Methods xxx (2014) xxx–xxx

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Methods

journal homepage: www.elsevier .com/locate /ymeth

Drosophila hematopoiesis: Markers and methodsfor molecular genetic analysis

http://dx.doi.org/10.1016/j.ymeth.2014.02.0381046-2023/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Department of Molecular, Cell and DevelopmentalBiology, University of California, Los Angeles, Los Angeles, CA 90095, USA. Fax: +1310 206 3987.

E-mail address: banerjee@mbi.ucla.edu (U. Banerjee).

Please cite this article in press as: C.J. Evans et al., Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.02.038

Cory J. Evans a, Ting Liu a, Utpal Banerjee a,b,c,d,⇑a Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USAb Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USAc Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USAd Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA

a r t i c l e i n f o

Article history:Received 15 February 2014Revised 26 February 2014Accepted 28 February 2014Available online xxxx

Keywords:DrosophilaBloodHematopoiesisLymph glandProtocolHemocytes

a b s t r a c t

Analyses of the Drosophila hematopoietic system are becoming more and more prevalent as developmen-tal and functional parallels with vertebrate blood cells become more evident. Investigative work on thefly blood system has, out of necessity, led to the identification of new molecular markers for blood celltypes and lineages and to the refinement of useful molecular genetic tools and analytical methods. Thisreview briefly describes the Drosophila hematopoietic system at different developmental stages, summa-rizes the major useful cell markers and tools for each stage, and provides basic protocols for practicalanalysis of circulating blood cells and of the lymph gland, the larval hematopoietic organ.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction specified and develop and how this relates to blood-forming pro-

Blood cells have been studied in invertebrate animal models formore than one hundred years, although how these cells relate tovertebrate blood cells in function and development has only re-cently begun at the molecular genetic level. This work is high-lighted by studies in the fruit fly Drosophila melanogaster, anestablished premier model system for genetic studies in other con-texts. The earliest studies of blood cells (known as hemocytes) inDrosophila date back to the late 1800s, however significant pro-gress in this area did not begin in earnest until the 1950s when Riz-ki and Rizki began their studies of blood cell types and associatedfunctions, particularly in the areas of innate immune responsesand self-/non-self-recognition, and established the nomenclaturefor the Drosophila blood cell system that is still in use today[1,2]. Furthermore, their observational work led to the earliest sug-gestion that the various Drosophila blood cell types arise from acommon precursor cell type, an idea similar to that which wouldbe established in the mouse within the next few years [3].

Since that time, with the advent of molecular genetics, quite abit has been discovered about how Drosophila blood cells are

cesses in vertebrate systems. Much of this knowledge has been ac-quired with the identification and use of many new cellularmarkers, genetic tools, and analytical methods. This review willbriefly describe the key elements of the Drosophila hematopoieticsystem and highlight useful reagents and approaches that are cur-rently available.

2. The Drosophila hematopoietic system

Mature Drosophila blood cells are found as at least three distincttypes known as plasmatocytes, crystal cells and lamellocytes. Theplasmatocyte behaves as a macrophage-like cell and is the predom-inant cell type comprising more than 90% of the hemocyte repertoire[1,4]. Crystal cells, named for their cytoplasmic paracrystallineprotein inclusions, represent approximately 5% of hemocytes incirculation while lamellocytes are rarely observed. Lamellocytescan be induced to differentiate, however, in the context of variousconditions including immune challenge by parasitic wasps [5–7].Several additional cell types have been described based on morpho-logical features or as precursor populations lacking differentiationmarkers [2,8–10], but their identification as independent cell typeswill require the discovery of new and unique markers.

A major function of hemocytes is in the provision of cellular in-nate immune responses, which is achieved primarily through thephagocytic clearance of microbial pathogens or the encapsulation

2 C.J. Evans et al. / Methods xxx (2014) xxx–xxx

of larger parasites. Additionally, plasmatocytes are known to pro-mote the humoral immune response by secreting cytokine-likeproteins and antimicrobial peptides [11–15]. Accordingly, flieswith impaired plasmatocyte function are more susceptible thanwild-type flies to microbial infection and are less effective in thepotentiation of humoral immune responses [16–19]. Hemocytesalso have roles during development where they secrete and remo-del extracellular matrix components as well as remove cellulardebris by phagocytosis [20–23]. These roles are highlighted bytheir requirement in the embryonic nervous system, the develop-ment of which is severely disrupted in the absence of plasmatocytefunction [24].

As professional phagocytes, plasmatocytes use a large reper-toire of receptors to identify and engulf different targets. Receptorsrecognizing dying cells include Croquemort (Crq), a CD36 familymember [25], PSR, a phosphatidyl serine receptor [26], and Draper,which is homologous to the CED-1 phagocytosis receptor in C. ele-gans [27,28]. Numerous receptors that bind microbial pathogensare also known and include the peptidoglycan recognition proteinPGRP-LC and the scavenger receptor D-SR-CI [29–31]. Additionally,the receptors Eater, Nimrod (NimC1, also known as the P1 antigen)and Dscam have been found to be important in bacterial clearance.Eater and Nimrod both contain multiple EGF-like motifs, calledNIM repeats, which, in the case of Eater, have been shown to bindbacteria and mediate an array of intermolecular interactions[32,33]. Nimrod belongs to a family of ten related proteins(grouped into three classes, NimA-NimC), the majority of whichare expressed by hemocytes [33]. Interestingly, the NimB proteinsappear to function as opsonins because they enter the secretorypathway but lack transmembrane and cytosolic domains. Severalisoforms of Dscam have also been identified in the hemolyph sug-gesting that it may also function to opsonize microbes [34]. Theextracellular domain of Dscam receptors is composed of repeatingimmunoglobulin-like domains, a subset of which are highly vari-able due to alternative splicing [35]. Though microarray analysishas shown that more than 18,000 alternatively-spliced isoformsare expressed by hemocytes, just five isoforms comprise 80–90%of Dscam mRNAs in these cells, suggesting a blood-specific rolefor these particular Dscam types [34].

