A Misexpression Screen to Identify Regulators of Drosophila Larval

Copyright � 2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.108.089094

A Misexpression Screen to Identify Regulators of DrosophilaLarval Hemocyte Development

Martin Stofanko, So Yeon Kwon and Paul Badenhorst1

Institute of Biomedical Research, University of Birmingham, Edgbaston B15 2TT, United Kingdom

Manuscript received March 14, 2008Accepted for publication July 14, 2008

ABSTRACT

In Drosophila, defense against foreign pathogens is mediated by an effective innate immune system, thecellular arm of which is composed of circulating hemocytes that engulf bacteria and encapsulate largerforeign particles. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. The mostabundant larval hemocyte type is the plasmatocyte, which is responsible for phagocytosis and is presenteither in circulation or in adherent sessile domains under the larval cuticle. The mechanisms controllingdifferentiation of plasmatocytes and their migration toward these sessile compartments are unclear. Toaddress these questions we have conducted a misexpression screen using the plasmatocyte-expressedGAL4 driver Peroxidasin-GAL4 (Pxn-GAL4) and existing enhancer-promoter (EP) and EP yellow (EY)transposon libraries to systematically misexpress �20% of Drosophila genes in larval hemocytes. The Pxn-GAL4 strain also contains a UAS-GFP reporter enabling hemocyte phenotypes to be visualized in thesemitransparent larvae. Among 3412 insertions screened we uncovered 101 candidate hemocyteregulators. Some of these are known to control hemocyte development, but the majority either haveno characterized function or are proteins of known function not previously implicated in hemocytedevelopment. We have further analyzed three candidate genes for changes in hemocyte morphology, cell–cell adhesion properties, phagocytosis activity, and melanotic tumor formation.

IN Drosophila, defense against foreign pathogens ismediatedby the innate immunesystem,which iscom-

posed of both a humoral and cellular arm. Humoralresponses include the rapid melanization and co-agulation reactions that accompany wound healing andthe production of antimicrobial peptides, principally bythe larval fat body. In larvae, the cellular arm consists ofcirculating hemocytes that engulf bacteria and apopto-tic cells and can encapsulate larger foreign particles.Three hemocyte cell types occur (reviewed in Lanot

et al. 2001; Evans et al. 2003; Meister and Lagueux

2003). The most abundant is the plasmatocyte, whichaccounts for 95% of circulating hemocytes. Plasmato-cytes are professional macrophages that can removeforeign material by phagocytosis and typically containresidual bodies or phagosomes containing lysosomalenzymes, reactive forms of oxygen, or nitric oxide(Jones et al. 1999). Less abundant are crystal cells(�5% of hemocytes), which are characterized by largecrystalline inclusions of prophenoloxidases (Fujimoto

et al. 1995) and are involved in melanin synthesis dur-ing pathogen encapsulation (Soderhall and Cerenius

1998) and wound healing (Lai-Fook 1966). The thirdhemocyte type is the lamellocyte. These large flattenedcells are rarely found in the larval hemolymph in the

absence of immune challenge. Lamellocytes encapsu-late foreign bodies and are responsible for the for-mation of melanotic tumors (Luo et al. 1995).

Two waves of hematopoiesis occur. The first occursduring embryonic development when a population ofhemocytes arises from the head mesoderm (reviewed inTepass et al. 1994; Ramet et al. 2002). These hemocyteseventually populate the whole embryo. Prior to larvalhatching, hemocytes are targeted to and concentrateat sites of cell death and phagocytose apoptotic cells(Abrams et al. 1993; Tepass et al. 1994). After larvalhatching, hemocytes of embryonic origin persist andreplicate in the larval hemolymph. At the same time, asecond wave of hematopoiesis occurs in the larval lymphgland (Lanot et al. 2001; Holz et al. 2003; Jung et al.2005). These hemocytes are liberated at metamorphosisand populate the pupa and adult fly (Holz et al. 2003).A number of transcription factors have been identifiedto control hemocyte development. All hemocytes ex-press the GATA factor Serpent (Srp) (Rehorn et al.1996; Lebestky et al. 2000), which is required for he-mocyte determination and maturation. Differentiationof plasmatocytes requires expression of Glial cellsmissing (Gcm) (Bernardoni et al. 1997; Alfonso andJones 2002). In turn, the transcription factors Lozenge(Bataille et al. 2005) and Collier (Crozatier et al.2004) are required for proper crystal cell and lamello-cyte development, respectively.

1Corresponding author: Institute of Biomedical Research, University ofBirmingham, Edgbaston B15 2TT, United Kingdom.E-mail: [email protected]

Genetics 180: 253–267 (September 2008)

Drosophila larvae have an open circulatory system.Circulation of the hemolymph (blood) is mediated bycontractions of the dorsal vessel and by peristalticmovements of the body. There are �5000 hemocytesin a mature third instar larva (Lanot et al. 2001). Abouttwo-thirds of these freely circulate in the hemocoel,but the remainder attach to the inner surface of theintegument (Lanot et al. 2001). These sessile cells formsegmentally repeated patches or ‘‘islets’’ of hemocytesand can also be found attached to the posterior of thelarva. The sessile hemocyte compartments are in directcontact with the cuticle and contain plasmatocytes andcrystal cells (Lanot et al. 2001).

Migration of hemocytes in the embryo is regulated bytwo mechanisms: chemotactic response to woundingand migration in response to signals from the platelet-derived growth factor/vascular endothelial growth fac-tor (PDGF/VEGF) ligands Pvf2 and Pvf3 (Wood et al.2006). However, the mechanisms controlling hemocyteadhesion and targeting to sessile islets in the larvae arenot completely understood. It is known that Rac1activation leads to release of cells from these domains(Williams et al. 2006). This release requires the activityof the Drosophila Jun N-terminal kinase Basket (Bsk)(Williams et al. 2006). In addition, sessile hemocytecompartments are disrupted at commencement of pu-pariation, suggesting that ecdysone signaling can mod-ify their adhesive properties.

In an effort to understand the mechanisms that controllarval hemocyte migration and differentiation, we haveconducted a misexpression screen using the GAL4-UASsystem (Brand and Perrimon 1993). We have used ahemocyte-specific GAL4 driver (Peroxidasin-GAL4) and alibrary of enhancer-promoter (EP) or EP yellow (EY)transposon insertion lines (Rorth 1996; Bellen et al.2004) to misexpress �20% of Drosophila genes in larvalhemocytes. The Pxn-GAL4 driver directs expression inplasmatocytes and crystal cells and also contains a UAS-GFPtransgene that allows visualization of hemocytes in thirdinstar larvae, which are semitransparent. Here we reportthe results of this screen. Among the 3412 insertionsscreened we identified 101 candidate genes that affecthemocyte development and migration. Detailed charac-terization of selected candidate genes is presented.

MATERIALS AND METHODS

Fly strains and genetic crosses: The w1118; Pxn-GAL4, UAS-GFP driver used is as described in Stramer et al. (2005) andwas a gift of M. Galko. The following UAS lines were used: UAS-nejire (Kumar et al. 2004) and UAS-Kruppel (Carrera et al.1998). hopTum-l is described in Luo et al. (2005). lozenge-lacZ(Bataille et al. 2005) was a gift of L. Waltzer. Lsp2-GAL4 wasobtained from the Bloomington Drosophila Stock Center. Thegain-of-function screen was performed with 567 P{EP} (EP)(Rorth 1996) and 2845 P{EPgy2} (EY) (Bellen et al. 2004)transposon insertion lines obtained from the Szeged andBloomington Drosophila Stock Centers, respectively. For each

cross, 5–10 virgin females of the w1118; Pxn-GAL4, UAS-GFPdriver line were independently crossed to 5 males of each EPand EY strain. For X chromosomal EP and EY insertions thatare male sterile, the cross was performed using 5–10 virgin EP/EY females and 5 males of the w1118; Pxn-GAL4, UAS-GFP driverline. Progeny larvae were staged using the blue gut method(Maroni and Stamey 1983) and 5–10 wandering third instarlarvae from each cross were scored for defects in hemocytedevelopment and distribution according to the parametersshown in Table 1. Hemocytes were visualized by GFP expres-sion using an Olympus SZX12 stereomicroscope with GFPfilter set. Candidate EP and EY lines that showed disruptedhemocyte development were retested to confirm that hemo-cyte phenotypes were reproducible. Lines that passed retestwere selected for further study. For each positive line, other EPand EY lines that contained transposon insertions in thevicinity of the positive insertion were tested for similar over-expression phenotypes. Typically, these were insertions withinthe same gene and/or insertions located up to 10 kbupstream/downstream from the original positive insertion.All overexpression phenotypes were recorded and listed insupplemental Table 1.

