Copyright 2004 by the Genetics Society of AmericaDOI: 10.1534/genetics.104.028639

Identification and Characterization of Genes Involved in Embryonic Crystal CellFormation During Drosophila Hematopoiesis

Allison B. Milchanowski,*,1 Amy L. Henkenius,*,1 Maya Narayanan,*Volker Hartenstein*,† and Utpal Banerjee*,†,‡,2

*Department of Molecular, Cell and Developmental Biology, ‡Department of Biological Chemistry and†Molecular Biology Institute, University of California, Los Angeles, California 90095

Manuscript received March 10, 2004Accepted for publication June 10, 2004

ABSTRACTParallels between vertebrate and Drosophila hematopoiesis add to the value of flies as a model organism

to gain insights into blood development. The Drosophila hematopoietic system is composed of at leastthree classes of terminally differentiated blood cells: plasmatocytes, crystal cells, and lamellocytes. Recentstudies have identified transcriptional and signaling pathways in Drosophila involving proteins similar tothose seen in human blood development. To identify additional genes involved in Drosophila hematopoie-sis, we have conducted a P-element-based genetic screen to isolate mutations that affect embryonic crystalcell development. Using a marker of terminally differentiated crystal cells, we screened 1040 P-element-lethal lines located on the second and third chromosomes and identified 44 individual lines that affectcrystal cell development. Identifying novel genes and pathways involved in Drosophila hematopoiesis islikely to provide further insights into mammalian hematopoietic development and disorders.

DESPITE the obvious differences in cell type and cells and debris as well as play a role in immune responseby eliminating pathogens (Tepass et al. 1994; Lanotfunctionality between vertebrate and Drosophilaet al. 2001). Crystal cells, which compose �5% of theblood systems, parallels in their developmental mecha-hemocyte population, participate in immune responsesnisms are remarkable (reviewed by Evans et al. 2003).and wound healing through melanization. The paracrys-Such shared developmental and functional mechanismstaline inclusions within the cells are thought to containhave prompted the use of Drosophila as a model organ-Pro-Phenoloxidase A1 (ProPO A1; Rizki et al. 1980), anism to further investigate the genetic control of hemato-enzyme that is similar to tyrosinase and is important inpoietic cell differentiation.the biosynthesis of melanin (Rizki et al. 1985). UnlikeBoth vertebrate and Drosophila hematopoiesis in-plasmatocytes and crystal cells, which are found in allvolve distinct, terminally differentiated lineages deriveddevelopmental stages, lamellocytes have been observedfrom common progenitor cells. Mammalian hematopoi-only in Drosophila larvae and increase in number dur-etic cells differentiate into two main branches: the lym-ing immune challenge (Lanot et al. 2001; Sorrentinophoid and myeloid lineages (reviewed by Dzierzak andet al. 2002).Medvinsky 1995). Differentiation, function, and lin-

Drosophila hemocytes have dual sites of origin. Earlyeage hierarchy of Drosophila blood cells, or hemocytes,hemocytes arising from the mesoderm of the embryonicare most similar to those of the vertebrate myeloid lin-head region are detected throughout development andeage (reviewed by Orkin 2000). The Drosophila hema-into adulthood (Holz et al. 2003). A split of the hemo-topoietic system is composed of at least three classescyte population into plasmatocytes and crystal cells oc-of terminally differentiated hemocytes: plasmatocytes,curs at an early stage. Crystal cells form a small, cohesivecrystal cells, and lamellocytes, which participate in devel-cell group that remains clustered around the embryonicopment and immune response (reviewed by Evans etproventriculus (Lebestky et al. 2000), whereas plasma-al. 2003; Meister and Lageaux 2003).tocytes migrate throughout the entire embryo (TepassPlasmatocytes are the most abundant hemocyte typeet al. 1994). A second population of hemocytes thatin Drosophila and are commonly referred to as macro-differentiate in the late larva and during metamorphosisphages. Accordingly, they function to engulf apoptoticto populate the pupa and adult are derived from asecond blood-forming tissue, the lymph gland, which issituated next to the dorsal blood vessel (aorta/heart)

1These authors contributed equally to this work. of the larva.2Corresponding author: Department of Molecular, Cell and Develop- Over 20 genes have been identified in mammalian

mental Biology, 2203 Life Sciences, 621 Charles E. Young Dr. S.,blood cell differentiation, including genes that encodeUniversity of California, Los Angeles, CA 90095.

E-mail: banerjee@mbi.ucla.edu transcription factors, recombinases, signaling mole-

Genetics 168: 325–339 (September 2004)

326 A. B. Milchanowski et al.

cules, transmembrane receptors, and secreted factors crystal cell development (Lebestky et al. 2000), whileUsh acts as a negative regulator of crystal cell differentia-(reviewed by Orkin 1996) that can act positively and/

or antagonistically in the regulation of hematopoiesis. tion (Fossett et al. 2003; Waltzer et al. 2003).srp-expressing cells that differentiate into plasmato-Many molecules play a role in both vertebrate and Dro-

sophila hematopoiesis, including transcriptional regula- cyte precursors also express the transcription factorGlial cells missing (Gcm) (Bernardoni et al. 1997),tors such as GATA, friend of GATA (FOG), and acute

myeloid leukemia-1 (AML-1), as well as the signaling trans- which is the primary regulator of glial cell differentia-tion in the nervous system (Hosoya et al. 1995; Jonesduction molecules Notch, Janus kinase/signal transducer

and activator of transcription (JAK/STAT), and NF�B of et al. 1995). There are two gcm genes in Drosophila, andGcm homologs have also been identified in vertebratesthe Toll/Cactus pathway (reviewed by Evans et al. 2003).