While plasmatocytes primarily function in phagocyticclearance, the main function of crystal cells is in the process of mel-anization, the darkening and hardening of tissue due to the local

Table 1Established genetic markers for the embryonic hematopoietic system.

Hematopoietic cells Marker Genetic reporters Antibody*

Embryoniccirculatingcells

srp srp-gal4srp-HEMO-gal4srpD-gal4

P

gcm gcm-lacZgcm-gal4

P

lz lz-gal4 Mcrq crq-gal4 PproPO-A1 PCgC25 Cg-gal4Pxn Pxn-gal4 P

Embryoniclymph gland

srp P

odd odd-gal4odd-lacZ

P

Hand Hand-gal4Hand-GFPHand-lacZ

Antp Antp-gal4 Mhth Pcol col-gal4 PDot Dot-gal4

* P = polyclonal; M = monoclonal.

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deposition of melanin. Melanization commonly occurs during im-mune responses in the context of barrier formation around patho-gens too large to remove by phagocytosis, as well as during theprocess of wound healing, particularly when the cuticular epitheliahas been breached. Phenoloxidase (PO) enzymes mediate the oxi-dation of phenols into quinones that then polymerize into melanin.Tightly-regulated serine protease cascades convert Prophenoloxi-dase (PPO) zymogens into the active PO form [36–39]. Experimen-tal evidence indicates that the large, cytoplasmic inclusions thatcrystal cells exhibit (and from which they derive their name) arecomposed primarily of PPO [2,5,10,40]. Furthermore, Drosophilamutant lines that lack crystal cells also lack PO activity in thecirculating hemolymph, identifying crystal cells as the primarysource of this activity [40–43]. Genetic analysis suggests thatcrystal cell rupture is the mechanism by which PO activity is deliv-ered to the hemolymph, a process mediated by JNK signaling andthe TNF homolog Eiger [41]. Despite the distribution of PPOthroughout the hemolymph, PO activity is tightly regulated andspatially restricted, such as at sites of coagulation and clotformation [44].

Lamellocytes are the most morphologically distinct blood celltype, being large (15–40 lm across), disc-shaped cells. These cellsfunction during the encapsulation response, where a cellular bar-rier, which includes plasmatocytes and crystal cells [45], formsaround foreign objects that cannot be removed by phagocytosis.Normally, very few lamellocytes are observed [46]; howevernumerous lamellocytes can be induced to differentiate inresponse to signals that include parasitic wasp infestation, injec-tion of foreign objects into the hemocoel, and sterile wounding[5,6,13,47].

With regard to understanding innate immune mechanisms,Drosophila has proven to be an exceptional model that has pro-vided key insights into systems, such as Toll-like receptor signal-ing, with relevance to human biology [48]. The majority of thiswork in flies has focused on understanding the humoral responseto microbial pathogens, however the role of hemocytes in the im-mune context is beginning to be explored in more detail. Method-ologies and protocols describing microbial challenge and themonitoring immune signaling have been previously published byLemaitre and colleagues [49,50]. A detailed protocol demonstratingwasp paristization and associated analytical methods has alsorecently been made available [51].

Cell type References

Pan Milchanowski et al. (2004) [63]Brückner et al. (2004) [109]Crozatier et al. (2004) [64]

PL Bernardoni et al. (1997) [110]Cho et al. (2002) [111]

CC Lebestky et al. (2000) [53]PL Franc et al. (1996) [25] and Olofsson and Page (2005) [112]CC Rizki et al. (1985) [40] and Waltzer et al. (2002) [113]PL Milchanowski et al. (2004) [63]; our observationPL Nelson et al. (1994) [95] and Stramer et al. (2005) [69]Pan Jung et al. (2005) [55]

Pan Ward and Skeath (2000) [114]Jung et al. (2005) [55]

Pan Han et al. (2005) [115]Evans et al. (2009) [116]

PSC Mandal et al. (2007) [65]Pan, non-PSC Mandal et al. (2007) [65]Pan, PSC Crozatier et al. (2004) [64] Krzemien et al. (2007) [79]Pan, PSC Kimbrell et al. (2002) [117] Jung et al. (2005) [55]

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Table 2Established genetic markers for the larval lymph gland.

Hematopoietic cells Marker Genetic reporters Antibody* Mature cell type References

Pan srp P Lebestky et al. (2000) [53]odd odd-lacZ P Jung et al. (2005) [55]He He-gal4 M Kurucz et al. (2003) [118]

Posterior Signaling Center (PSC) Antp Antp-gal4 M Mandal et al. (2007) [65]Ser Ser9.5-lacZ

Ser9.6-gal4P Lebestky et al. (2000) [53]

Jung et al. (2005) [55]hh hh-GFP P Mandal et al. (2007) [65] and Tokusumi et al. (2009) [82]col col-gal4 Crozatier et al. (2004) [64] and Krzemien et al. (2007) [79]Pvf1 P Mondal et al. (2011) [85]

Progenitors/Medullary Zone dome dome-gal4dome-gal4; ELAV-gal80dome-MESO-lacZdome-MESO-EGFPdome-MESO-EBFP2

Jung et al. (2005) [55]Krzemien et al. (2007) [79]Mondal et al. (2011) [85]this work

DE-Cad M Jung et al. (2005) [55]upd3 upd3-gal4 Jung et al. (2005) [55]ptc M Mandal et al. (2007) [65]wg M Sinenko et al. (2009) [86]ci M Mandal et al. (2007) [65]ROShigh Owusu-Ansah and Banerjee (2009) [90]bam M Tokusumi et al. (2011) [89]p-CamK-II P Mondal et al. (2011) [85]p-Akt P Shim et al. (2012) [88]GABA P Shim et al. (2013) [119]GABA-Rhigh P Shim et al. (2013) [119]TepIV TepIV-gal4

TepIV-gal4; ELAV-gal80Irving et al. (2005) [102] and Krzemien et al. (2007) [79]Avet-Rochex et al. (2010) [87]

Pvrlow P Jung et al. (2005) [55] and Mondal et al. (2011) [85]Differentiating cells/cortical zone NimC1 (P1) M PL Kurucz et al. (2007) [33] and Kurucz et al. (2007) [97]