Immunohistochemistry: Circulating hemocytes were iso-lated from 10 wandering third instar larvae, staged as above.Larvae were ripped on ice in 200 ml of HyQ CCM3 culturemedium (HyClone) containing protease inhibitors (Com-plete, Boehringer). Hemocytes were pelleted by centrifuga-tion at 260 g for 10 min at 4�, the cells resuspended in 50 ml ofHyQ CCM3 culture medium with protease inhibitors, andtransferred to individual wells on a Multispot slide (PH-001; C.A. Hendley). Slides were incubated at room temperature for30 min in a humid chamber to allow cells to attach to the slidesand then fixed in 3.7% paraformaldehyde (Sigma) for 10 min.After fixation, slides were washed in phosphate-buffered saline(PBS) containing 0.1% Saponin (Sigma) for 10 min and thenblocked overnight at 4� in blocking buffer (PBS containing0.1% Saponin and 1% FCS). Primary antibody incubationswere performed for 1 hr at room temperature in blockingbuffer. All subsequent steps were performed in PBS containing0.1% Saponin. Slides were washed three times for 10 min,followed by secondary antibody incubation for 30 min. Washeswere repeated and the slides were mounted in Vectashieldmounting media with DAPI (Vector Laboratories). Sampleswere observed using a Zeiss Axiovert 100M confocalmicroscope.

To prepare sessile hemocytes, individual larvae were cut toremove circulating hemocytes. Portions of cuticle that con-tained adherent hemocyte sessile patches were transferred tomultispot slides in HyQ CCM3 containing protease inhibitors.

TABLE 1

Scoring criteria used in the gain-of-function screen

Criterion Scoring system

Hemocytes present Y/NTotal hemocyte number under the cuticle [/YTotal hemocyte number in circulation [/YDisrupted dorsal sessile compartment 1 (disrupted)�5

(wild type)Inappropriate targeting of hemocytes Y/NAccumulation along the dorsal vessel Y/NHemocytes spread throughout the cuticle Y/NLymph gland size [/YHemocyte shape changes N/A

254 M. Stofanko, S. Y. Kwon and P. Badenhorst

Sessile hemocytes were dislodged from the cuticle usinga tungsten needle. The hemocytes were allowed to attach for30 min and were fixed and processed for immunofluoresenceas described above.

The following antibodies were used at the listed dilutions:polyclonal chicken anti-GFP antibody (1:400, Upstate Bio-technology), mouse MAb L1b (1:60, Sinenko et al. 2004),mouse MAb H2 anti-Hemese (1:50, Kurucz et al. 2003), mouseMAb CF.6G11 anti-Mys (1:20, Manseau et al. 1997), rabbit antib-Int-y (1:2000, Yee and Hynes 1993), rabbit anti-b-galactosi-dase (1:2000, Cappel), mouse MAb 40-1a anti-b-galactosidase(1:100). Secondary antibodies from Jackson Immunoresearchwere: Cy3-conjugated anti-mouse IgG (H 1 L), FITC-con-jugated anti-chicken IgY (H 1 L), and Cy3-conjugated anti-rabbit IgG (H 1 L), all used at 1:1000.

Live imaging microscopy: Wandering third instar w1118; Pxn-GAL4, UAS-GFP larvae were washed and attached at theirdorsal cuticle to transparent adhesive tape. The tape was thenattached to a glass slide. Time-lapse images of GFP-expressinghemocytes were taken using a Zeiss Axiovert 100M microscopeconnected to a Hamamatsu C742-95 digital camera. Time-lapse images were analyzed using SimplePCI (Compix).

Molecular analysis: For selected lines we verified that theobserved blood phenotypes were due to Pxn-GAL4-mediatedoverexpression of genes flanking the EP or EY insertion sitesby analyzing gene expression using RT–PCR. Hemocytes wereisolated from a total of 300 third instar larvae of eachgenotype. Larvae were bled into HyQ CCM3 culture media con-taining protease inhibitors and hemocytes pelleted by centri-fugation at 260 g for 5 min at 4�. Hemocytes were washed twicewith PBS. mRNA was isolated from hemocytes using a mMACSmRNA isolation kit (Miltenyi Biotec) according to the manu-facturer’s instructions. cDNA was generated by reverse tran-scription using Superscript II (Invitrogen) at 42�. PCR wasperformed for 25–32 cycles using gene-specific primers de-scribed in Table 2.

Phagocytosis assay: Phagocytosis was monitored by injec-tion of India ink (black drawing ink; Stephens) into thehemocoel of third instar larvae. India ink was aliquoted into a1.5 ml microfuge tube and centrifuged at maximum speed for5 min to precipitate large particles. The supernatant wastransferred to a drawn glass injection needle (TW100F-4;World Precision Instruments). Staged wandering third instarlarvae were immobilized on double-sided tape (Scotch 665;3M) attached to a glass slide. Larvae were injected with Indiaink using an Eppendorf Femtojet microinjector and LeitzLabovert inverted microscope with injection stand and needleholder (Leitz). Injections were made laterally through thelarval cuticle into the body cavity. Larvae were incubated for40 min at room temperature and hemocytes prepared formicroscopy as described above.

RESULTS

Characterization of the Pxn-GAL4 driver line: ThePeroxidasin-GAL4 (Pxn-GAL4) driver line expresses GAL4in embryonic macrophages and has proven to be a usefultool for live imaging of macrophages during embryonicwound healing (Stramer et al. 2005). We reasoned thatPxn-GAL4 would be a suitable GAL4 driver to be used in again-of-function genetic screen for regulators of larvalhemocyte development. To confirm the suitability of Pxn-GAL4, we first established its expression profile in larvalhemocytes. As shown in Figure 1A, we observed that Pxn-GAL4 was able to drive UAS-GFP expression in larvalhemocytes marked by the pan-hemocyte marker anti-Hemese. GFP expression was detected in 96% of circu-lating hemocytes of third instar larvae (data not shown).Expression was detected in both plasmatocytes (Figure1A) and crystal cells (Figure 1C), but was not detected inmature lamellocytes (Figure 1D).

As Drosophila larval cuticles are transparent, thedistribution of Pxn-GAL4-expressing cells in live animalscould be examined by viewing expression of a UAS-GFPreporter. As shown in Figure 2, hemocyte expression ofPxn-GAL4 could be detected in all larval instars. Expres-sion was largely restricted to hemocytes althoughweak expression could be observed in the fat bodyof third instar larvae. From the second larval instar,GFP-expressing hemocytes could also be detected in thelymph gland, consistent with previous reports ( Jung

et al. 2005). Expression was noted in circulating hemo-cytes but two populations of adherent or sessile cells

Figure 1.—Hemocyte expression of the Pxn-GAL4 driver.(A) Circulating and (B) sessile hemocytes were isolated fromwandering-stage Pxn-GAL4, UAS-GFP larvae and stained withantibodies against GFP (green) and the pan-hemocyte markerHemese (red). (C) Pxn-GAL4 directed expression of GFP(green) overlaps expression of the crystal cell marker lozenge-lacZ (lz-lacZ) (red). (D) Lamellocyte overproduction was trig-gered by introducing one copy of the hopTum-l mutation intothe Pxn-GAL4, UAS-GFP background. Lamellocytes (arrow-heads, revealed by MAb L1b staining in red) do not expressGFP (green). GFP-expressing plasmatocytes (asterisks) arenot MAb L1b positive. Bar, 20 mm.