Serpent (srp) is one of five GATA factors in Drosophila (gcm-1 and gcm-2); however, no hematopoietic functionhas been associated with these (Altshuller et al. 1996;and is required for both embryonic and larval blood

development (reviewed by Rehorn et al. 1996; Lebes- Kim et al. 1998; Gunther et al. 2000; Schreiber et al.2000). Drosophila gcm-1 and gcm-2 double mutants showtky et al. 2000; Fossett and Schulz 2001). A second

GATA factor, Pannier (Pnr), is required for develop- only a 40% reduction in presumptive plasmatocytes,and only a fraction of mature plasmatocyte markers arement of the heart (Rehorn et al. 1996; Gajewski et al.

1999) and larval blood (Mandal et al. 2004). Srp is the detected in the remaining cells (Akiyama et al. 1996;Alfonso and Jones 2002), suggesting gcm is unlikely toearliest known factor expressed in hemocyte precursors

as its expression is first detected in the procephalic be the only determinant of plasmatocyte differentiation.Signal transduction pathways common to both Dro-mesoderm (Tepass et al. 1994; Rehorn et al. 1996).

Expression of srp is necessary for the differentiation of sophila and mammalian hematopoiesis include Notch,JAK/STAT, NF�B, and receptor tyrosine kinases (RTKs).plasmatocytes and crystal cells in the embryo.

FOG is a zinc finger protein that functions as a tran- The Notch pathway is important in many Drosophiladevelopmental processes, including cell fate decisionsscriptional coregulator. Mammalian FOG1 binds di-

rectly to GATA-1 and has a similar loss-of-function phe- and cell proliferation in the nervous system, mesoderm,and imaginal discs (Artavanis-Tsakonas et al. 1999).notype as GATA-1 (reviewed by Cantor and Orkin

2001 and Fossett and Schulz 2001; Chang et al. 2002). Notch signaling has also been shown to be required forthe development of embryonic and larval crystal cellsThe corepressor C-terminal binding protein (CtBP) and

FOG together regulate hematopoietic lineage commitment and in the proliferation of hemocytes (Duvic et al. 2002;Lebestky et al. 2003). A specific and distinct role forin mammals. The Drosophila FOG ortholog, U-shaped

(Ush), is expressed in all hemocyte precursors throughout individual Notch receptors has been identified in mam-malian hematopoiesis as well (Walker et al. 2001;embryonic and larval hematopoiesis (Fossett and Schulz

2001). Physical and genetic interaction between Ush and Kumano et al. 2003; Saito et al. 2003). While it hasbeen well established that both the JAK/STAT and Toll/Srp has been demonstrated to repress crystal cell fate

in prohemocytes (Fossett et al. 2003; Waltzer et al. Cactus pathways are involved in blood cell productionduring immune response, it also appears that each par-2003). Accordingly, ush is downregulated in crystal cell

precursors and ush mutants exhibit an increase in crystal ticipates in normal blood development as well (Mathey-Prevot and Perrimon 1998; Qiu et al. 1998). JAK/cell number.

AML-1, or Runx1, was first isolated as a fusion partner STAT signaling seems to play a specific role in bloodcell differentiation and NF�B plays a role in blood cellin a chromosomal translocation associated with AML

(reviewed by Lutterbach and Hiebert 2000; Rab- proliferation (Luo et al. 1997). The RTK, vascular andendothelial growth factor receptor (VEGFR), mediatesbitts 1994) and is necessary for definitive hematopoie-

sis in mammals (Okuda et al. 1996; Wang et al. 1996). blood cell migration in mammals (reviewed by Traverand Zon 2002), while another RTK, c-Kit, is importantDrosophila Lozenge (Lz), a transcription factor that has

71% identity to the Runt domain of the human protein for the proliferation and maintenance of myeloid pro-genitors (Kelly and Gilliland 2002). Similarly, theAML-1, is necessary for crystal cell development during

embryonic and larval hematopoiesis (Daga et al. 1996; Drosophila platelet-derived growth factor and VEGF-receptor-related (PVR) protein is expressed in hemo-Lebestky et al. 2000). lz has been shown to function

downstream of srp ; however, additional interactions be- cytes and is required for the proper migration (Heinoet al. 2001; Cho et al. 2002) and survival (Bruckner ettween these transcription factors initiate hemocyte com-

mitment to the crystal cell lineage (Fossett et al. 2003; al. 2004) of plasmatocytes.The parallels between vertebrate and Drosophila he-Waltzer et al. 2003) by stage 11 of Drosophila embry-

onic development. An interesting parallel between matopoiesis have proven flies to be a useful model sys-tem in which to dissect the role of the many signalingmammals and Drosophila is the relationship between

AML-1 and FOG. In mammals, AML-1 is a positive regu- pathways involved in blood development. Drosophilagenetics allows for comprehensive screens for mutationslator of myeloid differentiation, while FOG1 is antago-

nistic. Similarly in Drosophila, Lz is required for proper that disrupt a particular biological process. To identify

327Crystal Cell Development in Drosophila

munohistochemistry protocols were as previously describedadditional genes involved in Drosophila hematopoiesis,(Tepass et al. 1994).we have conducted a P-element screen to isolate muta-

Counting methods: Crystal cells from 10 embryos for eachtions that affect crystal cell development in embryos. line were counted at 40� magnification on a Zeiss compoundCrystal cells, composing only 5% of the total hemocyte microscope. The average number of cells hybridized to ProPO

A1 per embryo was calculated and graphed using Excel (Micro-population (amounting to �36 cells in a wild-type em-soft) and Delta Graph (SPSS, Chicago). The number of cellsbryo), are ideal for a screen of this nature because thestaining for �-Phospho-H3 was determined in the head regiontotal number can be accurately determined. In the cur-of stage 14 embryos (n � 3). The head region for this purpose

rent screen we utilized the expression pattern of ProPO was defined as the tissue demarcated by the anterior edge ofA1, a highly specific and sensitive marker for crystal the amnioserosa and the anterior end of the embryo.