Hml Hml-gal4Hml-DsRed

PL Sinenko et al. (2004) [120] andMakhijani et al. (2011) [59]

eater eater-gal4eater-GFP

PL Tokusumi et al. (2009) [82]

Pvrhigh P Jung et al. (2005) [55] and Mondal et al. (2011) [85]Pxn Pxn-gal4 P PL Jung et al. (2005) [55] and Stofanko et al. (2008) [61]Collagen(gal4)

Cg-gal4 M PL Jung et al. (2005) [55]

GABA-Rlow P Shim et al. (2013) [119]lz lz-gal4 M CC Lebestky et al. (2000) [53] and Jung et al. (2005) [55]proPO-A1 Bc1

Bc-GFP, -BFP, -RFPP CC Rizki et al. (1980) [121], Jung et al. (2005) [55] and

Tokusumi et al. (2009) [82]Sima P CC Muhkerjee et al. (2011) [122]ItgaPS4 P LM Crozatier et al. (2004) [64] and Irving et al. (2005) [102]msn msn-lacZ

msn-GFP, msn-RFPLM Braun et al. (1997) [99] and

Tokusumi et al. (2009) [100]atilla (L1) atilla-GFP M LM Honti et al. (2009) [103]mys M LM Irving et al. (2005) [102]

* P = polyclonal; M = monoclonal.

C.J. Evans et al. / Methods xxx (2014) xxx–xxx 3

Much like in vertebrate systems, hematopoietic developmentin Drosophila is biphasic with regard to timing and location[8,52,53]. Drosophila blood cells are first specified in the headmesoderm and migrate throughout the developing embryo [54].At later stages of embryogenesis, a second group of hematopoieticprecursors are specified within the cardiogenic mesoderm(thoracic segments 1–3) and form the lymph gland, a specializedorgan that supports the hematopoietic process throughout subse-quent larval development [55,56]. At the end of the larval stage,the lymph gland breaks down and releases blood cells into circu-lation [57,58]. Several reports have also suggested that hemato-poietic cell populations exist as sessile cells in association withthe internal larval body wall and/or the dorsal vessel (heart),however a better understanding of the blood-forming potentialof these cells will require further exploration [59–61]. It has beendemonstrated that circulating blood cells derived from both theembryonic head mesoderm and the lymph gland persist intothe adult stage [57], however a major unanswered question is

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whether any adult blood cells arise from de novo hematopoiesisduring the pupal or adult stages.

3. Analysis of embryonic hemocytes

Molecular genetic analysis of embryonic blood cells has madeuse of standard in situ hybridization and immunostaining methodsin combination with specific markers. The number of genes withexpression patterns overlapping with embryonic hemocytes hasgrown in the last several years, however with regard to early devel-opmental analysis of blood cells derived from the head mesoderm,several markers stand out: serpent (srp), glial cells missing (gcm),lozenge (lz), Prophenoloxidase A1 (ProPOA1), collagen (Cg25c), andPeroxidasin (Pxn; see Table 1). The onset of Srp (a GATA transcrip-tional regulator) expression in the head mesoderm defines bloodcell identity, while the expression of Gcm and Lz (a Runx familytranscriptional regulator) mark the plasmatocyte and crystal cell

oi.org/10.1016/j.ymeth.2014.02.038

4 C.J. Evans et al. / Methods xxx (2014) xxx–xxx

lineages, respectively [8,53]. Maturing macrophages express colla-gen and Pxn, while ProPOA1 is a marker of mature crystal cells. Thelamellocyte cell type has not been reported in the embryo. Each ofthe described markers has been analyzed by in situ hybridizationand/or antibody staining, and some excellent examples describingtheir specific use, either alone or in combination, can be found inWaltzer et al. (2003) [62] and Milchanowski et al. (2004) [63],among others.

Embryonic lymph gland cells express srp, similar to head meso-derm blood cells, however the lymph gland lineage also expressesodd skipped (odd) [55,64]. Because of the existence of Odd antibodyand the reporters odd-lacZ and odd-gal4, odd expression has been amarker of choice for identifying lymph gland cells in late-stage em-bryos. Expression of the homeotic gene Antennapedia (Antp) alsodefines the earliest subdivision (stage 11) of the lymph gland byspecifying Posterior Signaling Center (PSC) cells, which behave asniche-like cells at later developmental stages [65]. The EBF factorCollier (Col, also known as Knot) is also an important regulatorand marker of the PSC (stage 11 onward) [64]. Collier functionsdownstream of Antp in the PSC and is important for its mainte-nance during larval development [65]. In col mutant animals, thePSC is lost, which causes lymph gland progenitors to differentiateprematurely [64]. Additional embryonic lymph gland markers aredescribed in Table 1.

Drosophila embryos have also been ideal for research groupsinterested in live imaging of blood cells. Because of their small size,relative transparency, and the vast genetic tools that are available,live imaging of embryonic hemocytes has been used to addressseveral basic developmental and cell biological questions, such ascell migration mechanics and chemotaxis [66–69]. Because of theversatility of this system, relatively recent detailed protocolsdescribing live imaging and in vivo tracking of embryonichemocytes have been published [70,71].

4. Analysis of larval hemocytes

Over the last several years, analysis of Drosophila hematopoie-sis has primarily focused on the larval stages. Recently, there hasbeen growing interest in characterizing larval circulating cells inboth functional and hematopoietic contexts, and several protocolsfor in vitro, ex vivo, and in vivo hemocyte analysis have been de-scribed [59,71–77]. A basic protocol for the collection and prepa-ration of circulating larval hemocytes for analysis is described inBox 1. Most larval analyses, however, have examined blood devel-opment in the lymph gland, which has proven to be a usefulsystem for understanding mechanisms of progenitor cell mainte-nance and differentiation [8,78]. Although the lymph gland isspecified in the embryo, its growth and differentiation into ma-ture blood cells occurs during the larval stages. Owing to a lackof good molecular markers, early work was limited mainly toobservation. However, many new markers and tools have beenidentified (see Table 2) that, in combination with the applicationof advanced microscopic techniques, have greatly enhanced thecurrent understanding of how hematopoiesis occurs in the lymphgland. In particular, these advances demonstrated that the pri-mary lobes of the lymph gland can be subdivided into distinctcellular populations that are spatially organized [55]. The periph-ery of each primary lobe contains maturing blood cells wasnamed the Cortical Zone (CZ), whereas the juxtaposed medial re-gion contains blood progenitor cells and was termed the Medul-lary Zone (MZ). The posterior tip of each primary lobe harborsthe Posterior Signaling Center (PSC), several dozen specializedlymph gland cells that do not behave as blood cells but rather

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form a niche-like group that supports hematopoietic development[65,79,80].

oi

Box 1 Collection and processing of larval circulating bloodcells.