Regulators of Hemocyte Development 255

were also detected. From the first larval instar onwards,GFP-positive cells accumulated at the posterior of thelarvae (arrowheads in Figure 2, A–C). From the secondlarval instar, GFP-expressing cells could also be observedin segmentally repeated patches along the dorsal mid-line on the inner surface of the integument (asterisks inFigure 2, B and C). These sessile hemocyte compart-ments bore superficial similarity to the hemocyte isletspreviously reported by Kitagawa and co-workers (Narita

et al. 2004).Antibody staining of these GFP-positive cells using

the pan-hemocyte marker anti-Hemese, confirmed theiridentity as hemocytes (Figure 1B). Time-lapse liveimaging of these sessile domains in Pxn-Gal4, UAS-GFPthird instar larvae showed binding of circulating hemo-cytes to these regions (supplemental Figure 1), suggest-ing that they arise by recruitment of hemocytes out ofcirculation. During the course of live imaging of thesesessile hemocyte patches, rearrangement of hemocyteswithin the compartment, and detachment of cells, wasalso observed (supplemental Figure 2), suggesting thatthey are dynamic. Indeed, shortly before pupariation,these sessile populations disappeared as the cells beganto detach from the regions under the cuticle (data notshown).

Gain-of-function screen: Prior to embarking on alarge gain-of-function screen we conducted a pilotscreen to establish a baseline for screening criteriaand the range of possible phenotypes. We selected anumber of UAS lines that express genes previouslyshown to affect hemocyte development, as well as anumber of random EP insertions in genes with nopreviously described hemocyte function. As a result, aset of nine criteria was established upon which to scoregain-of-function hemocyte phenotypes (Table 1). Theseincluded changes in hemocyte number; disruption of

dorsal sessile hemocyte compartments; inappropriatetargeting of blood cells, including accumulation alongthe dorsal vessel and spreading of hemocytes through-

TABLE 2

Primer pairs used for RT–PCR analysis

Gene Primer name Primer sequence (59 to 39) Product length (bp)

CG32813 CG32813_5P ACTCACAGGATGCCTTGGTC 167CG32813_3P CTCCTGGGGATCCTGATTCT

CG11448 CG11448_5P CCGGCCAGTCTAAATCAAGG 163CG11448_3P GAACGAGGATCGGACTGAGA

CG15321 CG15321_5P TGTGTCTGCTGCTGTTCGAC 180CG15321_3P CCGCTTGACCAGTACCTCTT

nejire nej_5P CCAGCACATCCTTGCCATAC 236nej_3P GCTGCTGGTTCATCATGTGC

buttonhead btd_5P GCCAGTTCATCCTCATCCTC 247btd_3P GATAACTGGCCGCATACTCG

Kruppel kr_5P ATGCTTGTTACCGCCAAACC 156kr_3P GCATGTTTAGAGCGCCGATT

Hemese He_5P TGGCACTCGGCGGAGCAGTTCACACTAAGT 491He_3P GGTACTTTCAATGGGCTTGCATTGCTCTTT

rp49 rp49_5P TCTTGAGAACGCAGGCGACCGTTGGGGTTG 399rp49_3P ATCCGCCACCAGTCGGATCGATATGCTAAG

Figure 2.—Temporalprofileofhemocyteaccumulation.GFP-expressing hemocytes were visualized in Pxn-GAL4, UAS-GFPfirst, second, and third instar larvae. (A) In first instar larvae asessilepopulationofhemocytes formsat theposteriorofthelarva(arrowhead). (B) By the second instar larval stage, this posterioraccumulation (arrowhead) is accompanied by the formation ofdistinct segmentally repeated dorsal patches or compartments(asterisks). (C) Third instar larvae show an increased numberof hemocytes forming distinct dorsal sessile patches (asterisks)and accumulations at the tail region (arrowhead).


out the cuticle; changes in lymph gland size; andchanges in hemocyte shape.

The full screen was then performed as shown inFigure 3. We screened 567 EP and 2845 EY lines (3412insertions in total) and recovered 108 insertions thatdisrupted hemocyte development according to at leastone of the criteria in Table 1. These insertions corre-sponded to 101 gene loci as we had recovered a numberof cases in which more than one selected EP/EYinsertion had targeted the same locus. For example,nejire (nej) had been identified on the basis of over-expression phenotypes of both nejEP1149 and nejEP1179. Asshown in Figure 4, the most commonly observed phe-notypes were disruption of dorsal sessile hemocytecompartments (58/108 insertions), changes in lymphgland size (54/108 insertions), changes in hemocytenumber (37/108 insertions), accumulation of sessilehemocytes along the dorsal vessel (23/108 insertions),and spreading of sessile hemocytes throughout thecuticle (10/108 insertions). In numerous cases a com-bination of these phenotypes was observed. For exam-ple, scabEY10270 (which overexpresses the a-integrinsubunit aPS3) results in decreased circulating hemo-cyte number and lymph gland size, disrupts sessiledorsal sessile hemocyte compartments, and causesaccumulation of hemocytes along the dorsal vessel. Afull list of positive insertions and their phenotypes isprovided in Table 3.

Disruption of dorsal sessile hemocyte compartments:We identified 58 lines representing 55 loci in which thedorsal sessile hemocyte patches were disrupted. This isthe largest class of EP/EY insertions and includes genesthat may regulate the adherent or migratory propertiesof hemocytes, for example, scab (scb, EY10270); C3G

(EP1613), a Ras family guanine nucleotide exchangefactor (Ishimaru et al. 1999); RhoGEF2 (EY08391),which has been shown to direct cell shape changes(Barrett et al. 1997) and control invagination ofmesodermal and endodermal primordia during gastru-lation (Hacker and Perrimon 1998); and tousled-likekinase (tlk, EP1200), which has been identified as aregulator of Rho signaling (Gregory et al. 2007).

This class is also composed of a number of transcrip-tion factors and chromatin modifying enzymes. Asshown in Figure 5B, overexpression of Kruppel (Kr) in

Figure 3.—Schematic of the screen. The Pxn-GAL4, UAS-GFP driver line was crossed to a set of EP and EY lines. TheGFP blood expression pattern was recorded and the lines thatshow deviations from the parental pattern were retained (pos-itives) and retested. For lines that pass the retest, EP/EY linesthat contain insertions in the same gene or in close proximitywere scored again for identical blood phenotypes.

Figure 4.—Observed hemocyte phenotype classes. (A) He-mocyte distribution in wild-type third instar larvae. Principaloverexpression phenotypes were (B) disruption of dorsal ses-sile hemocyte compartments; (C) increases in lymph glandsize; (D) increases in hemocyte number; (E) relocalizationof hemocytes to the dorsal vessel; and (F) spreading of hemo-cytes through the cuticle. Hemocytes are indicated as circles,lymph glands (lg) are shaded in the anterior of the larva, thedorsal vessel (dv) runs the length of the animal. Thoracic andabdominal segments are indicated (T1–A8).