Molecular analysis: The sequence of genes interrupted bycells. Out of 1040 lines screened, we identified 44 withP-element inserts was obtained from the Berkeley Drosophilaan abnormal number and/or distribution of crystalGenome Project (BDGP; http://www.fruitfly.org/p_disrupt/).cells. These lines were subjected to further phenotypic The locations of the inserts of four P lines not sequenced by

characterization to establish if other developmental de- BDGP, P10412, P10596, P10675, and P11066, were identifiedfects exist in these mutants. Using antibodies against using inverse PCR and sequencing. Blast searches were per-

formed using the standard nucleotide-nucleotide BLASTthe Engrailed and Twist proteins we established that[blastn] program provided by the NCBI database (http://none of the 44 mutations had generalized defects inwww.ncbi.nlm.nih.gov:80/BLAST/).germ-band patterning and gastrulation, respectively.

Collagen IV in situ hybridization allowed visualizationof plasmatocytes and revealed the subset of mutations RESULTS AND DISCUSSIONwith defects in both crystal cells and plasmatocytes.

Screen for crystal cell mutants: In Drosophila, mostcomponents of the hematopoietic developmental hier-archy are still unknown. We have conducted a geneticMATERIALS AND METHODSscreen to identify genes involved in this process. This

Fly stocks: All stocks and crosses used were maintained at screen utilizes in situ hybridization with ProPO A1, a25�, unless mentioned otherwise. A collection of lethal lines marker specific for terminally differentiated crystal cells.with P-element insertions on the second and third chromo-

A collection of 1040 P-element lethal lines generatedsomes from the Bloomington P-lethal collection was screened.and mapped by the Drosophila Genome Project, cov-The lines are referred to by their P-line numbers throughout

the study. All available genotypes are listed in Table 1. P lines ering the second and third chromosomes, was screened.were balanced over either CyO (Pw� Kr-Gal4, UAS-GFP) or To distinguish the homozygous mutant embryos fromTM3 (Pw� Kr-Gal4, UAS-GFP) balancer chromosomes (Casso those of other genotypes, the P-element lines were rebal-et al. 1999). The GFP balancers allowed the identification of

anced with balancer chromosomes containing Kruppelhomozygous mutant embryos, which were analyzed in all sub-(Kr)-Gal4 driving UAS-GFP (Casso et al. 1999). The screensequent experiments except where indicated. The followingidentified 44 mutants that affect crystal cell develop-stocks were obtained from the Bloomington Stock Center:

Oregon-R, Canton-S, ft 4, stg 4, lab 2, twr 1, put 135, osp 29, neur 1, ment (Figure 1). Forty-two demonstrate a reductioncbx 05704, Catsup 1, syt N6, Gug 03928, dpp H46, scw 11, tkv 7, sax4, wit B11, while crystal cell mislocalization is apparent in one lineMed 5, shn1, twi-Gal4, and UAS-nuclacZ. Temperature-sensitive (P11622), and there is one line (P10555) with both aalleles for twr1 and put 135 were collected at the nonpermissive

reduction and mislocalization of crystal cells.temperature of 30�. Sec61� [EP(2)2567] was obtained fromThe number of crystal cells present in wild-type fliesthe Szeged Stock Center. Mutant alleles obtained from other

laboratories include: srp neo45 and srp-Gal4 from R. Reuter, pap 53 was determined by counting the number of ProPO A1-from J. Botas, U2af38 �E18 from Donald Rio, corto420 from F. positive cells in Ore-R, Canton-S, heterozygous nonmu-Peronnet, smt3 l(2)k01211 from J. Schnorr, FRT(3R)CtBP Rev19 from tant 10579/CyO, Kr-GFP, and homozygous nonmutantS. Parkhurst, Sin3A from G. Rubin, hrg 1 from T. Murata, Mad 10

P10579 embryos. P10579, used as a control, contains aand Mad 12 from R. Padgett, brk M68 and UAS-brk from C.P-element insertion that does not affect crystal cells.Rushlow, ebi E4 from S. L. Zipursky, and UAS-Rab5 S43N from M. A.The combined average of the control fly lines estab-Gonzalez-Gaitan.