1. Collect larvae and wash thoroughly. This is important to

ensure that the circulating cell preparation is not contam-

inated with food debris, yeast, and bacteria (which will

obscure observation of the cells) from the culture.

2. Place a 12 (4 mm diameter)-well glass slide on the stereo

microscope and illuminate with the transmitted light

base.

3. Place a 20 lL drop of 1XPBS on one well spot of a 12 (4

mm diameter)-well glass slide. Do not place a drop on

each well at the beginning as evaporation will signifi-

cantly decrease the buffer volume as you proceed.

Instead, use one drop at a time.

4. Place one larva in the drop of buffer.

5. Using two pairs of forceps, pinch the body wall/cuticle at

the posterior of the larva. Keep one forceps in place

while tearing the cuticle down the length of the larva

toward the anterior. As the hemocoel is opened, hemo-

lymph will begin to stream into the 1XPBS buffer; this

should be relatively easy to observe because of differen-

tial fluid densities. To obtain a clean preparation of iso-

lated blood cells, it is essential to grab and tear only

the cuticle. Avoid damaging internal organs particularly

the gut, the contents of which will significantly contami-

nate the sample and will be difficult to wash away from

the slide surface.

6. Once the larval hemocoel is open, use forceps to gently

move the larva through the buffer to evenly distribute

the hemolymph and hemocytes throughout.

7. Using single tines from two different forceps, gently

remove the carcass from the drop of buffer containing

the blood cells. Do this slowly so that minimal buffer is

removed. Do not use one pair of forceps to pick up the

carcass as too much buffer will also be removed due to

capillary action between the tines.

8. Repeat for remaining samples.

9. Carefully place slide into humidified chamber. Incubate

for 30 min to let blood cells settle onto the glass surface.

10. Remove slide from the humidified chamber.

11. Under the microscope, carefully remove the 1XPBS buf-

fer from each drop using a pipette.

12. Add 20 lL of freshly prepared fixative to each well and place

into humidified chamber for 10 min at room temperature.

13. Under the microscope, remove fixative and replace with

20 lL of 1XPBS for short term storage at 4 �C or proceed

with subsequent assay of choice, such as immunostain-

ing. For steps using buffers with detergents, volumes of

5–10 lL are recommended to avoid mixing between wells

due to loss of surface tension. All steps should be carried

out in a humidified chamber to avoid evaporation.

14. After processing, place a small drop of VECTASHIELD on

each well, followed by a glass cover slip.

15. Seal with nail polish around the edges and proceed to

imaging.

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Box 2 Lymph gland dissection.

1. Using a large transfer pipette, place several large drops

of 1XPBS on the silicone dissection plate.

2. Place one larva in each drop. As technique improves, several

larvae can be placed in each drop without interference.

3. Place the plate on a standard stereo dissecting micro-

scope with a transmitted light base.

4. Illuminate the sample. If lighting makes use of standard

light bulbs (instead of a cold LED source), take care to

work quickly so that the larvae/samples do not overheat.

Dissect for a maximum of 30 min, with samples stored

on ice, before fixation.

5. Using forceps, orient larva dorsal-side up, anterior to the

right (for a right-handed person).

6. Using the left hand, gently hold larvae at 75% length

(A = 0%, P = 100%).

7. Using the right hand, pinch the dorsal cuticle (only, no

internal organs) at 70–75% length, and slowly pull toward

the anterior. Pull the entire length, stopping at the mouth

hooks. This will open the body cavity without damaging

internal structures. Sometimes the dorsal cuticle will com-

pletely detach or the ventral cuticle also; either is ok as long

as the body cavity is open without disrupting internal

organs. Alternative method: Using both pairs of forceps,

rip the larva in half, then invert the anterior part by pushing

the mouth hooks into the body cavity longitudinally (imag-

ine inverting a sock by pushing your hand inward at the

toes). Once inverted, proceed with step 10 below. Note: this

method opens the gut, which can make subsequent steps

more difficult because food debris, yeast, and bacteria

cloud the buffer.

8. Use both forceps to sever the larvae (internal organs and

ventral cuticle) completely at 75% length (near where the

larva was being held by the left hand).

9. Stabilize the anterior portion of the larva with the left

hand by grabbing the ventral cuticle. Using the right

hand forceps, reach inside the body cavity and directly

grasp the mouth hooks (do not grab them extenally as

this will include cuticle).

10. Gently pull the mouth hooks out, separating them from

the body wall and cuticle. Removing the mouth hooks

in this manner will bring along a complex of structures

that includes the eye imaginal discs, the brain, the dorsal

vessel, the prothoracic (ring) gland, and the lymph gland;

salivary glands are also commonly included.

11. Gently separate these lymph gland-containing com-

plexes from any residual tissues such as fat body.

12. Carefully remove the complex in a small drop of 1XPBS

retained between the tines of a single forceps) to a

microfuge tube containing cold 1 mL 1XPBS + 1 drop

1XPBST on ice. The PBST break the surface tension of

the buffer, allowing samples to sink to the bottom of

the tube.

13. Collect as many lymph gland complexes as needed.

14. Replace buffer with 1 mL of freshly prepared fixative.

15. Place microfuge tube on mixer (Nutator, orbital shaker,

or similar) for 30 min.

16. Proceed to immunostaining (described below) or other

analytical method.