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Y146

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ased

Dec

reas

ed3

YY

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orm

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CG

3318

2P

{EP

gy2}

EY1

0737

2RY

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ease

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ease

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CG

3363

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NC

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}EP

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ease

dN

CG

3624

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2}E

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ased

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reas

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orm

alY

(con

tin

ued

)


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(Co

nti

nu

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Gen

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CG

3654

P{E

Pgy

2}E

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ased

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ease

dN

CG

4807

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2}E

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ease

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sed

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4284

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Incr

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rmal

NC

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Dec

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2RY

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ease

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CG

5104

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rmal

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0181

3RY

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YN

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ease

dN

CG

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orm

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crea

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3RY

Dec

reas

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NN

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rmal

NC

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{EP

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2LY

Incr

ease

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crea

sed

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NN

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rmal

NC

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0183

3LY

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rmal

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NN

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ease

dN

CG

6114

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743L

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ased

Dec

reas

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crea

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NC

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crea

sed

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gy2}

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Dec

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ased

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NN

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rmal

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{EP

gy2}

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8303

3LY

No

rmal

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rmal

5N

YN

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ease

dN

CG

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P{E

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sed

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rmal

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crea

sed

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{EP

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2LY

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chn

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2}E

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2}E

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2LY

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3RY

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NN

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2RY

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sed

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NN

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P{E

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2}E

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orm

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orm

al4

YY

NN

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27C

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P74

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orm

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NN

NN

orm

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InR

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2}E

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813R

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crea

sed

No

rmal

4Y

NN

No

rmal

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gr1

P{E

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2}E

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crea

sed

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crea

sed

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3LY

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(con

tin

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)


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rmal

NN

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ased

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hemocytes using EY11357 disrupted sessile dorsal com-partments and resulted in isolated large flattened cells(Figure 5B9). Kr is a segmentation gene, which is alsoknown to regulate muscle identity (Ruiz-Gomez et al.1997). Two insertions in the Drosophila CBP homolognejire (EP1149 and EP1179) were also observed todisrupt sessile hemocyte domains as well as reducecirculating and sessile hemocyte numbers (Figure 5, Cand C9). Other transcription regulators that displayedthis phenotype included escargot (esg), which regulatescell adhesion and motility during tracheal branchfusion (Tanaka-Matakatsu et al. 1996); charlatan(chn), a zinc finger repressor of Delta and regulator ofproneural gene expression (Escudero et al. 2005;Tsuda et al. 2006); and distal antenna (dan).

Disruption of the dorsal sessile hemocyte compart-ments was often accompanied by relocalization ofhemocytes to ectopic locations. Two main classes ofphenotype were observed: (i) hemocytes accumulatedalong the dorsal vessel and (ii) hemocytes were spreadthroughout the larval cuticle.

Hemocytes accumulate along the dorsal vessel: Weidentified 23 lines representing 21 loci in which sessilehemocytes were targeted to the dorsal vessel. One ofthe most striking examples was generated by overex-pression of CG32813 using EY07727 (Figure 5, D andD9). The functions of CG32813 are unclear, but it is ex-pected that this phenotype could be induced by changesin adherent or migratory properties of hemocytes.This is confirmed by other insertions that display thesame phenotype. These included raspberry (EP1519),which has been implicated in the control of axonguidance (Long et al. 2006), and Rho signaling (Gregory

et al. 2007), and as described previously, scb, C3G, and tlk.Spreading of hemocytes throughout the cuticle: In

10 instances sessile hemocytes were observed to spreadthroughout the larval cuticle. This group comprised anumber of known transcription factors such as esg orproteins that are predicted to function as transcriptionregulators such as the zinc finger protein CG12701 andCG2034, the Drosophila homolog of DERP6 (Yuan et al.2006). An additional member of this class included Nelf-E, which encodes the RNA-binding subunit of NELF, anegative regulator of the RNA polymerase II transcrip-tion elongation (Yamaguchi et al. 1999). There is someevidence that regulation of transcript elongation by

Figure 5.—Selected overexpression phenotypes and geneontology classification of candidate genes. (A) Hemocyte dis-tribution in control Pxn-GAL4, UAS-GFP third instar larvae.(A9) Detail of the posterior of the same larva showing the dis-tinct dorsal sessile compartments. (B) Pxn-GAL4 directed ec-

topic expression of Kr changes hemocyte distribution andmorphology. (B9) Large flattened cells are observed. (C) Ec-topic expression of nej results in generally weaker GFP expres-sion in most hemocytes in both circulating and sessilepopulations apart from a few highly expressing cells. (D) Ec-topic expression of CG32813 results in inappropriate hemo-cyte targeting along the posterior of the dorsal vessel(enlarged detail shown in D9). GO classification of the 101identified candidate genes according to (E) biological pro-cess, and (F) molecular function. GO annotations were ob-tained from FlyBase.


NELF may be used to control cell fate determination inDrosophila (Wang et al. 2007).

Increased hemocyte number: We identified 37 linesrepresenting 36 loci in which hemocyte number wasincreased. This class includes genes that have previouslybeen linked to cell proliferation, for example, bantam(EY09041), a microRNA that controls proliferation andapoptosis (Brennecke et al. 2003); the DrosophilaCHK1 homolog grapes (EY09600); the Drosophila In-sulin-like receptor (EY00681), which can control cell sizeand number (Brogiolo et al. 2001); and kayak(EY12710), the Drosophila Fos homolog. This class alsoincluded a number of nucleic acid-binding and chro-matin-modifying enzymes that have not previously beenlinked to cell proliferation or apoptosis, for example,bruno-3 (EY08487), an RNA-binding protein that canbind to the EDEN translational repression sequence(Delaunay et al. 2004). Other genes of interest includethe histone H3K9 demethylase CG33182 (EY10737),the Drosophila SMARCAD1 chromatin remodelingenzyme CG5899 (EP701), the transcriptional regulatoreagle (EY00149), and the JAK/STAT pathway repressorjim (EY14392) (Mukherjee et al. 2006).

Increased lymph gland size: This was the second mostcommonly observed phenotype and included 54 inser-tions corresponding to 53 loci. Genes that resulted inincreased hemocyte number were often included in thiscategory, for example, bantam, bruno-3, CG33182, eagle,kayak, and jim. Additional genes that may control cellproliferation or survival that were identified includedcdc14 (EY10303), the EGF receptor ligand Keren(EY11963), and CG11134 (EP514), the Drosophilahomolog of the APIP inhibitor of cell death. In additionto the histone H3K9 demethylase CG33182, the Dro-sophila Jumonji homolog CG3654 was also identified asa regulator of lymph gland size.

Functional classification of screen selected genes:Gene ontology (GO) classification of the 101 identifiedgenes according to biological process and molecularfunction (validated by FlyBase) revealed that morethan half of the genes identified in the screen hadno previously described function. The next most abun-dant GO category was transcription factors (biologicalprocess, Figure 5E) or nucleic acid binding proteins(molecular function, Figure 5F). Examples includedKr, esg, chn, and broad (br), a primary responder to ec-dysone signaling that is also required for proliferationand differentiation of lamellocytes and crystal cells(Sorrentino et al. 2002).

Another abundant class of genes included those in-volved in signal transduction. Examples included C3Gand the Drosophila homolog of Transducer of ERBB2(Tob). Tob proteins contain a conserved N-terminalBTG domain and were initially identified as negativeregulators of cell proliferation, although have now beenshown to participate in a number of signaling pathways( Jia and Meng 2007). The observed phenotype with

decreased hemocyte number is consistent with a func-tion in hemocyte proliferation. The gene Hormonereceptor-like in 38 (Hr38) appears to mediate an atypicalEcdysteroid signaling pathway (Baker and Zitron

1995). When overexpressed in blood cells using theEY14161 responder, sessile hemocyte patches are dis-rupted. It is known that the ecdysone signaling pre-ceding pupariation disrupts sessile hemocyte patches.

Other categories of genes that were detected in-cluded regulators of cell adhesion, for example, scband Fasciclin 1; cell cycle regulators, for example, grapesand cdc14; and genes controlling cytoskeleton dynam-ics. The latter were of particular interest given thatidentification of modifiers of hemocyte migration andadhesion was an objective of this screen. Examplesincluded RhoGEF2 which has been shown to direct cellshape changes (Barrett et al. 1997) and controlinvagination of mesodermal and endodermal primor-dia during gastrulation (Hacker and Perrimon 1998)and microtubule star.