Phenotypic analysis: Embryo collections and in situ hybrid- lished that wild-type flies have a relatively invariant num-izations were performed in 30-well collection boxes as pre- ber (36 � 2.2) of crystal cells located in two bilateralviously described (Hummel et al. 1997). Antisense ProPO A1, clusters in the head region of stage 14 embryos. TheGFP, and Collagen IV (CIV) digoxygenin-labeled RNA probes

number of crystal cells in the P-line mutants ranges fromwere made from 2.3 kb ProPO A1, 0.74 kb GFP, and 1.6 kb3 to 57% of wild type, with the median number of crystalCIV cDNAs as previously described (Daga et al. 1996). Rabbitcells in a mutant embryo being 12.5 (Figure 1A). P11622antibodies to -Gal, GFP, Phospho-Histone H3, and Twist were

used. Mouse antibody to Engrailed was used. The Engrailed was different from the rest in that it showed wild-typeantibody developed by C. Goodman was obtained from the numbers of crystal cells that were grossly mislocalizedDevelopmental Studies Hybridoma Bank developed under the over the entire embryo.auspices of the National Institute of Child Health and Human

Of the 44 mutants found, 24 of the lines have P-elementDevelopment. The GFP and the Phospho-Histone H3 antibodyinsertions that are within previously characterized geneswere obtained from Sigma (St. Louis) and Upstate Innovative

Signaling Solutions, respectively. In situ hybridization and im- (Table 1). To verify the location of each P-element inser-

328 A. B. Milchanowski et al.

Figure 1.—Mutations affecting crystalcell development. (A) Quantitation ofthe effect of P-line mutants on crystalcell development. The chart shows theaverage number of crystal cells per em-bryo (n � 10) as marked by ProPO A1riboprobe. The wild-type average of 36is the combined averages of Ore-R, Can-ton-S, heterozygous nonmutant P10579/Cyo, Kr-GFP, and homozygous nonmu-tant P10579 embryos. (B) Stage 13–14embryos of wild-type (Ore-R) and mu-tant P lines hybridized with ProPO A1riboprobe marking terminally differenti-ated crystal cells. Wild-type mature crys-tal cells can be detected by ProPO A1hybridization as early as stage 12 and aregrouped in two clusters of 18 each inthe head mesoderm. Lines P10198 toP12350 show a reduction in the numberof crystal cells present. P10555 andP11622 show a mislocalization of the ma-ture crystal cells.

tion and its effect on crystal cell development, we tested three are P10527, P10664, and P10676, which mappedto the Sec61�, crossbronx (cbx), and hiiragi (hrg) loci, re-available loss-of-function mutant alleles of the known

genes identified in our screen. Of the 24 known genes, spectively. Mutant alleles of these genes all show a reduc-tion in crystal cells consistent with the corresponding18 had available loss-of-function alleles, all of which also

demonstrate a reduction in crystal cell number, consistent P line mutations (Figure 2).Eleven of the 44 P lines behave as enhancer traps andwith results from the P lines (Figure 2).

To identify genes disrupted in the remaining 20 P demonstrate expression in hemocytes and/or crystal cells(Figure 3). Heterozygous and homozygous embryos werelines, the P-element flanking region sequences were

obtained from the Drosophila Genome Project when examined in this experiment. P10604, P10664, P10848,P11121, P10675, P11066, P10586, P10676, P12059, andavailable or were determined in this study. These se-

quences were used in BLAST searches to identify candi- P10756 contain a lacZ within the P element that is ex-pressed in embryonic hemocytes in the head region ofdate genes for each of the 20 unknown P lines. One to

two candidate genes were identified for 18 of the 20 stage 11 embryos. As examples, P11066 and P10586 areshown (Figure 3, A and B). LacZ expression in P10756unknown P lines (Table 1). As with the known genes,

the three candidate genes with available loss-of-function and P10382 is detected in crystal cells of stage 14 em-bryos (Figure 3, C and D). These enhancer trap linesalleles were tested for recapitulation of the reduced

crystal cell phenotype observed in the P lines. These nicely illustrate an expression pattern consistent with

329Crystal Cell Development in Drosophila

TABLE 1

Summary of the mutants identified in the crystal cell screen

Avg. no.P line Map position Gene Candidate gene Description of cc

P10198 78 A2–5 pap L7062 Component of transcription mediatorcomplex (TRAP240) 9.9

P10199 78 A5–6 l(3)L5541 L5541 CG10581 DUF265, unknown function 12.9CG32434 sec7, PH domain

P10382 24 A1–2 for k04703 Protein ser/thr kinase 13.8P10412 98 C No CG in area 13.4P10419 27 C7 smt3 k06307 Ubiquitin like, nuclear protein tag 7.6

(SUMO-1)P10448 21 B7–8 U2af38 k14504 Component of splicesome 12.5P10492 29 C1–2 snRNA:U6atac:29B k01105 Nuclear mRNA splicing via U12-type 10.8

spliceosomeP10527 26 D6–9 l(2)k03201 k03201 Sec61� Protein transport, cell death 10.7P10542 46 F1–2 l(2)k04308 k04308 CG30011 pnt/SAM transcription factor 12.3P10555 55 C9–10 l(2)08770 k04808 CG30118 Mth_Ecto domain 21.2P10572 37 B8–12 Catsup k05424 Regulator of catecholamine 17.5

metabolismP10586 23 B1–2 syt k05909 Ca2� phospholipid binding, synaptic 10.8

vesicle fusionP10596 21 B4–6 l(2)k06019 k06019 kis myb-binding domain, DEAD/DEAH 9

box helicaseP10604 47 F1–2 Fpps k06103 Sterol biosynthesis, cell division and 10.5

enlargementP10664 46 C1–2 l(2)k07237 k07237 cbx Ubiquitin-conjugating enzyme 11.8