17. For short term storage, wash fixed tissue with 1 mL

C.J. Evans et al. / Methods xxx (2014) xxx–xxx 5

Among lymph gland markers there are several that stand out asbeing particularly useful for genetic analysis (see Table 2 for theseand others). The first lymph gland zone to be discovered was thePSC through the expression of Serrate (Ser9.5-lacZ), encoding aNotch receptor ligand. The Serrate enhancer, which is active inthe PSC from the second instar onward, was subsequently utilizedto generate a Gal4 driver line utilizing a fluorescent reporter (UAS-GFP) and as a genetic tool to manipulate PSC cells [55,80]. Later re-search demonstrated that Antennapedia, as described previously, isexpressed in and specifies PSC cells [65]. Antennapedia expressionin the PSC is maintained throughout lymph gland development,making it a marker and driver of choice for PSC-relatedexperiments (both Antennapedia-gal4 and a monoclonal antibodyare available, see Table 2). Similarly useful reagents are antibodiesagainst Collier as well as the col-gal4 reporter line, both of whichmark PSC cells throughout larval stages [79,81]. PSC cells alsoexpress and secrete Hedgehog protein, which is sensed bymedullary zone progenitor cells and is important for their long-term maintenance within the lymph gland [65]. A direct hedge-hog-GFP reporter is available that allows for direct visualizationof PSC cells [82], and can be placed in the context of other Gal4drivers for analysis of non-autonomous PSC regulation (somethingnot possible when using Antennapedia-gal4 as a reporter, forexample).

The medullary and cortical zones were identified simulta-neously through their mutually exclusive expression ofprogenitor and mature cell markers, respectively. The classicmedullary zone marker is domeless-gal4 (domeless encodes areceptor that activates the JAK/STAT pathway), which was firstisolated as an enhancer trap [55,83]. Subsequently the domelessenhancer responsible for medullary zone expression was identi-fied and has been used as a Gal4-independent lacZ (domeless-MESO-lacZ) reporter [79,84]. Recent analysis demonstratinglymph gland regulation by the central nervous system (CNS)found that domeless-gal4 is also expressed by various neuronsin the brain. To circumvent CNS effects when using domeless-gal4 to manipulate medullary zone blood progenitors, ELAV-gal80 has been placed in combination with domeless-gal4 [85].The ELAV enhancer is neuron-specific and expresses high levelsof Gal80, a natural protein inhibitor of Gal4, thereby mitigatingany brain effects due to domeless-gal4 expression. Additionaluseful genetic markers for lymph gland medullary zone progen-itors include E-cadherin [55], Cubitous interruptus [65], Wingless[86], TepIV-gal4 [87], phospho-Akt (p-Akt) [88], and Bag of mar-bles [89] (see Table 2).

Previous analysis of mitochondrial function in the lymph glanddemonstrated that blood progenitor cells exhibit relatively highlevels of reactive oxygen species (ROS), and that these ROS estab-lish a critical signaling threshold for proper progenitor mainte-nance and differentiation [90]. Since then, fluorescent staining forROS levels has become a standard analytic procedure for assessinghow genetic change or immune challenge affects ROS withinlymph glands and circulating cells. Many oxidation-sensitive dyesare available, however dihydroethidium (DHE) has been the mar-ker of choice because of its high specificity for superoxide radicalsand its ability to freely permeate cell membranes. Upon oxidation,fluorescent DHE metabolites are well retained by cells and toleratemild fixation [91], which may be critical for simultaneous analysisof other markers such as GFP. Brief protocols for DHE analysis inboth lymph glands and circulating blood cells are included here(see Boxes 5 and 6), with a more detailed protocol available asan online resource [92].

1XPBS for 5 min; repeat; store at 4 �C.

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Box 4 Mounting fixed lymph glands for microscopy.

Dissection of lymph glands itself can be difficult even for peo-ple with lots of practice with other tissues such as imaginaldiscs, however mounting them successfully after processingis just as difficult. Fewer things are more frustrating that dis-secting high quality lymph gland samples, only to destroythem during the mounting process. As with dissection, manyapproaches will work depending on the skill level of the inves-tigator. Below is the primary method taught to individuals inour laboratory.

1. Transfer lymph gland complexes (in VECTASHIELD)

from the microfuge tube to a glass slide using a small

transfer pipette or a pipette with a 200 lL tip cut by a

razor blade to enlarge the opening.

2. Place a drop of VECTASHIELD on a new slide.

3. Using forceps, move lymph gland complexes from the

first slide to the second, taking care to minimize the

transfer of mounting medium.

4. Once transferred, use forceps to form the VECTASHIELD

into a square or rectangle shape, leaving 5–8 mm of dry

area before the side edges.

5. Carefully separate the lymph gland complexes and dis-

tribute them in the rectangle of medium. Use of single

tines of the forceps is recommended because surface

tension of the medium will cause it to flow readily

between both tines of a single forceps, which will disrupt

your tissues on the slide.

6. Place one tine of one forceps underneath the posterior

end of the dorsal vessel, lift slightly, and gently pull the

lymph glands (and anterior structures) toward the

straight, perpendicular edge of the medium where it

meets the glass slide. The brain and discs create drag

that straightens out the lymph glands (dorsal vessel) as

you move through the medium.

7. Draw the dorsal vessel/lymph glands out past the perpen-

dicular edge of the medium and onto the dry part of the

slide. As this occurs, the volume of medium around the

lymph gland decreases, causing the lymph gland and other

structures to ‘‘sit down’’ onto the glass surface, instead of

just floating around. It is critical that the tissues make con-

tact with the slide, otherwise the samples will dislodge and

become contorted upon coverslipping.

8. Repeat this process for each lymph gland around the

perimeter of the medium, such that each one is well

spaced and straight.

9. Separate the brain and imaginal discs from the lymph

glands of each complex. This is most easily achieved by

using a scissoring motion with single tines of two different

forceps (avoid placing both tines of a pair of forceps into

6 C.J. Evans et al. / Methods xxx (2014) xxx–xxx

Pl

Box 3 Lymph gland immunostaining.

For this procedure, lymph glands are left attached to themouth hook/eye disc/brain complex, 1) to provide a ‘‘handle’’so that lymph glands are not damaged because of physicalmanipulation and 2) because these tissues are often conve-nient immunostaining controls.

