe defects in a lack of bristles, multiple socket cells (arrows) (b), and a low number of ommatidia (b, c, and d).Additionally, defects were seen in missing sockets (asterisks), a lack of resolution of dividing ommatidia (c and d).a) Scale bar ∼20 μm, b) scale bar ∼10 μm, c) scale bar ∼10 μm, and d) scale bar ∼5 μm.

Table 1 .
List of 3rd chromosome deficiency lines modification of the Jagn-RNAi eye phenotype.