CG12744 Zinc finger transcription factorP10675 38 B4 l(2)k07614 k07614 fok 9.9

Klp38B Chromokinesin, bipoplar spindleassembly

P10676 43 E15–16 l(2)07619 k07619 hrg Adenlyltransferase 11.4P10692 24 D7–E1 fat k07918 Contains 34 cadherin repeats 16P10756 51 D3–5 l(2)k08015 k08015 CG10228 Regulation of nuclear pre-mRNA 10.3P10770 39 F1–3 l(2)k08110 k08110 CG11628 Guanyl-nt exchange factor, 17.1

intracellular signalingP10786 22 E1–2 Rab5 k08232 Small monomeric GTPase, Dpp 11.4

traffickingP10791 46 C6–8 l(2)k08601 k08601 PKa-R2 cAMP-dep protein kinase R2 14.1P10848 30 E1–3 FKBP59 k09010 Peptidyl isomerase, protein folding 17.3P11066 53 D11–13 l(2)k12701 k12701 CG6301 15.6P11121 26 F3–5 l(2)k14206 k14206 CG11098 Putative SMC chromosome 10.3

segregation ATPaseCG13769 Putative leucine-rich ribonuclease

inhibitor type domainP11123 28 F1–2 l(2)k14308 k14308 CG8451 Sodium-dependent multivitamin 14.2

transporterCG8419 B-box zinc, C3HC4 ring finger

transcription factorP11166 21 C2–3 ebi k16213 WD40 repeats, F-box, binds E3 13.6

ubiquitin ligasesP11174 36 A4–5 l(2)k16215 k16215 CG5953 MADF domain, transcription factor 13.2P11342 43 D1–2 ALDH-III 03610 Oxidizes fatty and aromatic aldehydes 20.5P11525 99 A5–7 stg 01235 Tyrosine phosphatase, controls 7.9

G2/M transition (Cdc 25)P11527 84 A1–2 lab 01241 Homebox transcription factor 9.8P11538 89 B1–3 srp 01549 GATA-type zinc finger transcription 1

factorP11590 87 D9–11 CtBP 03463 Transcription corepressor 13.3P11622 66 C13/73 D3 l(3)04069b 04069b CG6983 35.9

Gug Transcription corepressor

(continued)

330 A. B. Milchanowski et al.

TABLE 1

(Continued)

Avg. no.P line Map position Gene Candidate gene Description of cc

P11663 84 A1–2 twr 05614 Affect eye and macrochaetae 14.1P11716 82 E5–7 corto 07128b RNA transcription factor, component 14.5

of the centrosomeP11745 88 C9–10 put 10460 ser/thr protein kinase, type II TGF 13.5

receptorP12045 34 A1–2 l(2)rK639 rK639 No CG in area 16.9P12046 35 B1–4 osp rJ571 Putative ligand carrier, component of 11.8

the cytoskeletonP12059 57 A3–4 l(2)s4831 s4831 CG13434 10.9

CG30153P12124 85 C5–9 neur j6B12 E3 type ubiquitin ligase 8.8P12196 59 E1–2 l(2)s4830 s4830 Or59a Olfactory receptor 14.1

CG5357 Putative component of ribosomeP12346 55 D1–2 Prp19 07838 Pre-mRNA splicing factor, component 12.8

of splicesomeP12350 49 B3–6 Sin3A 08269 Transcription corepressor 14.1

P lines have been listed in order of their line numbers and are referred to as such throughout this study. Correspondingalleles of each line are listed under Gene. Unknown lines with more than one candidate gene listed have a P-element insertionmapped between the loci of two genes. Avg no. of cc, the average number of crystal cells found in homozygous P-line embryosat stage 13–14.

the likely role of these genes in one or more stages of ployed the �-Phosphohistone-H3 (Phospho-H3) anti-body, which identifies cells in late G2 through anaphasehematopoietic development.

Characterization of isolated mutants: Crystal cells when chromatin is condensed (Hendzel et al. 1997).To quantitate the number of cells entering mitosis weconstitute a class of hemocytes derived from the head

mesoderm. Genes that, when mutated, cause a change chose the head region of stage 14 embryos in whichonly neuroblasts continue dividing. Choosing this laterin the number of crystal cells could act directly on the

specification of this cell type; alternatively, they could stage maximizes our chance of detecting general prolif-eration defects in mutants with significant maternalfunction at an earlier step during the formation of he-

mocytes or of mesoderm in general. For example, a components, and the relatively small number of cellsdividing at this time allows accurate quantitation. Sixdouble mutation of twist and snail that has no mesoderm

also lacks hemocytes, including crystal cells (V. Harten- mutant lines were identified with �-Phospho-H3 as hav-ing cell-cycle defects (Figure 6): P10198, P11166,stein, unpublished data); likewise, in bicaudal, where

early events in the specification of the anterior body P11525, P11663, P12045, and P12196. These were allshown to have a significant increase in the number ofaxis fail to take place, head structures, including head

mesoderm and all hemocytes, are missing (Tepass et al. �-Phospho-H3-positive cells, indicating that cells were ar-rested in G2 or mitosis at a higher than normal rate.1994). Finally, changes in the number of crystal cells

could be due to a generalized effect on cell division. Alternatively, the elevated number of �-Phospho-H3-posi-tive cells may elicit an escalated apoptotic response lead-To distinguish between these different steps at which

the genes uncovered in this screen might act, we used ing to a decrease in the number of differentiated crystalcells. Included in this group are two previously knownthe markers Collagen IV (plasmatocytes; Yasothorns-

rikul et al. 1997), Twist (mesoderm; Furlong et al. cell cycle regulators, ebi and stg. Ebi functions in normalG1 arrest of cells in the peripheral nervous system and2001), Engrailed (metamerically reiterated expression

along antero-posterior axis; Bejsovec and Wieschaus central nervous system. Boulton et al. (2000) discov-ered an increase in BrdU incorporation of cells in ebi1993), and Phosphohistone-H3 (marker for cells enter-

ing mitosis; Hendzel et al. 1997). mutants. Failure to arrest in G1 results in an increasednumber of cells that are Phospho-H3 positive in thisAll mutations showed grossly normal staining with

anti-Twist and anti-Engrailed antibodies, indicating that mutant. stg mutants have been shown to arrest cells inG2 phase, thus accounting for the increase in �-Phos-changes in crystal cell number were not caused by a

global defect in axis formation, gastrulation, or segmen- pho-H3 staining (Neufeld et al. 1998).We found that six crystal cell-reduction mutant linestation (Figures 4 and 5). To assess if the mutations

contribute to general cell proliferation defects, we em- also demonstrate a significant reduction in the number