1. With lymph glands either in fix or 1XPBS in a 1.5 mL

microfuge tube, discard buffer.

2. Add 1 mL 1XPBST (1XPBS + 0.4% Triton), place on mixer

(Nutator or similar) for 15 min; repeat twice.

3. During the washing stage, prepare 10% normal goat

serum (NGS)/1xPBST blocking solution.

4. Discard wash buffer from lymph glands and replace with

1 mL blocking solution; block in for 30 min.

5. Dilute primary antibody in blocking solution at desired

concentration.

6. Discard blocking solution from lymph glands and replace

with 100 lL or greater volume of primary antibody and

mix well. For smaller volumes and/or limited primary anti-

body, 0.6 mL microfuge tubes, PCR tubes, or Terasaki

microtiter plates (with 10 lL wells) can be used.

7. Place tubes in a rack. For short term incubations at room

temperature, intermittent mixing or placement on an

orbital shaker is helpful.

8. Incubate for 3 h at room temperature or overnight at 4 �C.

9. Remove primary antibody.

10. Add 1 mL 1XPBST, place on mixer for 15 min; repeat twice.

11. Discard wash buffer from lymph glands and replace with

1 mL blocking solution; block 10–30 min.

12. Dilute secondary antibody (usually 1:250 for lymph

glands) in blocking solution at desired concentration.

13. Discard blocking solution from lymph glands and replace

with 100 lL or greater volume of primary Incubate in sec-

ondary antibody (diluted usually 1:250) in block for 3 h at

room temperature or overnight at 4 �C.

14. Remove secondary antibody.

15. Add 1 mL 1XPBST, place on mixer for 15 min, discard

buffer; repeat once.

16. Add 1 mL 1XPBST + DNA stain (DAPI, etc.), place on

mixer for 15 min, discard buffer.

17. Add 1 mL 1XPBS, place on mixer for 15 min, discard buf-

fer; repeat once (these wash steps remove detergent and

excess DNA stain).

18. Use pipette and dissection microscope to remove as

much buffer as possible.

19. Add a drop of VECTASHIELD to the lymph glands in the

1.5 mL microfuge tube.

20. Store in refrigerator or begin mounting procedure.

the medium). Place the tines in a ‘‘Figure X’’ over the dor-

sal vessel anterior to the primary lobes, usually at or near

the location of the ring gland. Hold one tine still while

drawing the other tine across the glass surface; as the

tines pass each other, the dorsal vessel is severed. Push

the brain complex away from the lymph gland, toward

the center of the medium pool.

10. Once the lymph glands and brain complexes have been

separated, discard or move most of the brain complexes

(and any other tissue) to a new slide. Place one brain

near each corner of the medium rectangle and one or

two in the center of the medium; these will serve as spac-

ers to protect the lymph glands from severe compres-

sion by the cover slip.

Differentiating cells of the lymph gland cortical zone expressseveral useful markers, most notably Hemolectin-gal4 and the P1antigen, which are both blood specific [55]. The Hemolectin-gal4driver is one of the earliest known markers defining the onset ofprogenitor differentiation within the lymph gland at the mid-sec-ond instar, and stays on in mature cells [55,93]. Expression of Per-oxidasin (both the protein and the Peroxidasin-gal4 reporter)[94,95] is also an early marker of cortical zone formation [55],however it is also expressed at various levels in other tissues suchas the fat body and brain. The Collagen-gal4 driver [96] is slightlylater than Peroxidasin expression in the cortical zone [55], but alsohas strong expression in the fat body and, at lower levels, in

ease cite this article in press as: C.J. Evans et al., Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.02.038

Box 4 (continued)

11. Place one short edge of an appropriately sized cover slip

down onto the glass slide near, but not in, the medium.

Take care to center it longitudinally. Place the tines of a for-

ceps under the opposite end and slowly lower the cover

slip onto the medium. To avoid air bubbles and misalign-

ment, do not drop it onto the medium. Wait for the medium

to distribute between the cover slip and the slide. Slight

pressure on the cover slip with the forceps can help distrib-

ute the medium, however be careful not to compress the

tissues. If more mounting medium is needed, it can be

added using a pipette tip at the edge of the cover slip.

Excess medium should be removed at the edge with a pip-

ette or wicked away using a Kimwipe.

12. Once the cover slip is down, seal the cover slip around

the edges using nail polish. Allow to air dry for a few

minutes, then store slides (horizontal is best) at 4 �C until

they can be imaged.

Box 5 Staining for Reactive Oxygen Species (ROS) in thelymph glands.

Special materials

� 1X Schneider’s Drosophila Medium + L-Glutamine (GIBCO,

cat. No. 11,720).

� Anhydrous dimethyl sulfoxide (DMSO) > 99.9% (Sigma–

Aldrich, cat. No. 276855).

� Dihydroethidium – special packaging (Molecular Probes,

cat No. D11347).

� VECTASHIELD Mounting Medium (Vector laboratories,

cat. No. H-1000).

� Micro Spot Plate – 3 well (Electron Microscopy Sciences,

cat. No. 71561-01).

� 12-well microscope slides with hydrophobic barrier (Erie

Scientific).

1. Dissect lymph glands in room temperature Schneider’s

medium. Do not use cold 1XPBS, which may inhibit

respiration, thereby interfering ROS production.

Because these samples are not kept on ice or immedi-

ately fixed, dissection times less than 30 min are opti-

mal for limiting tissue degradation.

2. Quickly reconstitute DHE in anhydrous DMSO (see

reagent set up). Reconstituted dye solution should

appear slightly pink in color; a more intense color such

as purple may be indicative of oxidation of the dye.

3. Make DHE staining solution by adding 1 lL of the recon-

stituted DHE/DMSO to 1 mL of Schneider’s medium (in a

microfuge tube) to give a final concentration of approx-

imately 30lM. Vortex sample 15–30 s, but not more.

4. Remove Schneider’s medium from lymph glands and

replace with the DHE staining solution. Incubate 5 min

in the dark, on an orbital shaker at room temperature.