Further analysis of selected EP/EY lines: On thebasis of the phenotypes obtained, we selected threegenes for further analysis: the segmentation gene Kr, thehistone acetyltransferase nej, and the gene of unknownfunction CG32813. In an initial series of experiments wevalidated that the observed overexpression phenotypesobtained using EP/EY insertions in these genes were infact due to overexpression of the tagged gene. A firstsimple test was to confirm that other EP/EY insertionsthat are inserted in a location and orientation tooverexpress the same gene yielded an identical mis-expression phenotype. Conversely, EP/EY lines that areinserted in the vicinity of the gene, but in an orientationthat does not allow overexpression of the gene shouldnot yield the same phenotype. An example is shown inFigure 6A where the EP insertions nejEP1149 and nejEP1179,which are both predicted to overexpress nej, bothgenerated the same overexpression phenotype. Con-versely, nejEP950 and nejEP1410, which are inserted in reverseorientation and should not overexpress nej, failed togive a phenotype when crossed to Pxn-GAL4.

Next we verified by RT–PCR that EP/EY lines selectedin the screen did indeed overexpress the putative targetgene. Hemocytes were isolated from third instar larvaeof the appropriate genotypes, mRNA was purified, andthe abundance of transcripts was determined usinggene-specific primers. As shown in Figure 6, in all casesanalyzed EP/EY insertions selected in the screen wereable to drive significant overexpression of the desig-nated target gene. Thus when crossed to Pxn-GAL4 bothnejEP1149 and nejEP1179 significantly increased expression ofnej relative to the parental Pxn-GAL4 control (Figure6A). Similarly, KrEY11357 and CG32813EY07727 also predom-inantly increased expression of their correspondingtarget genes (Figure 6, B and C).

Finally, phenotypes were confirmed using existingUAS lines, which express only the cDNA encoding the


gene of interest. Thus, phenotypes obtained after crossingnejEP1149 or UAS-nej (Kumar et al. 2004) to Pxn-GAL4 wereidentical (data not shown). Similarly, overexpressionphenotypes obtained using KrEY11357 and UAS-Kr (Carrera

et al. 1998) were indistinguishable.Hemocyte phenotypes of selected EP/EY lines: Next

we determined the changes that occurred in hemocytesafter overexpression of Kr, nej, or CG32813 that couldhave accounted for the observed phenotypes. Hemo-cytes were isolated from wandering-stage third instarlarvae and stained first with the pan-hemocyte markeranti-Hemese to reveal alterations in hemocyte morphol-ogy. As shown in Figure 7B, overexpression of Kr inhemocytes resulted in differentiation of many largehemocytes together with smaller hemocytes of irregularprofile, unlike the parental Pxn-GAL4/1 driver controlin which a uniform population of round plasmatocyteswas observed (Figure 7A). After overexpression of nej(Figure 7C) and CG32813 (Figure 7D) most hemocytesappeared wild type, although a few larger cells couldalso be detected.

These larger cells appeared similar to lamellocytes, ahemocyte type that is rarely found in circulation. Toestablish if overexpression of Kr, nej, or CG32813 led tothe production of lamellocytes, isolated hemocytes werestained with the lamellocyte-specific antibody MAb L1b.As shown in Figure 7, E–H, overexpression of any ofthese proteins was able to trigger increased lamellocyte

number. Counts of lamellocyte number as a proportionof total hemocyte number revealed that lamellocytefrequency increased from 0.5% in the parental Pxn-GAL4/1 driver control to 9.5% after Kr overexpression,5.5% after nej misexpression, and 3.8% after CG32813overexpression.

However, differentiation of the alternative lamello-cyte cell type could only partially explain the changesin hemocyte distribution observed with these lines, aslamellocyte frequencies were still comparatively low. Toascertain, whether adhesive properties of hemocyteswere changed, we examined expression of the Drosoph-ila b-integrin subunit Myospheroid (Mys). Weak expres-sion of Mys was detected in the parental Pxn-GAL4/1

driver control (Figure 7M). In contrast, strong upregu-lation of Mys in hemocytes .15 mm was seen in alloverexpression experiments (Figure 7, N–P). Thissuggested that adhesive properties of these cells hadaltered. Consistent with this, staining of filamentousactin (F-actin), using rhodamine-phalloidin, revealedchanges in actin polymerization following overexpres-sion of Kr, nej, and CG32813. Weak F-actin staining wasdetected in plasmatocytes of the parental Pxn-GAL4/1

driver control (Figure 7I). However strong F-actinstaining was detected in Kr (Figure 7J), nej (Figure7K), and CG32813 (Figure 7L) overexpressing hemo-cytes, suggesting that these hemocytes are activelypolymerizing and depolymerizing their actin cytoskele-

Figure 6.—Validation of selected EP/EY inser-tions. Expression of genes flanking EP/EY inser-tions identified in the screen was determined byRT–PCR. Insertions identified in the screen areindicated in boldface type while those that failedto give a hemocyte phenotype are shaded. (A)Two EP insertions in the nejire (nej) locus,EP1149 and EP1179 were selected as positivesin the screen. RT–PCR on isolated hemocytes re-veals that both EP1149 and EP1179 drive signifi-cant overexpression of nej but not of theflanking genes, buttonhead (btd) and CG15321.The EP insertions EP950 and EP1410 (shaded),which are inserted in an opposite orientationto EP1149 and EP1179 and are not predicted tooverexpress nej, do not give a hemocyte overex-pression phenotype. (B) RT–PCR confirms thatthe EY11357 insertion drives overexpression ofKr when crossed to Pxn-GAL4. No other predictedgenes occur within 10 kb upstream or down-stream of EY11357. (C) Two EY insertions inthe CG32813 locus, EY07727 and EY14694, wereidentified in the screen. RT–PCR indicates thatEY07727 drives overexpression of CG32813 whencrossed to Pxn-GAL4. CG11448, which flanksCG32813, shows slight increase in expression.However, the EY06476 insertion, which is pre-dicted to drive overexpression of CG11448, doesnot give a hemocyte overexpression phenotype.In A–C, He and rp49 are used as loading controlsand to assess mRNA purity.


ton network, consistent with changes in their adherentproperties.

As Pxn-GAL4 is expressed at low levels in the larval fatbody, the other principal immune-competent tissue, thepossibility existed, albeit slight, that the hemocytephenotypes observed above were triggered indirectly.Potentially, low-level misexpression of Kr, nej, orCG32813 in the fat body may have disrupted the fatbody, in turn eliciting a response in hemocytes. Toexclude this possibility, we overexpressed Kr, nej, orCG32813 exclusively in the fat body using the Lsp2-GAL4driver and found no change in any of the hemocyteproperties examined (data not shown). This confirmedthat Pxn-GAL4 hemocyte phenotypes were cellautonomous.

Finally, we tested whether hemocytes were still func-tional after overexpression of either Kr or nej. Hemo-cytes in third instar larvae play a key role in innateimmunity by phagocytosing foreign material. Normallythis includes invading bacteria, but particles of India ink(a mixture of carbon particles of heterogeneous size)can also be recognized and engulfed by hemocytes.Forty minutes after injection of India ink into wander-ing-stage third instar larvae, particles of India ink can bedetected in lysosomes in hemocytes of the parental Pxn-GAL4/1 driver control (Figure 8A). Hemocytes thatoverexpressed nej were also able to engulf India ink

particles, suggesting that they are still competent toengage in phagocytosis (Figure 8C). In contrast, hemo-cytes that overexpressed Kr did not engulf India inkparticles. Instead hemocytes can be observed to adhereto larger particles of India ink (Figure 8B). It appearedthat these cells had switched from a pathway in whichsmall particles are engulfed to one in which largerparticles are encapsulated.