331Crystal Cell Development in Drosophila

Figure 2.—Loss-of-function alleles of known and candidate genes of P-line mutants. (A) Quantitation of the effect of loss-of-function alleles of known and candidate genes on crystal cell development. The chart shows the average number of crystal cellsper embryo as marked by ProPO A1 riboprobe (n � 10). The wild-type average of 36 is the combined averages of Ore-R, Canton-S,heterozygous nonmutant P10579/Cyo, Kr-GFP, and homozygous nonmutant P10579 embryos. The complete Rab5 DN genotype issrp-Gal4/UAS-Rab5DN. for06860 is the strongest allele available; however, is not a null allele, thus accounting for the reduced crystalcell effect as compared to line 10382. (B) Stage 13–14 mutant embryos were hybridized with ProPO A1 riboprobe. All recapitulatethe crystal cell reduction of their corresponding P line except for Gug, which is unlikely to be the insertion site of P11622, as itdoes not recapitulate the mislocalized phenotype.

332 A. B. Milchanowski et al.

Figure 3.—Enhancer trapexpression patterns of P lines.Four examples of P lines con-tain P-lacZ inserts express-ing lacZ in hemocytes. Em-bryos of all stages werestained with �--gal anti-body. Arrows indicate a rep-resentative hemocyte ineach embryo. (A and B)Stage 11 embryos in whichthe enhancer trap linesshow expression in hemo-cytes. (C and D) Stage 14embryos in which enhancertrap expression is evident inmature crystal cells.

of plasmatocytes (Figure 7). These are P10848, P11525, plasmatocytes were clustered in the anterior one-thirdof this mutant. Similarly, P11622 also exhibits a mis-P11538, P11716, P11745, and P10676. Plasmatocytes

were found to be mislocalized in P10555, where instead localization of plasmatocytes into large clumps through-out the embryo (Figure 7).of an even distribution throughout the entire embryo,

Figure 4.—Characterization of the effect of crystal cell mutants on mesoderm development. Stage 8 embryos of wild-type(Ore-R) and mutant P lines stained with �-Twi antibody are shown. All 44 P-line mutants demonstrate proper Twist expressionand mesoderm patterning.

333Crystal Cell Development in Drosophila

Figure 5.—Characterization of the effect ofcrystal cell mutants on anterior/posterior segmenta-tion. Stage 10 embryos of wild-type (Ore-R) andmutant P lines stained with �-En antibody areshown. All 44 P-line mutants demonstrate wild-type expression of Engrailed.

Classes of genes that reduce crystal cell development: sion pattern of several of the enhancer trap lines inembryonic hemocytes and the fact that eight of theWith the exception of the P line interrupting srp, we

did not observe a complete elimination of crystal cells mutants identified affect both crystal cells and plasmato-cytes also indicate that these P-element insertions inter-in any of our mutants although they show significant

reductions. There can be a number of reasons for this rupt genes involved in blood development. Known andcandidate genes isolated in this screen can be placedpartial loss. First, approximately half of the P lines that

interrupt previously characterized genes have a mater- into four groups: transcriptional regulators, signalingmolecules, cell proliferation regulators, and other mis-nal complement of RNA. Future analysis of germline

clones for each of the P-line mutants will determine if cellaneous factors.Transcriptional regulators: srp, a GATA factor homo-maternal contribution is responsible for the observed

partial development of crystal cells. As a test case, we log required for all hematopoiesis in Drosophila, wasfound in the screen to ablate crystal cells. This is reason-found that ebi germline clones have a significantly more

severe defect in crystal cells than that seen with the able as Srp is necessary for the expression of Gcm andLz, the transcription factors that define the two mainzygotic loss-of-function P line (data not shown). Second,

P-line insertions tend to cause hypomorphic rather than branches of Drosophila hematopoiesis (Lebestky et al.2000; Fossett et al. 2003; Waltzer et al. 2003). Muta-complete null mutations. However, we tested all avail-

able null alleles corresponding to our mutants and these tions in genes encoding transcriptional corepressors,CtBP and Sin3A, cause a significant reduction in the num-still eliminate only a fraction of the crystal cell popula-

tion. Finally, the incompleteness in the number of crys- ber of crystal cells. CtBP plays several roles as a corepressorthat binds short-range repressors such as Knirps, Snail,tals cells lost could reflect the flexible nature of the

blood development system, allowing multiple develop- Hairless, and Kruppel (Nibu et al. 1998). In Xenopus,CtBP interacts with FOG to suppress GATA factors andmental signal response pathways that lead to cell differ-

entiation. thereby block erythroid development. U-shaped has alsobeen shown to repress crystal cell development; how-By excluding general defects in the patterning of the

embryo, we were able to show that the P-line mutants ever, loss of CtBP does not cause an increase in crystalcell numbers as with Ush. CtBP also interferes with bothlikely have a direct role in crystal cell development. Of

course, this does not preclude the function of these Dpp and Notch signaling pathways (reviewed by Morelet al. 2001; Turner and Crossley 2001). Sin3A formsgenes in other tissues. In fact, many genes identified in

the screen are pleiotropic. Our results do suggest that a complex with histone deacytelases, RpAp46/48, his-tone-binding proteins, and with many other proteins tothe observed defects in hematopoiesis are not secondary

consequences of gross patterning defects. The expres- act as a transcriptional repressor (reviewed by Ahringer