5. Wash three times, 5-min each, with Schneider’s medium

in the dark, on an orbital shaker at room temperature.

6. Lightly fix for 5 min in 7% formaldehyde/1XPBS.

7. Wash once in 1XPBS.

8. Immediately mount lymph glands on a glass slide in

VECTASHIELD medium.

9. Image immediately using fluorescence microcopy.

C.J. Evans et al. / Methods xxx (2014) xxx–xxx 7

Please cite this article in press as: C.J. Evans et al., Methods (2014), http://dx.d

various cell types [96]. The P1 antigen is specific to mature plas-matocytes [33,97] and, by comparison, is considered to be a rela-tively late marker [55]. The P1 antigen was identified as one ofseveral proteins interacting with monoclonal antibodies derivedfrom Drosophila blood cell preparations [97]. The P1 antigen wassubsequently identified as the protein product of the Nimrod C1gene (NimC1), described previously [33]. While the anti-P1 mono-clonal antibody remains among the first in the choice of tools to beused for blood analysis given its specific expression in late plas-matocytes, ease of use, and widespread availability, it should benoted that several commonly used Drosophila parental stocks areP1-negative [98]. This recessive expression polymorphism can leadto serious problems with experimental interpretation of progenyphenotypes, making it imperative to analyze P1-expression inparental strains. The P1-negative genetic background of any impor-tant experimental stock can easily be corrected through standardchromosome mechanics [98].

Crystal cells in the cortical zone can be identified by expres-sion of the determinant gene lozenge (both the protein and thelozenge-gal4 reporter) and mature marker Prophenoloxidase A1(ProPOA1; protein). The gene encoding ProPOA1 is the Black cells(Bc) gene, and the crystal cell enhancer has been used to generateseveral different fluorescent, Gal4-independent Bc-expressionreporters [82]. Lamellocytes also now have several useful markersavailable. A classic marker of lamellocyte differentiation is amisshapen (a JNK activator)-lacZ reporter gene (msn-lacZ) [99], ini-tially isolated as an enhancer trap. The msn lamellocyte enhancerhas subsequently been identified and used to generate severaluseful Gal4-independent fluorescent reporter lines [100]. Usefullamellocyte protein markers, for which antibodies are available,include L1 (identified along with P1 described above) [97], Filam-in-240 [101], a-PS4 integrin [79], and Myospheroid (bPS-integrin)[102]. A GFP enhancer trap of the L1 gene (atilla) is also available[103].

Box 6 Staining for reactive oxygen species (ROS) in circulat-ing cells.

1. Bleed larvae into 20 lL Schneider’s medium. Mix thor-

oughly and take care to remove as little liquid volume

as possible when removing the carcass.

2. Let hemocytes settle 20–30 min in a humidified chamber

at room temperature.

3. Toward the end of the settling period, reconstitute DHE

and prepare DHE staining solution as described above

for lymph glands.

4. Wash settled hemocytes by gently removing Schneider’s

medium from the edge of each well using a pipette, and

replace with 20 lL Schneider’s medium.

5. Remove Schneider’s medium from each well and replace

with 20 lL DHE staining solution. Incubate 5 min in a

dark, humidified chamber.

6. Remove DHE staining solution and wash each well of

cells twice, 5 min each, with 20 lL Schneiders medium.

7. Lightly fix cells with 20 lL of 4% formaldehyde/1XPBS

for 5 min.

8. Remove fixative and wash once with 1XPBS.

9. Remove 1XPBS and add a small volume (�2 lL) of VEC-

TASHIELD to each well. Coverslip the slide.

10. Image immediately using fluorescence microscopy.

oi.org/10.1016/j.ymeth.2014.02.038

Box 7 Collection of adult hemocytes by perfusion.

1. Anesthetize adults of interest on a standard carbon diox-

ide pad under the dissecting microscope.

2. Using forceps and microscissors, cut a small slit or hole

in the posterior abdomen; this should be slightly lateral

so that internal organs, genitalia, and the gut are not

disrupted.

3. Place a 10 lL drop of 1XPBS onto a well of a 12-well

hydrophobic slide.

4. Using a mouth pipette, draw 1XPBS into a drawn glass

capillary needle.

5. Grasp the adult fly by the wing using forceps and gently

push the tip of the needle into the lateral thorax.

6. Place cut posterior end of the adult fly in or near the drop

of 1XPBS and gently perfuse buffer through the adult.

7. Monitor the drop of buffer on the slide for hemolymph

streaming into it; when the drop approximately doubles

in size, halt perfusion and remove the adult carcass from

the drop.

8. Place slide in a humidified chamber for 30 min.

9. Carefully remove buffer using a pipette and replace with

20 lL of freshly prepared fixative.

10. Return to humidified chamber for 10 min.

11. Carefully remove fixative and replace with 20 lL of

1XPBS, or continue with processing as described

previously.

8 C.J. Evans et al. / Methods xxx (2014) xxx–xxx

5. Development of new tools for lymph gland analysis

Although several new markers for lymph gland analysis havebeen identified, their relative utility varies. As described, somemarkers are antibody based (many of which are not monoclonal)and their availability and quality can vary widely. Genetic report-ers such as Hml-gal4, while extremely useful, can be difficult forexperiments in which it is necessary to discern whether Gal4 is acell identity marker (through UAS-GFP expression, for example)or is a tool to drive other UAS-transgenes (e.g., dsRNA) for geneticanalysis; simultaneous use can make phenotypic interpretationsdifficult. Another key issue common to the use of Gal4-based driv-ers in Drosophila is that it is nearly impossible to perform and/orinterpret results from experiments that combine multiple Gal4reporters into the same background, as expression patterns willcombine. In short, continued progress in characterizing the flyhematopoietic system will require the implementation of new ormodified genetic technologies that can complement existing tools.In the past few years, several groups have expanded the Drosophilamolecular genetic tool kit through the generation of new bipartitegene expression systems [104], such as the LexA-lexAop [105]and QF-QUAS systems [106]. Once adapted for use in thehematopoietic system (through the creation of appropriate driverlines such as Hml-QF or dome-QF), it will be possible to use suchlines in combination with the Gal4/UAS system to enhance andrefine analyses.