Figure 7.—Hemocyte phenotypes follow-ing Pxn-GAL4 mediated overexpression ofKr, nej, and CG32813. (A–D) Hemocytemorphology was revealed using the pan-hemocyte marker anti-Hemese (MAb H2).In the parental Pxn-GAL4 driver a uniformpopulation of round plasmatocytes is de-tected. Overexpression of nej, Kr, orCG32813 results in the appearance of lamel-locytes (arrowheads) and some hemocyteswith irregular profiles, presumably activatedhemocytes. (E–H) Antibody staining usingthe lamellocyte marker MAb L1b, reveals in-creases in lamellocyte number followingPxn-GAL4-mediated overexpression of nej,Kr, and CG32813. (I–L) Filamentous actin(F-actin) was revealed using rhodamine-phalloidin. (I) Weak F-actin staining is de-tected in plasmatocytes of the Pxn-GAL4driver line. Strong F-actin staining is de-tected in large hemocytes in ( J) Kr-, (K)nej-, and (L) CG32813-overexpressing he-mocytes, suggesting that these contain anactively polymerizing cytoskeleton network.(M–P) Antibody staining using antibodiesagainst the Drosophila b-integrin Myosphe-roid (Mys) shows weak expression of Mys incontrol hemocytes (M), but strong expres-sion in (N) Kr, (O) nej, and (P) CG32813overexpressing hemocytes. Upregulationof Mys was most apparent in hemocytes.15 mm. In A–P DNA (visualized usingDAPI) is shown in white and antibody- andphalloidin-staining in green. Bar, 20 mm.

Figure 8.—Phagocytic properties of hemocytes. Hemo-cytes were isolated from wandering-stage third instar larvae40 min after injection of India ink particles and visualized us-ing phase contrast microscopy. (A) Small particles of India inkwere detected in lysosomes (arrowhead) of hemocytes of theparental Pxn-GAL4, UAS-GFP driver indicating that phagocyto-sis was normal. (B) Larger hemocytes present after Kr overex-pression did not engulf India ink particles but rather adheredto large particles of India ink (arrowheads). (C) Hemocytesoverexpressing nej were still capable of engulfing India inkparticles, detected in lysosomes (arrowhead). Bar, 20 mm.


This confirmed our earlier results in which we ob-served increased lamellocyte numbers in Pxn-GAL4-xUAS-Kr larvae. An encapsulation response is typical oflamellocytes and differentiation of lamellocytes is animportant component of the innate immune responseto larger pathogens such as parasitic wasp eggs. How-ever, ectopic differentiation of lamellocytes also canlead to the inappropriate targeting and encapsulationof host tissue, leading to the production of inflamma-tory melanotic tumors. Pxn-GAL4xUAS-Kr larvae areviable and survive to the adult stage. However, �40%of these flies exhibit melanotic tumors, consistent withderegulated lamellocyte function (data not shown).

DISCUSSION

In this study, we have conducted a misexpressionscreen to identify factors that regulate Drosophila larvalhemocyte development and function. Using a hemocytespecific GAL4 driver, Pxn-GAL4, we screened 3412 EPand EY insertions and uncovered 108 insertions corre-sponding to 101 candidate genes that may be regulatorsof hemocyte development. This corresponds to a hitrate of 3.2% and compares favorably with recovery ratesobserved previously in gain-of-function screens in othertissues using EP elements. For example, 4.6% of EPinsertions generate a phenotype when overexpressed inadult sensory organs (Abdelilah-Seyfried et al. 2000).Similarly, 1.5% of EP insertions screened affectedmuscle pattern formation (Staudt et al. 2005) and 9%of EP insertions screened generated a phenotype inthe adult dorsal thorax (Pena-Rangel et al. 2002). Wefound that hit rates obtained using EP and EY lines weresimilar. Thus, 4% of EP lines screened affected hemo-cyte development while 3% of EY lines screened werepositive.

As part of preparatory work for the screen weperformed detailed characterization of the Pxn-GAL4driver at all larval instars, showing that it expresses in twoDrosophila hemocyte lineages: plasmatocytes and crys-tal cells. This analysis also revealed that two distinctgroups of plasmatocytes—a circulating and a sessilepopulation—exist at all larval stages. The latter sessilehemocytes are distributed predominately at the poste-rior part of the larva and as segmentally repeatedpatches or compartments on the inner dorsal surfaceof the integument of most larval segments. These resultsare consistent with previous analyses of larval hemocytesthat indicated the presence of sessile hemocyte islets(Lanot et al. 2001; Narita et al. 2004). However in thisstudy we have been able to examine dynamics of thesesessile hemocyte compartments and show they arisepartly as a result of hemocyte recruitment from thecirculating hemolymph, although cell division can alsobe detected within the sessile islets (data not shown). Atthis stage it is not obvious what features of the in-

tegument in these regions induce recruitment ofhemocytes. It is clear, however, that these compartmentsare dynamic as hemocytes can be observed to detachfrom these regions, and they are disrupted in prepupalstages possibly as a result of rising ecdysone titers.Currently little is known about the function of theseadherent hemocytes. One possibility is that these re-gions may simply act as a depot or reserve of hemocytesthat can be liberated as required, either upon infectionor at pupariation to take part in tissue remodeling.Alternatively they may provide a specialized function,for example, acting as sensors or sentinels for infections.Two hemocyte cell types, macrophages and crystal cells,can be detected in these regions, suggesting that bothare responsive to cues from these regions. However,lamellocytes are not detected.

Taking advantage of the transparent nature of theDrosophila larval cuticle and by incorporating a UAS-GFP responder in the genetic background of the Pxn-GAL4 driver line, we were able to screen for genes thataffected the distribution of the sessile hemocyte com-partment, altered hemocyte morphology and number,or which caused inappropriate targeting of hemocytesand changes in the lymph gland size. Among theobserved phenotypes, disruption of the sessile dorsalhemocyte compartments was the most frequent cate-gory recorded. It is reasonable to expect that thisphenotype is associated with defects in hemocyteadhesion properties, for example, altered productionof cell adhesion molecules by hemocytes or changes incytoskeleton organization in hemocytes. Consistentwith this, we recovered insertions that misexpress scb(the Drosophila a-integrin aPS3), C3G (a Ras familyguanine nucleotide exchange factor), and RhoGEF2within this category. In addition, we have been able toshow that misexpression of Kr, nej, and CG32813 ledto elevated expression of the Drosophila b-integrinsubunit Mys in hemocytes and that these hemocytesshowed increased staining of F-actin using rhodamine-phalloidin, both consistent with changes in their adher-ent properties.

Enlargement of the larval lymph gland was the secondmost frequent phenotype observed. Given the similar-ities that have been proposed between lymph glandhematopoiesis in Drosophila and mammalian aorta-gonadal-mesonephros mesoderm development (Mandal

et al. 2004), genes identified here may add importantinsights into developmental regulation of mammalianhematopoiesis. In principle, the lymph gland overgrowthphenotypes we recorded may have been caused by in-creases in hemocyte proliferation or by changes in hemo-cyte survival. Banerjee and co-workers ( Jung et al. 2005)have shown that proliferation in the third larval instarlymph gland is restricted to the cortical zone. Importantly,expression of Pxn-GAL4 in the lymph gland is limited tomature hemocytes also located in the cortical zone. Assuch, factors recovered in this screen are likely to regulate


the expansion phase of lymph gland development, whenmature hemocytes divide to increase numbers. One factorthat we have identified with an enlarged lymph gland phe-notype is the microRNA bantam, which has been shown tocontrol both proliferation and apoptosis (Brennecke

et al. 2003) and which is a target of the Hippo pathway(Nolo et al. 2006; Thompson and Cohen 2006). In-terestingly misexpression of two histone demethylases, thehistone H3K9 demethylase CG33182 (also known asJMJD2C) and the Drosophila Jumonji homolog CG3654,also gave a lymph gland overgrowth phenotype. In hu-mans JMJD2C has been found to be amplified in cell linesderived from esophageal squamous carcinomas whileknockdown of JMJD2C leads to decreased cell prolifera-tion (Cloos et al. 2006).