334 A. B. Milchanowski et al.

Figure 6.—Quantitationof the effect of crystal cell mu-tants on the cell cycle. (A)The average number of �-Phospho-H3-positive cells inthe head region per embryo(n � 3). (B) �-Phospho-H3staining in a stage 14 wild-type (Ore-R) embryo. (C)Stage 14 stg01235 embryo shownhere as an example of in-creased �-Phospho-H3-posi-tive cells. In B and C, the lineindicates the defined head re-gion for this analysis as theanterior-most edge of the am-nioserosa to the anterior endof the embryo.

2000). Thus, Sin3A could play a role in repressing a signal- Notch signaling in a non-cell-autonomous manner bytargeting Delta for internalization and subsequent deg-ing factor, allowing for the preferential development of

the crystal cell lineage over plasmatocytes. radation (Lai et al. 2001). Loss of Notch signaling, whichregulates Lz expression in crystal cell precursors, is oneSignaling pathways: One factor with a relationship to

the Toll pathway (Smt3) and two with roles in Notch possible explanation for the decrease in crystal cellscaused by neur j6B12. hiiragi (hrg), a candidate gene forsignaling (Neur and Hrg) were detected in the screen.

smt3k06307 and neuralized (neur j6B12) were found to cause 10676, encodes an adenyltransferase that is importantin the regulation of the Notch pathway (Bachmann anda particularly strong reduction of crystal cells. Smt3 is

the Drosophila homolog of SUMO-1, an ubiquitin-like Knust 1998). Hrg downregulates the transcription ofSerrate during larval wing development (Murata et al.protein, which may conjugate with other proteins and

alter their function. One such role of Smt3 is to regulate 1996). Therefore, Hrg may be important in the tempo-ral regulation of Notch signaling during crystal cell de-the nuclear localization and transcriptional activation

of Dorsal, a well-characterized component of the Toll velopment. Note that previous work has established thatthe Notch pathway is essential for crystal cell develop-signaling pathway, which is involved in both hematopoi-

esis and immunity (Qiu et al. 1998; Wu and Anderson ment (Lebestky et al. 2003).Four mutants could be linked to the Decapentaplegic1998; Bhaskar et al. 2000, 2002). The loss of smt3 may

lead to the loss of Dorsal import and interfere with (Dpp) pathway: punt (put), Rab-protein 5 (Rab5), labial (lab),and poils aux pattes (pap). Drosophila Dpp is a TGF-normal NF�B-like signaling, which, one may speculate,

is needed for proper crystal cell development. The E3 family member, and the TGF- pathway has been knownto play a pleiotropic role in all stages of mammaliantype ubiquitin ligase, Neur, was discovered as a neuro-

genic gene required to direct neuroectodermal cells hematopoiesis (reviewed by Ruscetti and Bartelmez2001); however, its role in Drosophila hematopoiesisfrom a neural to an epidermal fate (Boulianne et al.

1991; Lai and Rubin 2001). Neur positively regulates has not been previously explored. Loss-of-function mu-

335Crystal Cell Development in Drosophila

crystal cell development when ectopically expressedthroughout the mesoderm using the twi-Gal4 driver, butnot when expressed in prohemocytes using a srp-Gal4driver (Figure 8, O and P). These studies support anindirect role of Dpp in crystal cell development and arequirement of Dpp signaling in the head mesodermadjacent to Srp-positive prohemocytes.

To investigate the effect of ectopic dpp throughoutthe mesoderm, we expressed UAS-dpp with a twi-Gal4driver. Ectopic Dpp signaling in the mesoderm causesmislocalization of crystal cells to further posterior withinthe embryo (Figure 8Q). A null allele of brk, which hasbeen shown to cause an upregulation of Dpp targetgenes (Jazwinska et al. 1999a,b; reviewed by Nakayamaet al. 2000), phenocopied this phenotype (Figure 8R),thereby indicating a role for Dpp target genes in thepatterning of the mesoderm to establish a backgroundin which normal crystal cell development may occur.Additionally, put10460 also causes a reduction in the num-ber of plasmatocytes in the embryo.