An alternative approach taken by our lab to overcome currentlimitations and to facilitate genetic analysis in the lymph glandhas been to generate a new line of flies that uses multipleGal4-independent fluorescent protein reporters to simultaneouslymonitor different lymph gland cell populations. To generate thisnew hematopoietic tool, we first re-tasked the dome-MESO

Please cite this article in press as: C.J. Evans et al., Methods (2014), http://dx.d

progenitor cell enhancer [84] to drive expression of enhancedblue fluorescent protein 2 (EBFP2, Ex/Em: 383/448 nm) [107]. Thistransgene was then recombined with differentiating-cell markerHemolectin-DsRed [59] and the PSC/niche cell marker hedgehog-GFP [82] onto the X chromosome. As can be seen in Fig. 1, each re-porter clearly identifies their respective cell in the lymph glandand, therefore, will be extremely useful for assessing phenotypesin various experimental contexts, such as genetic screening. Thistriple recombinant marker line, as well as the new, individual fluo-rescent dome-MESO transgenic lines, will be made freely availableto the research community.

In contrast to other organs such as the imaginal discs, brain, gut,and fat body, the lymph gland is likely to be unfamiliar to mostDrosophilists. Because of its relatively small size and delicate nat-ure, the lymph gland presents a significant challenge to anyoneinterested in analyzing it by dissection and microscopy. To provideguidance in this area, a protocol for lymph gland dissectionis described, and is followed by a standard immunostainingprocedure and a summary of how to mount high quality, intactlymph glands on glass slides for microscopic analysis (see Boxes2 and 3, and 4).

6. Analysis of adult hemocytes

As mentioned, relatively little is known about adult hemocytesor hematopoiesis, owing primarily to a lack of useful tools andmethods. In contrast to larvae, which are akin to fluid-filled bal-loons, adult bodies are rigid and have a relatively lower hemo-lymph volume, making analysis of circulating cells by ‘‘bleeding’’more challenging. This becomes even more difficult in light ofthe fact that many, if not most, adult hemocytes are sessile, associ-ated with the adult body wall and internal organs and structures.Many such adult hemocytes can be visualized through the cuticlein whole-mount preparations using markers such as Hml-gal4UAS-GFP, however approach this is not very amenable to more de-tailed analyses of blood cell fate, proliferation, or function. It isclear that more work needs to be done to develop reagents andtechniques to make the adult blood system more accessible tomolecular genetic analysis. To date, the most commonly appliedmethod for visualizing and analyzing adult hemocytes is to perfusethe adult hemocoel with aqueous buffer, which flushes hemocytesout of the adult and onto a slide for analysis. An adult perfusionprotocol is described in Box 7. Recently, such a method wasused in the identification of the first adult-specific blood marker,Ad1 [108], and further demonstrated that adult hemocytesmaintain NimC1 (P1) expression but lose expression of Hemese(He), the blood-specific pan hemocyte marker found in larvalhemocytes.

7. Summary

As with any developing field of study, continued effort on thepart of researchers will enhance the ability to manipulate and ana-lyze the fly hematopoietic system. This advancement will undoubt-edly rely in large part on the identification of improved molecularmarkers offering higher resolution identification of cell types with-in the hematopoietic lineages, as well as on improved technologiesfor genetic analysis and imaging. Of particular importance will beapproaches that allow for analyses in vivo during the larval, pupal,and adult stages, which may mitigate current difficulties inextracting blood cells from animals for ex vivo analyses. This re-view has highlighted some of the more useful genetic markersand analytic methods, both by pointing toward expertise in thepublished literature where appropriate and by providing here a

oi.org/10.1016/j.ymeth.2014.02.038

Fig. 1. A new triple-fluorescence reporter line identifies mature, progenitor, and niche cells within the hematopoietic lymph gland. (A) Differentiated blood cells in thecortical zone of a third-instar larval lymph gland are marked by DsRed protein (red) expression driven by the Hemolectin enhancer (Hml-DsRed). (B) Blood progenitor cells inthe medulary zone express EBFP2 protein (cyan) under the control of the domeless MESO enhancer (dome-MESO-EBFP2). (C) PSC cells express EGFP protein (green) under thecontrol of the hedgehog PSC enhancer (hh-EGFP). (D) A combined image (Merge) showing differential marking of blood cell populations juxtaposed in the lymph gland primarylobe. DNA (blue) is labeled with TO-PRO-3 (Molecular Probes) in each panel. (E) Overview of the generation of the dome-MESO-EGFP and dome-MESO-EBFP2 constructs. The2.8 kb dome-MESO enhancer (1) was amplified by PCR from Drosophila genomic DNA, along with an added 50 CACC nucleotide sequence for Gateway� Directional TOPO�

cloning into the pENTR/D-TOPO� entry vector (2; Life Technologies). Upon directional cloning of the dome-MESO enhancer into the Gateway entry vector (3), the enhancer wasrecombined with the Ganesh-G2 Gateway destination vector (4), thereby creating the dome-MESO-EGFP Drosophila transformation vector (5). The DNA coding sequence fornuclear-localized Enhanced Blue Fluorescent Protein 2 (EBFP2-NLS) was PCR amplified from the expression vector pLV-EBFP2-nuc (Addgene) as an AgeI/MfeI fragment (6). Thedome-MESO-EBFP2 expression vector (7) was subsequently created by removing the EGFP-NLS DNA sequence from the dome-MESO-EGFP vector by digesting with AgeI andMfeI restriction endonucleases and then ligating in the EBFP2-NLS DNA sequence into the vector using the same restriction sites.

C.J. Evans et al. / Methods xxx (2014) xxx–xxx 9

set of basic protocols for dealing with the larval and adult bloodsystem.

Acknowledgments

Because of space limitations, the review could not be exhaus-tive in its description of the hematopoietic system. We apologizeto those whose important work in the field is not cited here. Thiswork was supported by National Institutes of Health Grants R01HL067395 and R21 AI094048 to U.B., and a China ScholarshipCouncil award to T.L.

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