A GAL4 screen for regulators of larval hemocytefunction has identified a number of critical signalingpathways that regulate hemocyte activation (Zettervall

et al. 2004). However, this screen was a directed screenthat altered function of a set of 33 genes that werepredicted to affect hemocyte development. In thisreport we have substantially extended this work byscreening �20% of Drosophila genes. Moreover, ourscreen has been performed using the Pxn-GAL4 driverthat expresses in mature plasmatocytes. This has al-lowed us to recover regulators of mature plasmatocyteadhesion and function without the complication ofaltering plasmatocyte determination by expression ofthese factors at earlier stages in the hemocyte lineage.

A final caveat that must be considered is that aproportion of the genes recovered in misexpressionscreens may not necessarily be normally expressed orfunction within the tissue assayed. As such, some of thecandidate genes identified here may not have a normalfunction in hemocyte development. Moreover, a num-ber of the genes identified appear to be general factorsinvolved in cell development and are not hemocytespecific. To confirm which genes are bona fide regulatorsof hemocyte function it will be necessary to determineexpression profile and loss-of-function phenotypes ofthese genes. Increasing availability of inducible RNAilines (Kambris et al. 2006; Dietzl et al. 2007) will makethis an important future avenue of research.

We thank I. Ando, M. Galko, D. Hultmark, R. Hynes, H. Jackle,J. Kumar, and L. Waltzer for generously providing flies and reagents.EP and EY transposons used in this screen were kindly provided bythe Bloomington and Szeged Drosophila Stock Centers. We thankC. Buckley, and M. Mortin for critical reading of the manuscript andM. Krajcovic for assistance preparing fly food. This work was partlysupported by grants from the Royal Society, the Rowbotham Bequest,and the Medical Research Council (United Kingdom).

LITERATURE CITED

Abdelilah-Seyfried, S., Y. M. Chan, C. Zeng, N. J. Justice, S.

Younger-Shepherd et al., 2000 A gain-of-function screen for

genes that affect the development of the Drosophila adult exter-

nal sensory organ. Genetics 155: 733–752.

Abrams, J. M., K. White, L. I. Fessler and H. Steller,1993 Programmed cell death during Drosophila embryogene-sis. Development 117: 29–43.

Alfonso, T. B., and B. W. Jones, 2002 gcm2 promotes glial cell dif-ferentiation and is required with glial cells missing for macro-phage development in Drosophila. Dev. Biol. 248: 369–383.

Baker, N. E., and A. E. Zitron, 1995 Drosophila eye development:Notch and Delta amplify a neurogenic pattern conferred on themorphogenetic furrow by scabrous. Mech. Dev. 49: 173–189.

Barrett, K., M. Leptin and J. Settleman, 1997 The Rho GTPaseand a putative RhoGEF mediate a signaling pathway for the cellshape changes in Drosophila gastrulation. Cell 91: 905–915.

Bataille, L., B. Auge, G. Ferjoux, M. Haenlin and L. Waltzer,2005 Resolving embryonic blood cell fate choice in Drosophila:interplay of GCM and RUNX factors. Development 132: 4635–4644.

Bellen, H. J., R. W. Levis, G. Liao, Y. He, J. W. Carlson et al.,2004 The BDGP gene disruption project: single transposon in-sertions associated with 40% of Drosophila genes. Genetics 167:761–781.

Bernardoni, R., V. Vivancos and A. Giangrande, 1997 glide/gcmis expressed and required in the scavenger cell lineage. Dev. Biol.191: 118–130.

Brand, A. H., and N. Perrimon, 1993 Targeted gene expression as ameans of altering cell fates and generating dominant pheno-types. Development 118: 401–415.

Brennecke, J., D. R. Hipfner, A. Stark, R. B. Russell and S. M.Cohen, 2003 bantam encodes a developmentally regulated mi-croRNA that controls cell proliferation and regulates the proa-poptotic gene hid in Drosophila. Cell 113: 25–36.

Brogiolo, W., H. Stocker, T. Ikeya, F. Rintelen, R. Fernandez

et al., 2001 An evolutionarily conserved function of the Dro-sophila insulin receptor and insulin-like peptides in growth con-trol. Curr. Biol. 11: 213–221.

Carrera, P., S. Abrell, B. Kerber, U. Walldorf, A. Preiss et al.,1998 A modifier screen in the eye reveals control genes forKruppel activity in the Drosophila embryo. Proc. Natl. Acad.Sci. USA 95: 10779–10784.

Cloos, P. A., J. Christensen, K. Agger, A. Maiolica, J. Rappsilber

et al., 2006 The putative oncogene GASC1 demethylates tri- anddimethylated lysine 9 on histone H3. Nature 442: 307–311.

Crozatier, M., J. M. Ubeda, A. Vincent and M. Meister,2004 Cellular immune response to parasitization in Drosophilarequires the EBF orthologue collier. PLoS Biol. 2: E196.

Delaunay, J., G. Le Mee, N. Ezzeddine, G. Labesse, C. Terzian et al.,2004 The Drosophila Bruno paralogue Bru-3 specifically bindsthe EDEN translational repression element. Nucleic Acids Res.32: 3070–3082.

Dietzl, G., D. Chen, F. Schnorrer, K. C. Su, Y. Barinova et al.,2007 A genome-wide transgenic RNAi library for conditionalgene inactivation in Drosophila. Nature 448: 151–156.

Escudero, L. M., E. Caminero, K. L. Schulze, H. J. Bellen andJ. Modolell, 2005 Charlatan, a Zn-finger transcription factor,establishes a novel level of regulation of the proneural achaete/scute genes of Drosophila. Development 132: 1211–1222.

Evans, C. J., V. Hartenstein and U. Banerjee, 2003 Thicker thanblood: conserved mechanisms in Drosophila and vertebrate he-matopoiesis. Dev. Cell 5: 673–690.

Fujimoto, K., N. Okino, S. Kawabata, S. Iwanaga and E. Ohnishi,1995 Nucleotide sequence of the cDNA encoding the proen-zyme of phenol oxidase A1 of Drosophila melanogaster. Proc.Natl. Acad. Sci. USA 92: 7769–7773.

Gregory, S. L., T. Shandala, L. O’Keefe, L. Jones, M. J. Murray

et al., 2007 Drosophila overexpression screen for modifiers ofRho signalling in cytokinesis. Fly 1: 13–22.

Hacker, U., and N. Perrimon, 1998 DRhoGEF2 encodes a memberof the Dbl family of oncogenes and controls cell shape changesduring gastrulation in Drosophila. Genes Dev. 12: 274–284.

Holz, A., B. Bossinger, T. Strasser, W. Janning and R. Klapper,2003 The two origins of hemocytes in Drosophila. Develop-ment 130: 4955–4962.

Ishimaru, S., R. Williams, E. Clark, H. Hanafusa and U. Gaul,1999 Activation of the Drosophila C3G leads to cell fatechanges and overproliferation during development, mediatedby the RAS-MAPK pathway and RAP1. EMBO J. 18: 145–155.


Jia, S., and A. Meng, 2007 Tob genes in development and homeo-stasis. Dev. Dyn. 236: 913–921.

Jones, S. L., F. P. Lindberg and E. J. Brown, 1999 Phagocytosis. J. B.Lippincott, Philadelphia.

Jung, S. H., C. J. Evans, C. Uemura and U. Banerjee, 2005 TheDrosophila lymph gland as a developmental model of hemato-poiesis. Development 132: 2521–2533.