Cell proliferation regulators: Two crystal cell mutantswith known roles in cell proliferation are fat and ebi. Amember of the Cadherin superfamily, Fat is importantin cell-cell adhesion and plays an autonomous role inthe regulation of cell proliferation (Mahoney et al.1991). Loss of fat function may cause a defect in theproliferation of crystal cells due to a misregulation ofproliferation signals. ebi, which encodes a GTP-bindingmolecule containing WD40 repeats and an F-box do-main, causes a significant reduction in crystal cells.WD40 repeats can bind E3 type ubiquitin ligases andbring them in contact with F-box-bound proteins thatare to be degraded. Ebi regulates cell proliferation bylimiting the entry of cells into the S-phase of the cellcycle during neuronal development (Boulton et al.2000) and we show, with �-Phospho-H3 staining, that ebialso affects cell cycle regulation during embryogenesis.However, in the regulation of Suppressor of Hairlessduring photoreceptor development, ebi functions down-

Figure 7.—Characterization of the effect of crystal cell mu- stream of EGF signaling in cell fate specification (Tsudatants on plasmatocytes. Stage 13–14 embryos of wild-type andet al. 2002). Given that ebi k16213 does not affect plasmato-mutant P lines hybridized with CIV riboprobe are shown.cytes, it is possible that the role of ebi in crystal cellP10676, P10848, P11525, P11538, P11716, and P11745 show

a reduction in the number of plasmatocytes. Crystal cell mislo- development is in patterning the precursor and not incalization mutants P10555 and P11622 also show a mislocaliza- the regulation of cell proliferation.tion of the plasmatocytes. Other bona fide cell cycle factors identified by the screen

include String (Stg), a Cdc25 homolog, Centrosomal andchromosomal factor (Ccf or Corto), and Twisted bristletants in the Dpp pathway, dppH46, scw11, tkv7, put135, Mad12,roughened eye (Twr). Stg is a nuclear tyrosine phospha-Med5, and shn1, all cause significant reductions in thetase that controls the G2/M transition of the cell cyclenumber of crystal cells while mutants in the secondby dephosphorylating Cdc2, a mitotic kinase (reviewedtype I Dpp receptor, sax, and the second type II Dppby Edgar 1994; Edgar et al. 1994a,b). stg 01235 causes areceptor, wit, do not (Figure 8, A–K).strong reduction in the number of crystal cells and alsoDpp acts in an indirect and non-cell-autonomousreduces the number of plasmatocytes. Little is knownmanner during Drosophila blood development. Activeabout twr except that hypomorphic alleles affect the eyeDpp signaling, marked by pMad staining, is detected inand macrochaetae (Lewis et al. 1980). Corto is necessarycells adjacent to, but not colocalizing with, Srp-positivefor proper condensation of mitotic chromosomes andcells in the head mesoderm (Figure 8, L–N). brk, a

negative regulator of Dpp target genes, is able to block the maintenance of chromosome structure during mito-

336 A. B. Milchanowski et al.

Figure 8.—Characterization of the effect of Dpp pathway mutants on crystal cells. (A–J) Stage 13–14 mutant embryos werehybridized with ProPO A1 riboprobe. The graph in K demonstrates the number of crystal cells found in each mutant (n � 10).(L–N) Double staining of stage 11 srp-Gal4 � UAS-Gal embryos with (L) �-pMAD (red) and (M) �-Gal (green) antibodies.(N) The merged image of �-pMAD and �-Gal staining shows �-pMAD-positive cells are adjacent to the �-Gal-positive cells.(O–R) Stage 14 embryos hybridized with ProPO A1 riboprobe. (O) Ectopic expression of brinker driven by twi-Gal4. (P) Ectopicexpression of brinker driven by srp-Gal4. (Q) Ectopic expression of dpp driven by twi-Gal4. (R) brkM68.

sis and interphase (Kodjabachian et al. 1998). Corto al. 1999) and is expressed in both the head mesodermand mature crystal cells, consistent with a potential roleis also a putative regulator of Hox genes (Lopez et al.

2001). papL7062 and two unknown mutant lines, P12045 in crystal cell development. Fpps encodes a protein re-quired for sterol biosynthesis, which in plants is impor-and P12196, were also identified by this study to affect

the cell cycle; however, their role in this process remains tant in membrane stability, cell growth, proliferation,and respiration (Gaffe et al. 2000). FKBP59 encodes ato be investigated.

Other factors: Eleven mutants were identified in the peptidylprolyl isomerase, which has been indicated tofunction as a molecular chaperone during protein fold-screen that correspond to genes that have interesting

functions, but do not fit into any one category: U2 small ing. Consistent with a role in crystal cell development,FKBP59 has been shown to have a unique expressionnuclear riboprotein auxiliary factor 38 (U2af38), Prp19, for-

aging (for), Farnesyl pyrophosphate synthase (Fpps), FK506 pattern in embryonic lymph glands (Zaffran 2000).Interestingly, FKBP59 is also expressed in a cell type-binding protein 59 (FKBP59), Aldehyde dehydrogenase type

III (Aldh-III), outspread (osp), synaptotagmin (syt), Sec61�, and developmental stage-specific pattern during mousemale germ cell differentiation (Sananes et al. 1998).Catecholamines up (Catsup), and crossbronx (cbx). RNA

splicesome components U2af38 and Prp19, both neces- Aldh-III, which causes a weak reduction of crystal cells,is known as the tumor-associated Aldh due to its upregu-sary for RNA splicing (Tarn et al. 1993; Rudner et al.

1996), were found to affect crystal cell development. lation in several human tumor types (Park et al. 2002).Aldh’s are considered to be general detoxifying enzymesLoss of RNA splicing can lead to the improper pro-

cessing of many important developmental proteins. that oxidize toxic biogenic and xenobiotic aldehydes(reviewed by Yoshida et al. 1998). osp encodes a putativeHowever, it is unclear why the development of hemato-

poietic cells should be particularly sensitive to the func- transmembrane receptor involved in developmentalprocesses (Ashburner et al. 1999). Syt is a calcium phos-tion of these proteins. for encodes a serine/threonine

kinase involved in larval feeding behavior (Renger et pholipid-binding protein that aids in synaptic vesicle

337Crystal Cell Development in Drosophila

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