Kambris, Z., S. Brun, I. H. Jang, H. J. Nam, Y. Romeo et al.,2006 Drosophila immunity: a large-scale in vivo RNAi screenidentifies five serine proteases required for Toll activation. Curr.Biol. 16: 808–813.

Kumar, J. P., T. Jamal, A. Doetsch, F. R. Turner and J. B. Duffy,2004 CREB binding protein functions during successive stagesof eye development in Drosophila. Genetics 168: 877–893.

Kurucz, E., C. J. Zettervall, R. Sinka, P. Vilmos, A. Pivarcsi et al.,2003 Hemese, a hemocyte-specific transmembrane protein, af-fects the cellular immune response in Drosophila. Proc. Natl.Acad. Sci. USA 100: 2622–2627.

Lai-Fook, J., 1966 The repair of wounds in the integument of in-sects. J. Insect Physiol. 12: 195–226.

Lanot, R., D. Zachary, F. Holder and M. Meister, 2001 Post-embryonic hematopoiesis in Drosophila. Dev. Biol. 230: 243–257.

Lebestky, T., T. Chang, V. Hartenstein and U. Banerjee,2000 Specification of Drosophila hematopoietic lineage by con-served transcription factors. Science 288: 146–149.

Long, H., S. Cameron, L. Yu and Y. Rao, 2006 De novo GMP syn-thesis is required for axon guidance in Drosophila. Genetics 172:1633–1642.

Luo, H., W. P. Hanratty and C. R. Dearolf, 1995 An amino acid sub-stitution in the Drosophila hopTum-l Jak kinase causes leukemia-like hematopoietic defects. EMBO J. 14: 1412–1420.

Mandal, L., U. Banerjee and V. Hartenstein, 2004 Evidence for afruit fly hemangioblast and similarities between lymph-gland he-matopoiesis in fruit fly and mammal aorta-gonadal-mesonephrosmesoderm. Nat. Genet. 36: 1019–1023.

Manseau, L., A. Baradaran, D. Brower, A. Budhu, F. Elefant et al.,1997 GAL4 enhancer traps expressed in the embryo, larvalbrain, imaginal discs, and ovary of Drosophila. Dev. Dyn. 209:310–322.

Maroni, G., and S. C. Stamey, 1983 Use of blue food to select syn-chronous, late third-instar larvae. Dros. Inf. Serv. 59: 142–143.

Meister, M., and M. Lagueux, 2003 Drosophila blood cells. Cell.Microbiol. 5: 573–580.

Mukherjee, T., U. Schafer and M. P. Zeidler, 2006 Identificationof Drosophila genes modulating janus kinase/signal transducerand activator of transcription signal transduction. Genetics 172:1683–1697.

Narita, R., H. Yamashita, A. Goto, H. Imai, S. Ichihara et al.,2004 Syndecan-dependent binding of Drosophila hemocytesto laminin alpha3/5 chain LG4–5 modules: potential role in ses-sile hemocyte islets formation. FEBS Lett. 576: 127–132.

Nolo, R., C. M. Morrison, C. Tao, X. Zhang and G. Halder,2006 The bantam microRNA is a target of the hippo tumor-suppressor pathway. Curr. Biol. 16: 1895–1904.

Pena-Rangel, M. T., I. Rodriguez and J. R. Riesgo-Escovar,2002 A misexpression study examining dorsal thorax formationin Drosophila melanogaster. Genetics 160: 1035–1050.

Ramet, M., P. Manfruelli, A. Pearson, B. Mathey-Prevot and R. A.Ezekowitz, 2002 Functional genomic analysis of phagocytosisand identification of a Drosophila receptor for E. coli. Nature416: 644–648.

Rehorn, K. P., H. Thelen, A. M. Michelson and R. Reuter, 1996 Amolecular aspect of hematopoiesis and endoderm developmentcommon to vertebrates and Drosophila. Development 122: 4023–4031.

Rorth, P., 1996 A modular misexpression screen in Drosophila de-tecting tissue-specific phenotypes. Proc. Natl. Acad. Sci. USA 93:12418–12422.

Ruiz-Gomez, M., S. Romani, C. Hartmann, H. Jackle and M. Bate,1997 Specific muscle identities are regulated by Kruppel dur-ing Drosophila embryogenesis. Development 124: 3407–3414.

Sinenko, S. A., E. K. Kim, R. Wynn, P. Manfruelli, I. Ando et al.,2004 Yantar, a conserved arginine-rich protein is involved inDrosophila hemocyte development. Dev. Biol. 273: 48–62.

Soderhall, K., and L. Cerenius, 1998 Role of the prophenoloxi-dase-activating system in invertebrate immunity. Curr. Opin. Im-munol. 10: 23–28.

Sorrentino, R. P., Y. Carton and S. Govind, 2002 Cellular im-mune response to parasite infection in the Drosophila lymphgland is developmentally regulated. Dev. Biol. 243: 65–80.

Staudt, N., A. Molitor, K. Somogyi, J. Mata, S. Curado et al.,2005 Gain-of-function screen for genes that affect Drosophilamuscle pattern formation. PLoS Genet. 1: e55.

Stramer, B., W. Wood, M. J. Galko, M. J. Redd, A. Jacinto et al.,2005 Live imaging of wound inflammation in Drosophila em-bryos reveals key roles for small GTPases during in vivo cell mi-gration. J. Cell Biol. 168: 567–573.

Tanaka-Matakatsu, M., T. Uemura, H. Oda, M. Takeichi and S.Hayashi, 1996 Cadherin-mediated cell adhesion and cell mo-tility in Drosophila trachea regulated by the transcription factorEscargot. Development 122: 3697–3705.

Tepass, U., L. I. Fessler, A. Aziz and V. Hartenstein,1994 Embryonic origin of hemocytes and their relationshipto cell death in Drosophila. Development 120: 1829–1837.

Thompson, B. J., and S. M. Cohen, 2006 The Hippo pathway regu-lates the bantam microRNA to control cell proliferation and ap-optosis in Drosophila. Cell 126: 767–774.

Tsuda, L., M. Kaido, Y. M. Lim, K. Kato, T. Aigaki et al., 2006 AnNRSF/REST-like repressor downstream of Ebi/SMRTER/Su(H)regulates eye development in Drosophila. EMBO J. 25: 3191–3202.

Wang, X., C. Lee, D. S. Gilmour and J. P. Gergen, 2007 Transcriptionelongation controls cell fate specification in the Drosophila em-bryo. Genes Dev. 21: 1031–1036.

Williams, M. J., M. L. Wiklund, S. Wikman and D. Hultmark,2006 Rac1 signalling in the Drosophila larval cellular immuneresponse. J. Cell Sci. 119: 2015–2024.

Wood, W., C. Faria and A. Jacinto, 2006 Distinct mechanisms reg-ulate hemocyte chemotaxis during development and woundhealing in Drosophila melanogaster. J. Cell Biol. 173: 405–416.

Yamaguchi, Y., T. Takagi, T. Wada, K. Yano, A. Furuya et al.,1999 NELF, a multisubunit complex containing RD, cooperateswith DSIF to repress RNA polymerase II elongation. Cell 97: 41–51.

Yee, G., and R. O. Hynes, 1993 A novel, tissue-specific integrin sub-unit, beta nu, expressed in the midgut of Drosophila melanogaster.Development 118: 845–858.

Yuan, J., W. Tang, K. Luo, X. Chen, X. Gu et al., 2006 Cloning andcharacterization of the human gene DERP6, which activates tran-scriptional activities of p53. Mol. Biol. Rep. 33: 151–158.

Zettervall, C. J., I. Anderl, M. J. Williams, R. Palmer, E. Kurucz

et al., 2004 A directed screen for genes involved in Drosophilablood cell activation. Proc. Natl. Acad. Sci. USA 101: 14192–14197.

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A Misexpression Screen to Identify Regulators of Drosophila Larval

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